WO2003029404A2

WO2003029404A2 - MAST CELL RasGRP4 COMPOSITIONS AND RELATED METHODS

Info

Publication number: WO2003029404A2
Application number: PCT/US2002/024499
Authority: WO
Inventors: Richard L. Stevens; Lixin Li; Yi Yang
Original assignee: The Brigham And Women's Hospital, Inc.
Priority date: 2001-08-02
Filing date: 2002-08-02
Publication date: 2003-04-10
Also published as: WO2003029404A3; AU2002356515A1

Abstract

The invention relates to the identification of new human and mouse genes and their transcripts that encode new mast cell-restricted proteins (designated hRasGRP4 and mRasGRP4) that are able to activate varied members of the Ras family of signaling proteins in a cation-dependent manner. The invention also relates to the identification of RasGRP4 as a new phorbol ester receptor/diacylglycerol-binding protein.

Description

MAST CELL RasGRP4 COMPOSITIONS AND RELATED METHODS

Related Applications

This application claims priority under 35 U.S.C. §119 from U.S. provisional application serial number 60/309,586, filed August 2, 2001.

Government Support

This invention was made in part with government support under grant number HL63284 from the National Institutes of Health (NIH). The government may have certain rights in this invention.

Field of the Invention

The present invention relates to novel RasGRP4 genes and transcripts that have been cloned from human and mouse. The invention is directed to the isolated RasGRP4 nucleic acids, the proteins encoded by these nucleic acids, agents that selectively bind thereto, and various diagnostic, therapeutic, and research uses of these compositions.

Background of the Invention

Mast cells release a diverse array of biologically active molecules (including cytokines, chemokines, leukotrienes, prostaglandins, amines, proteoglycans, and proteases) when activated via their high-affinity IgE, complement, or protease-activated receptors. Mast cells play important roles in bacteria infections (Malaviya, R. et al., Nature 381 :77-80, 1996; Echtenacher, B. et al., Nature 381 :75-77, 1996; Prodeus, A.P. et al., Nature 390:172-175, 1997) due, in part, to their release of tumor necrosis factor-α (Wershil, B.K. et al., J. Clin. Invest. 87:446-453, 1991) and varied granule tryptases (Huang, C. et al., J. Immunol.

160:1910-1919, 1998; Huang, C. et al., J Biol. Chem. 276(29)26276-84, 2001) that work in concert to induce the extravasation of neutrophils that kill bacteria. While these and other findings indicate that mast cells play beneficial immunosurveillance and effector roles in the body, it has been known for some time that mast cells also exhibit adverse roles in numerous inflammatory disorders including asthma, chronic urticaria, and systemic anaphylaxis.

Recent studies also have revealed that these effector cells participate in the pathogenesis of AIDS. (Patella, V. et al., J. Immunol. 164:589-595, 2000; Marone, G. et al., J Allergy Clin. Immunol. 107:22-30, 2001; Li, Y. et al., Blood 97:3484-3490, 2001.

Because mast cells play such prominent roles in inflammation, an intense effort has been made to identify the regulatory proteins that act relatively early in this cell's varied intracellular signaling pathways to dampen mast cell development and/or function. Some progress has been made in our understanding of how different populations of mast cells signal through their immunoglobulin, complement, and cytokine receptors, even though no signal-transduction protein has been identified to date that is highly specific to this cell type. The Ras superfamily of small GTP-binding proteins plays pivotal roles in virtually every cell. GTP activates the varied Ras family members, whereas GDP inactivates them. Guanine nucleotide exchange factors (GEFs) are of immense importance because they activate these signal-transducing proteins by dissociating bound GDP. Those GEFs that have been identified to date in mast cells are not highly restricted to this cell type. For example, mast cells and many other cells express the Ras GEF Sos (Turner, H. et al., J. Biol. Chem. 270:9500-9506, 1995) and the Rac GEF Vav. (Song, J.S. et al., J. Immunol. 163:802-810, 1999; Song, J.S. et al., J. Biol. Chem. 271:26962-26970, 1996).

Nevertheless, a few GEFs that reportedly are more restricted in their cellular expression have been identified recently. Three such relatively restricted regulatory proteins are Ras guanine nucleotide releasing protein (RasGRP) 1 (also known as CalDAG-GEFII), RasGRP2 (also known as CalDAG-GEFI), and RasGRP3 (also known as KIAA0846 and CalDAG-GEFIII). Kedra, D. et al, Hum. Genet. 100:611-619, 1997; Ebinu, J.O. et al, Science 280:1082-1086, 1998; Kawasaki, H. et al., Proc. Natl. Acad. Sci. U.S.A. 95:13278- 13283, 1998; Nagase, T. et al, DNA Res. 5:355-364, 1998; Bottorff, D. et al., Mamm. Genome 10:358-361, 1999; Yamashita, S. et al., J. Biol. Chem. 275:25488-25493, 2000. RasGRP 1 , RasGRP2, and RasGRP3 , have additional Ca²⁺ and phorbol ester/diacylglycerol (DAG) binding motifs in their C-terminal domains which suggest possible roles in multiple signaling pathways.

Progress has been made toward understanding mast cell regulation. Although increased information about mast cell development, function, and fate have been obtained, aspects of these processes remain unclear. Elucidation of mechanisms of mast cell regulation, their association with wide-spread diseases such as asthma, allergies, and mastocytosis, and the development of agents that can modulate such mechanisms, would be beneficial.

Summary of the Invention The invention provides novel human and mouse mast cell-restricted molecules

(nucleic acid molecules and proteins) designated hRasGRP4 and mRasGRP4. The proteins are able to activate varied members ofthe Ras family of signaling proteins.

According to one aspect ofthe invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the group consisting of: (a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 1 and which code for a human RasGRP4 protein,

(b) deletions, additions and substitutions ofthe nucleic acid molecules of (a), which code for a human RasGRP4 protein, (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy ofthe genetic code, and (d) complements of (a), (b) or (c).

In one embodiment, the isolated nucleic acid molecule comprises SEQ ID NO: 1, or the coding region thereof. According to another aspect ofthe invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the group consisting of:

(a) a unique fragment ofthe nucleotide sequence set forth as nucleotides 1-2634 of SEQ ID NO: 1 between 12 and 2633 nucleotides in length, and

(b) complements of (a), wherein the unique fragments exclude nucleic acids consisting of nucleotide sequences that are contained within SEQ ID NO: 1, and that are known as ofthe filing date of this application.

According to another aspect ofthe invention, an expression vector is provided. The expression vector includes any ofthe isolated nucleic acid molecules ofthe invention, operably linked to a promoter. According to another aspect ofthe invention, a host cell transformed or transfected with the expression vector ofthe invention is provided, hi some embodiments, the host cell is a CD14⁺ cell or an HMC-1 cell.

According to still a further aspect ofthe invention, a transgenic non-human animal comprising the expression vector ofthe invention is provided. Such transgenic animals are capable of expressing a variable level of hRasGRP4, Mutant hRasGRP4, mRasGRP4, or Mutant mRasGRP4 (described below). In some embodiments, the transgenic non-human animal is a transgenic V3 -mastocytosis animal.

According to another aspect ofthe invention, an isolated protein encoded by any of the foregoing isolated nucleic acid molecules ofthe invention is provided. Preferably the isolated protein comprises the amino acid sequence of SEQ LD NO: 2 or SEQ ID NO: 21. According to another aspect ofthe invention, binding polypeptides that selectively bind to the forgoing isolated protein or nucleic acid molecules ofthe invention are provided. In some embodiments, the binding polypeptides are antibodies or an antigen-binding fragments thereof. In some embodiments, the antibodies or antigen-binding fragments specifically bind to the N-terminus ofthe isolated protein comprising an amino acid sequence of SEQ ID NO: 2 or SEQ LD NO:21. In other embodiments, the binding polypeptide selectively binds to the polypeptide sequence set forth as SEQ LD NO: 21.

According to a further aspect ofthe invention, pharmaceutical compositions are provided. The pharmaceutical compositions include an active agent selected from any ofthe foregoing nucleic acids ofthe invention and a pharmaceutical acceptable carrier. Additional pharmaceutical compositions provided include an active agent selected from any ofthe foregoing proteins ofthe invention or any ofthe foregoing binding polypeptides ofthe invention and a pharmaceutically acceptable carrier. According to another aspect ofthe invention methods for making a medicament, are provided. The methods include placing an active agent selected from the group consisting of any ofthe foregoing nucleic acids ofthe invention, any ofthe foregoing proteins ofthe invention or any ofthe foregoing binding polypeptides ofthe invention in a pharmaceutically acceptable carrier. In some embodiments, placing includes placing a therapeutically effective amount ofthe active agent in the pharmaceutically acceptable carrier to form one or more doses. According to yet another aspect ofthe invention, methods of making a human mast cell line in vitro are provided. The methods include transfecting CD 14 cells with any ofthe foregoing isolated nucleic acids ofthe invention that encodes an hRasGRP4 protein. In some embodiments, the isolated nucleic acid comprises the nucleic acid sequence set forth as SEQ H> NO: l.

According to another aspect ofthe invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the group consisting of:

(a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 58 and which code for a Mutant human RasGRP4 protein,

(b) deletions, additions and substitutions ofthe nucleic acid molecules of (a), which code for a Mutant human RasGRP4 protein,

(c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy ofthe genetic code, and (d) complements of (a), (b) or (c).

In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO:58.

According to yet another aspect ofthe invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the group consisting of:

(a) a unique fragment ofthe nucleotide sequence set forth as nucleotides 1-2749 of SEQ ID NO: 3 between 12 and 2748 nucleotides in length, or set forth as nucleotides 1-2592 of SEQ ID NO: 5 between 12 and 2591 nucleotides in length, or set forth as nucleotides 1- 883 of SEQ LD NO:58 between 12 and 882 nucleotides in length, and (b) complements of (a), wherein the unique fragments exclude nucleic acids consisting of nucleotide sequences that are contained within SEQ LD NO: 3, SEQ LD NO: 5, or SEQ ID NO:58, and that are known as ofthe filing date of this application.

According to another aspect ofthe invention, an expression vector is provided. The expression vector includes any ofthe foregoing isolated nucleic acid molecules ofthe invention operably linked to a promoter. According to yet another aspect ofthe mvention, a host cell transformed or transfected with the forgoing expression vector is provided. In some embodiments, the host cell is a CD14⁺ cell. In other embodiments, the host cell is a HMC-1 cell.

According to another aspect ofthe invention, a transgenic non-human animal is provided. The transgenic non-human animal includes the foregoing expression vector. In some embodiments, the transgenic non-human animal expresses a variable level of Mutant hRasGRP4. In some embodiments, the transgenic non-human animal is a transgenic V3- mastocytosis animal.

According to another aspect ofthe invention, an isolated Mutant hRasGRP4 protein is provided. The isolated protein is encoded by any ofthe foregoing isolated Mutant hRasGRP4 nucleic acid molecules ofthe invention. In some embodiments, the isolated protein includes an amino acid sequence selected from the group consisting of SEQ ED NO: 4, SEQ ED NO: 6, and SEQ ED NO:59.

According to another aspect ofthe mvention, binding polypeptides that selectively bind to the foregoing isolated Mutant hRasGRP4 protem ofthe invention are provided. In some embodiments, the binding polypeptides are antibodies or antigen-binding fragments thereof. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to the N-terminus ofthe isolated protein comprising an amino acid sequence of SEQ DD NO:4, SEQ ID NO:6, or SEQ DD NO:59. According to another aspect ofthe invention, pharmaceutical compositions are provided. The pharmaceutical compositions include an active agent selected from the group consisting of: any ofthe foregoing Mutant hRasGRP4 nucleic acids ofthe invention, any of the foregoing Mutant hRasGRP4 proteins ofthe invention encoded by the isolated nucleic acid molecules ofthe invention, or any ofthe foregoing binding polypeptides ofthe invention, and a pharmaceutically acceptable carrier.

According to yet another aspect ofthe invention, method for making a medicament are provided. The methods include placing an active agent selected from the group consisting of: (a) any ofthe foregoing isolated Mutant hRasGRP4 nucleic acid molecules of the invention, (b) any ofthe forgoing isolated Mutant hRasGRP4 proteins ofthe invention, or (c) any ofthe foregoing binding polypeptides ofthe invention in a pharmaceutically acceptable carrier. In some embodiments, placing includes placing a therapeutically effective amount ofthe active agent in the pharmaceutically acceptable carrier to form one or more doses.

According to yet another aspect ofthe invention, method for diagnosing a disorder characterized by aberrant expression of an hRasGRP4 molecule are provided. The methods include detecting in a first biological sample obtained from a subject, expression of an hRasGRP4 molecule or a Mutant hRasGRP4 molecule, wherein a difference in expression level ofthe hRasGRP4 molecule compared to an hRasGRP4 control or an increase in expression level ofthe Mutant hRasGRP4 molecule compared to a Mutant hRasGRP4 control indicates that the subject has a disorder characterized by aberrant expression of an hRasGRP4 molecule. In some embodiments, the methods also includes the steps of detecting in a second biological sample obtained from the subject, expression of an hRasGRP4 molecule or a Mutant hRasGRP4 molecule, and comparing the expression ofthe hRasGRP4 molecule or the Mutant hRasGRP4 molecule in the first biological sample and the second biological sample. In some embodiments, the disorder is a bacterial infection, wherein an increase in expression level ofthe lιRasGRP4 in the second biological sample compared to the expression level in the hRasGRP4 in the first biological sample indicates progression.of the , disorder characterized by aberrant expression of hRasGRP4. In certain embodiments, the disorder is a bacterial infection, wherein a decrease in expression level ofthe hRasGRP4 in the second biological sample compared to the expression level in the hRasGRP4 in the first biological sample indicates regression ofthe disorder characterized by aberrant expression of hRasGRP4. In some embodiments, the disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and AIDS, and wherein an increase in expression level ofthe hRasGRP4 in the second biological sample compared to the expression level in the hRasGRP4 in the first biological sample indicates progression ofthe disorder characterized by aberrant expression of hRasGRP4. In some embodiments, the disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and AIDS, and wherein a decrease in expression level ofthe lιRasGRP4 in the second biological sample compared to the expression level in the hRasGRP4 in the first biological sample indicates regression ofthe disorder characterized by aberrant, expression of hRasGRP4. In some embodiments, the disorder is a bacterial infection, wherein an increase in expression level ofthe Mutant hRasGRP4 in the second biological sample compared to the expression level ofthe Mutant hRasGRP4 in the first biological sample indicates progression ofthe disorder characterized by aberrant expression of hRasGRP4. In some embodiments, the disorder is a bacterial infection, wherein a decrease in expression level ofthe Mutant hRasGRP4 in the second biological sample compared to the expression level ofthe Mutant hRasGRP4 in the first biological sample indicates regression ofthe disorder characterized by aberrant expression of hRasGRP4. In certain embodiments, the disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and AIDS, and wherein an increase in expression level ofthe Mutant hRasGRP4 in the second biological sample compared to the expression level ofthe Mutant hRasGRP4 in the first biological sample indicates progression ofthe disorder characterized by aberrant expression of hRasGRP4. In some embodiments, the disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and AEDS, and wherein a decrease in expression level ofthe Mutant hRasGRP4 in the second biological sample compared to the expression level ofthe Mutant hRasGRP4 in the first biological sample indicates regression ofthe disorder characterized by aberrant expression of hRasGRP4. In some embodiments, the disorder characterized by aberrant expression of an hRasGRP4 molecule is a bacterial infection. In certain embodiments, the mast cell disorder characterized by aberrant expression of an hRasGRP4 molecule is a mast cell disorder. In certain embodiments, the mast cell disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ALDS. In some embodiments, the mast cell disorder is asthma. In some embodiments, the mast cell disorder is mastocytosis. In certain embodiments, the method includes detecting expression of an hRasGRP4 nucleic acid molecule. In some embodiments, the hRasGRP4 nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ED NO: 1. In certain embodiments, the method includes detecting expression of a Mutant hRasGRP4 nucleic acid molecule. In certain embodiments, the Mutant hRasGRP4 molecule is a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO:58. hi certain embodiments, the Mutant hRasGRP4 molecule is a nucleic acid comprising a nucleotide sequence set forth as SEQ DD NO: 1 with one or more nucleic acid additions, deletions, or substitutions which affect the functional activity ofthe hRasGRP4 nucleic acid molecule. In some embodiments, the method includes detecting expression of an hRasGRP4 protem. In some embodiments, the hRasGRP4 protein comprises a nucleotide sequence set forth as SEQ DD NO: 2. In certain embodiments, the method includes detecting expression of a Mutant hRasGRP4 protein. In certain embodiments, the Mutant hRasGRP4 protein comprises an amino acid sequence selected from the group consisting of SEQ DD NO: 4, SEQ ED NO: 6, and SEQ DD NO: 59. Dn some embodiments, the Mutant hRasGRP4 protein comprises an amino acid sequence selected from the group consisting of SEQ ED NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ED NO:59 with one or more amino acid additions, deletions, or substitutions which affect the functional activity of a hRasGRP4 protein. In some embodiments, detecting includes contacting the biological sample with an agent that selectively binds to the hRasGRP4 molecule or to the Mutant hRasGRP4 molecule. In some embodiments, the hRasGRP4 molecule or the Mutant hRasGRP4 molecule is a nucleic acid and wherein the agent that selectively binds to the hRasGRP4 molecule or to the Mutant hRasGRP4 molecule is a nucleic acid that hybridizes to SEQ ID NO: 1, SEQ ID NO: 3, SEQ DD NO: 5, or SEQ ID NO:58 under high stringency conditions. In some embodiments, the hRasGRP4 molecule or the Mutant hRasGRP4 molecule is a protein and wherein the agent that selectively binds to the hRasGRP4 molecule or to the Mutant hRasGRP4 molecule comprises a binding polypeptide that selectively binds to an amino acid sequence selected from the group consisting of SEQ DD NO: 2, SEQ DD NO: 4., SEQ DD NO: 6, and SEQ ED NO:59. In certain embodiments, the biological sample is selected from the group consisting of: a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract.

According to yet another aspect ofthe invention, methods for evaluating the effect of candidate pharmacological agents on the regulation of mast cells are provided. The method includes, administering a candidate pharmacological agent to a subject; determining the effect ofthe candidate pharmacological agent on the expression level of hRasGRP4 relative to the expression level of hRasGRP4 in a subject to which no candidate pharmacological agent is administered, wherein a relative increase or relative decrease in the expression level of hRasGRP4 indicates regulation of mast cells by the candidate pharmacological agent. According to another aspect ofthe invention, methods for evaluating the effect of candidate pharmacological agents on the regulation of mast cells are provided. The methods include contacting a candidate pharmacological agent with a mast cell sample; determining the effect ofthe candidate pharmacological agent on the expression level of hRasGRP4 relative to the expression level of hRasGRP4 in a mast cell sample not contacted with the candidate pharmacological agent, wherein a relative increase or relative decrease in the expression level of hRasGRP4 indicates regulation of mast cells by the candidate pharmacological agent. Dn some embodiments, the mast cell sample includes RasGRP4⁺ HMC-1 cells. According to another aspect ofthe invention, kits for diagnosing a disorder associated with aberrant expression of an hRasGRP4 molecule are provided. The kits include one or more nucleic acid molecules that hybridize to an hRasGRP4 nucleic acid molecule or to a Mutant hRasGRP4 nucleic acid molecule under high stringency conditions, and instructions for the use ofthe nucleic acid molecules, and control agents in the diagnosis ofa disorder associated with aberrant expression of an hRasGRP4 molecule. In some embodiments, the one or more nucleic acid molecules consist of a first primer and a second primer, wherein the first primer and the second primer are constructed and arranged to selectively amplify at least a portion of an isolated hRasGRP4 nucleic acid molecule comprising SEQ ED NO: 1 or an isolated Mutant hRasGRP4 nucleic acid molecule comprising SEQ ID NO: 3, SEQ DD NO: 5, or SEQ ID NO:58. Dn certain embodiments, the nucleic acids and the one or more control agents are bound to a substrate.

According to yet another aspect ofthe invention, kits for diagnosing an hRasGRP4- associated disorder in a subject are provided. The kits include one or more binding polypeptides that selectively bind to an hRasGRP4 protein or a Mutant hRasGRP4 protein, and instructions for the use ofthe binding polypeptides, and control agents in the diagnosis of a disorder associated with aberrant expression of an hRasGRP4 molecule. Dn some embodiments, the one or more binding polypeptides are antibodies or antigen-binding fragments thereof. In certain embodiments, the antibodies or antigen-binding fragments thereof and the one or more control agents are bound to a substrate. In some embodiments, the binding polypeptides bind to the Mutant hRasGRP4 protein but do not bind to the hRasGRP4 protein. According to another aspect ofthe invention, methods for treating a subject with a disorder characterized by decreased expression of an hRasGRP4 nucleic acid are provided. The methods include administering to the subject an effective amount of an hRasGRP4 nucleic acid molecule to treat the disorder. According to another aspect ofthe invention, methods for treating a subject with a disorder characterized by increased expression of an hRasGRP4 nucleic acid molecule are provided. The methods include administering to the subject an effective amount of an antisense or RNAi molecule to an hRasGRP4 nucleic acid molecule to treat the disorder.

According to another aspect ofthe mvention, methods for treating a subject with a disorder characterized by decreased expression of an hRasGRP4 protein are provided. The methods include administering to the subject an effective amount of an hRasGRP4 protein to treat the disorder.

According to another aspect ofthe invention, methods for treating a subject with a disorder characterized by increased expression of an hRasGRP4 protein are provided. The methods include administering to the subject an effective amount of a binding polypeptide to an hRasGRP4 protein to treat the disorder. In some embodiments, the binding polypeptide agent is an antibody or an antigen-binding fragment thereof, hi certain embodiments, the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated hRasGRP4 protein or Mutant hRasGRP4 protein. Dn some embodiments, the antibodies or antigen-binding fragments are labeled with one or more cytotoxic agents.

According to another aspect ofthe invention, methods for producing an hRasGRP4 protein are provided. The methods include providing an isolated hRasGRP4 nucleic acid molecule operably linked to a promoter, wherein the hRasGRP4 nucleic acid molecule encodes the hRasGRP4 protein or a fragment thereof, and expressing the hRasGRP4 nucleic acid molecule in an expression system. In some embodiments, the method also includes isolating the hRasGRP4 protein or a fragment thereof from the expression system. In certain embodiments, the hRasGRP4 nucleic acid molecule comprises the nucleotide sequence set forth as SEQ DD NO: 1. According to another aspect ofthe invention, methods for producing a Mutant hRasGRP4 protein are provided. The methods include providing an isolated Mutant hRasGRP4 nucleic acid molecule operably linked to a promoter, wherein the Mutant hRasGRP4 nucleic acid molecule encodes the Mutant hRasGRP4 protein or a fragment thereof, and expressing the Mutant hRasGRP4 nucleic acid molecule in an expression system. n some embodiments, the method also includes isolating the Mutant hRasGRP4 protein or a fragment thereof from the expression system. In certain embodiments, the Mutant hRasGRP4 nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ED NO: 5, and SEQ ED NO:58. In some embodiments, the Mutant hRasGRP4 nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ DD NO: 3, SEQ DD NO: 5, and SEQ ID NO: 58 with one or more point mutations or deletions to encode a Mutant hRasGRP4 protein.

(a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 9, and which codes for an mRasGRP4 protein,

(b) deletions, additions and substitutions ofthe nucleic acid molecules of (a), which code for an mRasGRP4 protem

Dn some embodiments, the isolated nucleic acid molecule comprises a nucleotide set forth as SEQ ID NO: 7.

According to another aspect ofthe invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the group consisting of: (a) a unique fragment ofthe nucleotide sequence set forth as nucleotides 1-2327 of

SEQ ID NO: 7 between 12 and 2326 nucleotides in length, and (b) complements of (a), wherein the unique fragments exclude nucleic acids consisting of nucleotide sequences that are contained within SEQ ID NO: 7 and that are known as ofthe filing date of this application. According to another aspect ofthe invention, expression vectors are provided. The expression vectors include any ofthe foregoing isolated nucleic acid molecules ofthe invention operably linked to a promoter.

According to another aspect ofthe invention, host cells transformed or transfected with any ofthe forgoing expression vectors ofthe invention are provided. In some embodiments, the host cell is a CD14⁺ cell. In certain embodiments, the host cell is a HMC-1 cell.

According to another aspect ofthe invention, transgenic non-human animal that include any ofthe foregoing expression vectors ofthe invention are provided. Dn some embodiments, the transgenic non-human animal expresses a variable level of mRasGRP4 or Mutant mRasGRP4. In some embodiments, the transgenic non-human animal is a transgenic V3 -mastocytosis animal.

According to another aspect ofthe invention, an isolated protein encoded by any of the foregoing isolated nucleic acid molecule ofthe invention is provided. Dn some embodiments, the isolated protein includes the amino acid sequence set forth as SEQ ID NO: 8.

According to another aspect ofthe invention, binding polypeptides that selectively bind to the foregoing isolated protein ofthe invention are provided. Dn some embodiments, the binding polypeptide is an antibody or an antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated protein that includes the amino acid sequence of SEQ DD NO: 8.

According to another aspect of the mvention, an antigen used to generate the any of the foregoing antibodies or antibody-binding fragments ofthe invention is provided. The antigen includes the amino acid sequence set forth as SEQ DD NO: 36. According to another aspect ofthe invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule selected from the group consisting of:

(a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ DD NO: 9 and which code for a Mutant mRasGRP4 protein, (b) deletions, additions and substitutions ofthe nucleic acid molecules of (a), which code for a Mutant mRasGRP4 protein (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy ofthe genetic code, and

(d) complements of (a), (b) or (c).

Dn some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence set forth as SEQ ED NO: 9.

According to another aspect ofthe invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid is selected from the group consisting of:

(a) a unique fragment ofthe nucleotide sequence set forth as nucleotides 1-2312 of SEQ DD NO: 9 between 12 and 2311 nucleotides in length, and (b) complements of (a), wherein the unique fragments exclude nucleic acids consisting of nucleotide sequences that are contained within SEQ DD NO: 9, and that are known as ofthe filing date of this application.

According to another aspect ofthe invention, expression vectors are provided. The expression vectors include the any ofthe foregoing isolated nucleic acid molecules ofthe invention operably linked to a promoter.

According to another aspect ofthe invention, host cells transformed or transfected with any ofthe foregoing expression vectors ofthe invention, hi some embodiments, the host cell is a CD14⁺ cell, h certain embodiments, the host cell is a HMC-1 cell. According to another aspect ofthe invention, transgenic non-human animal comprising any ofthe expression vectors ofthe invention are provided. In some embodiments, the non-human animal expresses a variable level of Mutant mRasGRP4. In certain embodiments, the transgenic non-human animal is a transgenic V3 -mastocytosis animal. According to another aspect ofthe invention, an isolated protein encoded by any of the foregoing isolated nucleic acid molecules is provided. In some embodiments, the isolated protein comprises the amino acid sequence set forth as SEQ DD NO: 10.

According to another aspect ofthe invention, binding polypeptides that selectively bind to any ofthe foregoing isolated protein ofthe invention are provided. Dn some embodiments, the binding polypeptide is an antibody or an antigen-binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment thereof specifically binds to the N-terminus ofthe isolated protein comprising the amino acid sequence of SEQ DD NO: 10.

According to another aspect ofthe invention, methods for preparing an animal model of a disorder characterized by aberrant expression of an hRasGRP4 molecule are provided. The methods include (a) introducing into a non-human animal, a mRasGRP4 molecule or a Mutant mRasGRP4 molecule; and (b) detecting in a first biological sample obtained from the non-human animal, expression ofthe mRasGRP4 molecule or a Mutant mRasGRP4 molecule. Dn some embodiments, the animal model is of a disorder that is a bacterial infection. Dn certain embodiments, the animal model is ofa disorder selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS. In some embodiments, the nιRasGRP4 molecule is an mRasGRP4 nucleic acid molecule or a Mutant mRasGRP4 nucleic acid molecule, hi some embodiments, the mRasGRP4 molecule is an mRasGRP4 protein or a Mutant mRasGRP4 protein. According to another aspect ofthe invention, methods for evaluating the effect of candidate pharmacological agents on the regulation of mast cells are provided. The methods include administering a candidate pharmacological agent to a subject; determining the effect ofthe candidate pharmacological agent on the expression level of mRasGRP4 relative to the expression level of mRasGRP4 in a subject to which no candidate pharmacological agent is administered, wherein a relative increase or relative decrease in the expression level of mRasGRP4 indicates regulation of mast cells by the candidate pharmacological agent.

According to another aspect ofthe invention, methods for evaluating the effect of candidate pharmacological agents on the regulation of mast cells, are provided. The methods include contacting a candidate pharmacological agent with a mast cell sample; determining the effect ofthe candidate pharmacological agent on the expression level of n RasGRP4 relative to the expression level of mRasGRP4 in a mast cell sample not contacted with a pharmacological agent, wherein a relative increase or relative decrease in the expression level of mRasGRP4 indicates regulation of cells by the candidate pharmacological agent.

According to another aspect ofthe mvention, kits for preparing a non-human animal model of a disorder associated with aberrant expression of an hRasGRP4 molecule are provided. The kits include one or more nucleic acid molecules that hybridize to an mRasGRP4 nucleic acid molecule or to a Mutant mRasGRP4 nucleic acid molecule under high stringency conditions, and instructions for the use ofthe nucleic acid molecules, and control agents in the preparation of a non-human animal model of a disorder associated with aberrant expression of an hRasGRP4 molecule. In some embodiments, the one or more nucleic acid molecules consist of a first primer and a second primer, wherein the first primer and the second primer are constructed and arranged to selectively amplify at least a portion of an isolated mRasGRP4 nucleic acid molecule comprising a nucleotide selected from the group consisting of SEQ DD NO: 7 and SEQ DD NO: 9. h certain embodiments, the one or more nucleic acids or control agents are bound to a substrate.

According to another aspect ofthe invention, kits for preparing a non-human animal model of a disorder of a hRasGRP4-associated disorder in a subject are provided. The kits include one or more binding polypeptides that selectively bind to an mRasGRP4 protein or a Mutant mRasGRP4 protein, and instructions for the use ofthe binding polypeptides, and control agents in the preparation of a non-human animal model of a disorder associated with aberrant expression of an hRasGRP4 molecule. In some embodiments, the one or more binding polypeptides are antibodies or antigen-binding fragments thereof. In certain embodiments, the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated protein of an mRasGRP4 protein or a Mutant mRasGRP4 protein. In some embodiments, the antibodies or antigen-binding fragments thereof, or one or more control agents are bound to a substrate. In some embodiments, the binding polypeptides bind to the Mutant nιRasGRP4 protein but do not bind to the mRasGRP4 protein.

According to another aspect ofthe invention, methods for preparing a non-human animal model of a disorder characterized by aberrant expression of an hRasGRP4 molecule are provided. The methods include administering to a non-human animal an effective amount of an antisense or RNAi molecule to a Mutant mRasGRP4 nucleic acid molecule or an anti-sense or RNAi molecule to a mRasGRP4 nucleic acid molecule to reduce expression ofthe Mutant mRasGRP4 nucleic acid molecule or the mRasGRP4 nucleic molecule in the non-human animal.

According to another aspect ofthe invention, methods for preparing a non-human animal model of a disorder characterized by aberrant expression of a RasGRP4 molecule are provided. The methods include administering to a non-human animal an effective amount of a binding polypeptide to a Mutant mRasGRP4 protein or to a mRasGRP4 protein to reduce expression ofthe Mutant mRasGRP4 protein or ofthe mRasGRP4 protein in the non-human animal. In some embodiments, the binding polypeptide agent is an antibody or an antigen- binding fragment thereof. In certain embodiments, the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated mRasGRP4 protein or Mutant mRasGRP4 protein. In some embodiments, the antibodies or antigen-binding fragments are labeled with one or more cytotoxic agents.

According to another aspect ofthe invention, methods for producing an mRasGRP4 protein are provided. The methods include providing an isolated mRasGRP4 nucleic acid molecule operably linked to a promoter, wherein the mRasGRP4 nucleic acid molecule encodes the mRasGRP4 protein or a fragment thereof, and expressing the mRasGRP4 nucleic acid molecule in an expression system. In some embodiments, the method also includes isolating the mRasGRP4 protein or a fragment thereof from the expression system. In some embodiments, the mRasGRP4 nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ DD NO: 7 and SEQ DD NO: 9.

According to another aspect ofthe invention, methods for producing a Mutant mRasGRP4 protein are provided. The methods include providing an isolated Mutant mRasGRP4 nucleic acid molecule operably linked to a promoter, wherein the Mutant mRasGRP4 nucleic acid molecule encodes the Mutant mRasGRP4 protein or a fragment thereof, and expressing the Mutant mRasGRP4 nucleic acid molecule in an expression system, hi some embodiments, the method also includes isolating the Mutant mRasGRP4 protein or a fragment thereof from the expression system. In some embodiments, the Mutant mRasGRP4 nucleic acid molecule comprises a nucleotide sequence set forth as SEQ DD NO: 9. hi certain embodiments, the Mutant mRasGRP4 nucleic acid molecule comprises a nucleotide sequence set forth as SEQ DD NO: 7 with one or more point mutations or deletions to encode a Mutant mRasGRP4 protein. According to another aspect ofthe invention, methods for treating a subject with a disorder characterized by a decreased amount of a prostaglandin D₂ (PGD₂) molecule and/or its synthase are provided. The methods include administering to the subject an effective amount of a RasGRP4 molecule to treat the disorder. In some embodiments, the RasGRP4 molecule is a RasGRP4 nucleic acid molecule. In certain embodiments, the RasGRP4 molecule is a RasGRP4 protein.

According to another aspect ofthe invention, methods for treating a subject with a disorder characterized by an increased amount of a prostaglandin D₂ (PGD₂) molecule and/or its synthase are provided. The methods include administering to the subject an effective amount of an inhibitor of a RasGRP4 molecule to treat the disorder. Dn some embodiments, the inhibitor of a RasGRP4 molecule is an antisense or RNAi molecule to a RasGRP4 nucleic acid molecule. Dn certain embodiments, the inhibitor of a RasGRP4 molecule is a binding polypeptide to a RasGRP4 protein. Dn some embodiments, the binding polypeptide agent is an antibody or an antigen-binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated RasGRP4 protein or the N terminus of a Mutant RasGRP4 protein. In some embodiments, the antibodies or antigen-binding fragments are labeled with one or more cytotoxic agents. According to another aspect ofthe invention, methods for increasing the amount ofa prostaglandin D₂ (PGD₂) molecule and/or its synthase in a tissue or cell are provided. The methods include increasing the amount of RasGRP4 in the tissue or cell.

According to another aspect ofthe invention, methods for decreasing the amount ofa prostaglandin D₂ (PGD₂) molecule and/or its synthase in a tissue or cell are provided. The methods include inhibiting RasGRP4 activity in the tissue or cell. Dn some embodiments, inhibiting RasGRP4 activity is interfering with RasGRP4 activity. In certain embodiments, inhibiting RasGRP4 activity is reducing the level of expression of RasGRP4.

According to another aspect ofthe invention, methods for diagnosing a disorder characterized by aberrant amounts of a prostaglandin D (PGD₂) molecule and/or its synthase are provided. The methods include determining in a first biological sample obtained from a subject, an amount of a RasGRP4 molecule, wherein a difference in the amount ofthe RasGRP4 molecule compared to a RasGRP4 control indicates that the subject has a disorder characterized by aberrant amounts of a PGD₂ molecule and/or its synthase. In some embodiments, the method also includes the steps of: detecting in a second biological sample obtained from the subject, an amount of a RasGRP4 molecule, and comparing the amount of the RasGRP4 molecule in the first biological sample and the second biological sample. In some embodiments, the disorder characterized by aberrant expression of a prostaglandin D₂ (PGD₂) molecule and/or its synthase is a bacterial infection. In certain embodiments, the disorder characterized by aberrant expression of a PGD₂ molecule and/or its synthase is a mast cell disorder. In some embodiments, the mast cell disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS. In some embodiments, the mast cell disorder is asthma. In certain embodiments, the mast cell disorder is mastocytosis. hi some embodiments, the biological sample is selected from the group consisting of: a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract.

According to another aspect ofthe invention, methods for diagnosing a disorder characterized by aberrant expression of a RasGRP4 molecule are provided. The methods include determining in a first biological sample obtained from a subject, an amount of a prostaglandin D₂ (PGD₂) molecule and/or its synthase, wherein a difference in the amount of the PGD₂ molecule and/or its synthase compared to a PGD₂ and/or its synthase control indicates that the subject has a disorder characterized by aberrant expression of a RasGRP4 molecule. In some embodiments, the method also includes the steps of detecting in a second biological sample obtained from the subject, an amount of a prostaglandin D₂ (PGD₂) molecule and/or its synthase, and comparing the amount ofthe PGD₂ molecule and/or its synthase in the first biological sample and the second biological sample. In some embodiments, the disorder characterized by aberrant expression of a RasGRP4 molecule is a bacterial infection. In certain embodiments, the disorder characterized by aberrant expression ' of a RasGRP4 molecule is a mast cell disorder. In some embodiments, the mast cell disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS. Dn certain embodiments, the mast cell disorder is asthma. Dn certain embodiments, the mast cell disorder is mastocytosis. In some embodiments, the biological sample is selected from the group consisting of: a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract.

According to another aspect ofthe invention, methods for treating a subject with a disorder characterized by a decreased amount ofa granule neutral protease are provided. The methods include administering to the subject an effective amount of a RasGRP4 molecule to treat the disorder. In some embodiments, the RasGRP4 molecule is a RasGRP4 nucleic acid molecule. In certain embodiments, the RasGRP4 molecule is a RasGRP4 protein.

According to another aspect ofthe invention, methods for treating a subject with a disorder characterized by increased amount ofa granule neutral protease are provided. The methods include administering to the subject an effective amount of an inhibitor of a RasGRP4 molecule to treat the disorder, h some embodiments, the inhibitor of a RasGRP4 molecule is an antisense or RNAi molecule to a RasGRP4 nucleic acid molecule. In certain embodiments, the inhibitor of a RasGRP4 molecule is a binding polypeptide to a RasGRP4 protein. Dn some embodiments, the binding polypeptide agent is an antibody or an antigen- binding fragment thereof. In some embodiments, the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated RasGRP4 protein or the N terminus of a Mutant RasGRP4 protein. In certain embodiments, the antibodies or antigen-binding fragments are labeled with one or more cytotoxic agents. According to another aspect ofthe invention, methods for increasing the amount of a granule neutral protease in a tissue or cell are provided. The methods include increasing the amount of RasGRP4 in the tissue or cell.

According to another aspect ofthe mvention, methods for decreasing the amount ofa granule neutral protease in a tissue or cell are provided. The methods include inhibiting RasGRP4 activity in the tissue or cell. Dn some embodiments, inhibiting RasGRP4 activity is interfering with RasGRP4 activity. Dn certain embodiments, inhibiting RasGRP4 activity is reducing expression of RasGRP4.

According to another aspect ofthe invention, methods for diagnosing a disorder . characterized by aberrant amounts of a granule neutral protease molecule are provided. The methods include determining in a first biological sample obtained from a subject, an amount of a RasGRP4 molecule, wherein a difference in the amount ofthe RasGRP4 molecule compared to a RasGRP4 control indicates that the subject has a disorder characterized by aberrant amounts of a granule neutral protease. In some embodiments, the method also includes the steps of: detecting in a second biological sample obtained from the subject, an amount of a

RasGRP4 molecule, and comparing the amount ofthe RasGRP4 molecule in the first biological sample and the second biological sample. In some embodiments, the disorder characterized by aberrant expression of a granule neutral protease is a bacterial infection. Dn certain embodiments, the disorder characterized by aberrant expression of a granule neutral protease is a mast cell disorder. In some embodiments, the mast cell disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS, hi certain embodiments, the mast cell disorder is asthma. In some embodiments, the mast cell disorder is mastocytosis. Dn some embodiments, the biological sample is selected from the group consisting of: a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract.

According to another aspect ofthe invention, methods for diagnosing a disorder characterized by aberrant expression of a RasGRP4 molecule are provided. The methods include determining in a first biological sample obtained from a subject, an amount ofa granule neutral protease, wherein a difference in the amount ofthe granule neutral protease compared to a granule neutral protease control indicates that the subject has a disorder characterized by aberrant expression of a RasGRP4 molecule. In some embodiments, the method also includes the steps of detecting in a second biological sample obtained from the subject, an amount ofa granule neutral protease, and comparing the amount ofthe granule neutral protease in the first biological sample and the second biological sample. In some embodiments, the disorder characterized by aberrant expression of a RasGRP4 molecule is a bacterial infection. In certain embodiments, the disorder characterized by aberrant expression of a RasGRP4 molecule is a mast cell disorder. In some embodiments, the mast cell disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS, hi certain embodiments, the mast cell disorder is asthma. Dn some embodiments, the mast cell disorder is mastocytosis. In certain embodiments, the biological sample is selected from the group consisting of: a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract. According to another aspect ofthe invention, methods for evaluating the effect of candidate pharmacological agents on the regulation of mast cells are provided. The methods include administering a candidate pharmacological agent to a subject; determining the effect ofthe candidate pharmacological agent on the amount of prostaglandin D₂ (PGD₂) relative to the amount of PGD₂ in a subject to which no candidate pharmacological agent is administered, wherein a relative increase or relative decrease in the amount of PGD₂ indicates regulation of mast cells by the candidate pharmacological agent. According to another aspect ofthe invention, methods for evaluating the effect of candidate pharmacological agents on the regulation of mast cells are provided. The methods include contacting a candidate pharmacological agent with a mast cell sample; determining the effect ofthe candidate pharmacological agent on the amount of prostaglandin D₂ (PGD₂) and/or its synthase relative to the amount of PGD₂ and/or its synthase in a mast cell sample not contacted with the candidate pharmacological agent, wherein a relative increase or relative decrease in the amount of PGD and/or its synthase indicates regulation of mast cells by the candidate pharmacological agent. In some embodiments, the mast cells are RasGRP4⁺ HMC-1 cells. All increases/decreases/differences in the foregoing aspects ofthe invention are preferably significant, e.g. statistically significant. In addition, as used herein the terms: "deletion", "addition", and "substitution" mean deletion, addition, and substitution changes to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleic acids of a sequence ofthe invention. These and other aspects ofthe invention, as well as various embodiments thereof, will become more apparent in reference to the drawings and detailed description ofthe mvention.

Brief Description of the Drawings

The descriptions include drawings that may reference color components. The figures are illustrative only and are not required for enablement ofthe mvention disclosed herein.

With respect to the figure descriptions, the term "variant" means a mutant form ofthe nucleic acid and/or protein.

Figure 1 shows digitized images of gels from polymerase chain reaction (PCR) evaluation of hRasGRP4 mRNA levels in the indicated fetal (Fig. 1A) and adult (Fig. IB, Fig. IC) human tissues and cells. The primers corresponded to sequences residing in coding exons 1 and 9 ofthe hRasGRP4 gene. The expected 900-bp product (arrow) is indicated. Results from analysis of pooled peripheral blood mononuclear cells from 4-36 individuals were sorted by Clontech based on their expression of CD4, CD8, CD14, and CD19 (c). Samples ofthe resulting cell populations were evaluated for their expression of hRasGRP4 mRNA immediately after their isolation (resting) or after their subsequent exposure to phytohemaggultinin, pokeweed mitogen, and/or concanavalin A (lectin activated). hRasGRP4 mRNA was only detected in the unfractionated and the "resting" CD 14 population of peripheral blood mononuclear cells. Glyceraldehyde-3-phosphate dehydrogenase-specific primers were used in a similar analyses to confirm the presence of intact RNA in all samples.

Figure 2 depicts the nucleotide sequence ofthe cDNA that corresponds to the major ~2.6-kb hRasGRP4 transcript (SEQ DD NO: 1) present in the human population, as well as the predicted amino acid sequence of its translated product (SEQ DD NO: 2). The ten indicated nucleotide differences found in the varied hRasGRP4 cDNAs that have been sequenced so far are presumed to be allelic polymorphisms of a single hRasGRP4 gene. The new amino acid is indicated ifthe nucleotide change results in a different amino acid. The putative polyadenylation regulatory site is underlined.

Figure 3 shows digitized photomicrographs of serial sections of human breast (Fig. 3 A, Fig 3B) and stomach (Fig. 3C, Fig. 3D) stained with anti-hRasGRP4 antibody (Fig. 3 A, Fig. 3C) or anti-tryptase antibody (Fig. 3B, Fig. 3D). The reaction product (arrows) indicates the presence of hRasGRP4 or tryptase depending on the antibody used in the analysis. Tryptase is a well-described mast cell marker.

Figure 4 is a diagram ofthe comparison ofthe amino acid sequences of hRasGRP4

(SEQ DD NO: 2) and mRasGRP4 (SEQ DD NO: 8). The putative REM domains (blue), CDC25-like catalytic domains (red), EF hands ofthe Ca²⁺-binding domains (yellow), and the phorbol ester/DAG-binding domains (green) of hRasGRP4 and mRasGRP4 are highlighted based on their respective locations in niRasGRPl, rRasGRPl, hRasGRPl, mRasGRP2, hRasGRP2, and hRasGRP3. The five residues that differ in the allelic variants of hRasGRP4 are indicated at positions 18, 120, 145, 261, and 335. A variant of mRasGRP4 has been identified that lacks the "VSTGP" (SEQ DD NO: 56) sequence at residues 561-565.

Fig. 5. (A) shows a schematic representation ofthe three-dimensional (3D) model of residues 34-445 of hRasGRP4 (grey) bound to H-Ras (blue). The two residues (Val³⁵⁹ and Glu³⁶³) in hRasGRP4 that are predicted to be important for its interaction with H-Ras are shown in ball and stick (green) representation. The 14-residue peptide that is lost in the variant 2 transcript isolated form the asthma patient is highlighted (red). Allelic differences in hRasGRP4 have been noted at residues 120, 145, 261, and 335 (yellow). Fig. 5B is a cartoon representation ofthe 3D model ofthe DAG-binding domain ofthe hRasGRP4 (residues 537-590). The conserved His and Cys residues in DAG binding proteins are indicated (blue). All representations were rendered using Molscript.

Figure 6 shows graphs illustrating the generation of recombinant hRasGRP4 in COS- 7 cells and fibroblasts, and the evaluation of its guanine nucleotide exchange activity. Fig. 6 A is a graph of evaluation of purified native (■) and heat-denatured (•) recombinant hRasGRP4 for their ability to transfer radioactive GTP to GDP -loaded H-Ras in a kinetic manner at room temperature. The level of immunoreactive hRasGRP4 in the conditioned media ofthe expressing cells always was below detection. The digitized image insert shows the SDS-PAGE/immunoblot analysis of lysates of fibroblasts transfected with the hRasGP4 construct; the depicted blot was probed with the anti-V5 antibody that recognizes the C- terminal epitope tag. Molecular weight markers are shown on the left. Recombinant hRasGRP4 is ~10 kDa larger than the native protein due to the epitope tag. Fig. 6B is a histogram illustrating the ability of purified recombinant hRasGRP4 to transfer radioactive GTP to GDP-loaded recombinant H-Ras, RhoA, Cdc42, Racl, Rab3A, and Ran after a 20- min incubation at room temperature in the absence (Fig. 6B) or presence of CaCl₂ (Fig. 6C). Fig. 6C indicates calcium is able to dominantly inhibit the GTP transfer activity of recombinant hRasGRP4 even ifthe reaction is carried out in the presence ofthe activating cation magnesium.

Figure 7 shows the sequences of aberrant hRasGRP4 cDNAs from patients that encode truncated proteins. Fig. 7 A shows variant 1 isolated from an asthma patient and a mastocytosis patient that contained a 117-bp insertion. The nucleic acid sequence in Fig. 7 A is nucleotides 215 tlirough 2351 of SEQ DD NO: 3). Shaded region is intron 5 in the precursor transcript. The 117-bp insertion (shaded sequence) causes an early translation- termination codon (*). Ifthe normal translation-initiation site is used at residues 215-217, the resulting protein from the variant 1 transcript (SEQ DD NO: 4) will not be able to activate H- Ras because it lacks -75% of its primary amino acid sequence, including the entire CDC25- catalytic domain. Fig. 7B shows nucleotides 215 through 2194 of variant 2 (SEQ DD NO:5), which encodes a truncated form of hRasGRP4 that lacks the Leu-Ser-Pro-Gly-Gly-Pro-Gly- Pro-Pro-Leu-Pro-Met-Ser-Ser (SEQ DD NO: 57) sequence that precedes the CDC 25-catalytic domain in the normal protein (SEQ DD NO: 1). Variant 2 is caused by a failure of the hRasGRP4-expressing cell to use the normal intron 5/exon 6-splice site to remove intron 5 in the precursor transcript. The use of a cryptic splice site in the middle of exon 6 to eliminate the intron and a portion of exon 6 causes the expressed protein (SEQ DD NO: 6) to be 14 amino acids shorter than normal hRasGRP4. The area of the transcript and protein that is affected by this post-transcriptional splicing event is indicated (#). Fig. 7C reverse transcriptase (RT) PCR analyses were carried out using different primer sets to evaluate the expression of the variant 1 (top panel) and variant 2 (bottom panel) forms of the hRasGRP4 transcript in a leukocyte preparation derived from 550 individuals and in the MC progenitors residing in the blood of four normal individuals, a patient with asthma, and a patient with systemic mastocytosis. Fig. 7D shows a diagram of the splicing events that result in the generation of the normal hRasGRP4 transcript and its two variants. The X in Fig. 7D identifies the position of a "translation-termination codon" in the expressed transcript. The variant 1 transcript in Fig.7D has two Xs. The first X identifies is the premature translation- termination codon that forms when intron 5 is not removed. The second X is the normal translation-termination codon.

Figure 8 depicts the nucleotide sequence of the BALB/c mouse bone marrow-derived mast cells (mBMMC) cDNA that corresponds to the ~2.3-kb mRasGRP4 transcript (SEQ DD NO: 7), as well as the predicted amino acid sequence of its translated product (SEQ ED NO: 8).

Figure 9A shows a diagram of chromosomal location of the mRasGRP4 gene as determined by FISH analysis. The location of the mRasGRP4 gene on this chromosome is indicated by the arrow in the digitized cartoon. Fig. 9B is a digitized photomicrograph image showing fluorescent-labeled, mRasGRP4-containing BAG clone F1231 hybridized specifically to a medium-sized chromosome (arrow) that was subsequently shown to be chromosome 7 using a probe specific for the telomeric region of this chromosome (arrow head). The location of the mRasGRP4 gene on this chromosome is more clearly indicated in the digitized cartoon shown on the left. Figure 10 shows digitized images of gels illustrating mRasGRP mRNA expression in n BMMCs and various adult and embryonic mouse tissues using an RT-PCR approach. Fig. 10A shows results of a semi-quantitative RT-PCR approach that was used to evaluate n RasGRPl, mRasGRP2, and mRasGRP4 transcript expression in BALB/c mBMMCs, C57BL/6 mBMMCs, and in BALB/c mouse brain. Fig. 10B shows results of a similar semi- quantitative PCR approach that was used to screen various cDNA libraries to identify those tissues (i.e. spleen and lung) that contain the highest levels of mRasGRP4 mRNA. mRasGRP4 mRNA expression also was evaluated in a peritoneal cavity cellular exudate.

Figure 11 shows digitized images of RNA blot analysis. Fig. 11A shows a blot containing total RNA from mBMMCs; the mast cell lines V3, C1.MC/C57.1, and RBL-1; the myelomonocytic/macrophage (MΦ) lines RAW and WEHI-3; the T cell (TC) hybridoma line MTC-1; and the fibroblast (Fib) line 3T3 was probed with a 197-bp mRasGRP4-gene specific probe (upper panel) to measure the steady-state levels ofthe mRasGRP4 transcript in the varied cell populations. The ethidium bromide-stained 18S and 28S rRNA present in each lane (lower panel) documents the presence of intact RNA in all samples. Fig. 1 IB shows results of a kinetic experiment to evaluate mRasGRP4 mRNA levels in mBMMCs derived by culturing BALB/c mouse bone marrow cells for 2-7 weeks in the media supplemented with IL-3.

Figure 12 shows digitized photomicrographs of frozen 5-μm sections of tongue (Fig. 12A , Fig. 12B), or plastic-embedded 1.5-μm serial sections of skin (Fig. 12C, Fig. 12D), spleen (g, h), or a peritoneal cavity-derived cell pellet (Fig. 12E, Fig. 12F) stained at the light level with toluidine blue (Fig. 12A, Fig. 12C, Fig. 12E, Fig. 12G) or with anti-mRasGRP4 antibody (Fig. 12B, Fig. 12D, Fig. 12F, Fig. 12 H). The spleen was obtained from a V3- mastocytosis mouse. Arrows and arrow-heads point to mast cells. As noted in Fig. 12A and Fig. 12B, the number of toluidine blue^"1" cells in the tongue of a normal BALB/c mouse are comparable to the number of mRasGRP4 cells. Although background staining is stronger when tissue samples are embedded in plastic, more conclusive evidence that mature mast cells selectively express mRasGRP4 was obtained using serial sectioned tissue and cell pellets Fig. 12C-Fig. 12H. Fig. 121 shows that frozen sections (100 nm) ofthe spleen also were stained with anti- mRasGRP4 antibody followed by gold-labeled secondary antibody to identify the two primary locations of mRasGRP4 inside those V3-mast cells that home to the spleen (Fig. 121). At the ultrastructural level, mRasGRP4 resides primarily in the cytoplasm (+) or plasma membrane (*). hnmunoreactive mRasGRP4 was not detected in the nucleus (N) or secretory granules (SG) of these mast cells.

Figure 13 shows a digitized image of SDS-PAGE/immunoblot analysis of native mRasGRP4. The proteins in the detergent extract ofthe cells isolated from the peritoneal cavity of a mouse were subjected to SDS-PAGE/immunoblot analysis to evaluate the levels and size of mRasGRP4 in mast cells, that have been differentiated in vivo. The arrow on the right points to the immunoreactive 75-kDa protein present in the sample.

Figure 14 is a diagram and sequence comparison ofthe amino acid sequences of mRasGRP4 with mouse, rat, and human RasGRP 1 , RasGRP2, and RasGRP3. Fig. 14A shows a dendrogram comparing mRasGRP4 with its closely related proteins was generated by the GCG program "Distances" using the unweighted pair group with arithmetic mean algorithm (UPGMA). Fig. 14B shows the amino acid sequence comparisons of mRasGRP4 (SEQ DD NO: 8) with those of mRasGRPl (SEQ DD NO: 49), rRasGRPl (SEQ DD NO: 50), hRasGRPl (SEQ ED NO: 51), mRasGRP2 (SEQ ED NO: 52), hRasGRP2 (SEQ ED NO: 53), and hRasGRP3 (SEQ DD NO: 54). The amino acid sequences ofthe previously cloned members of this family of GEFs were extracted from GenBank. Gaps (dashes) are indicated. The REM domains (blue), CDC25-like catalytic domains (red), EF hands ofthe Ca²⁺-binding domains (yellow), and the phorbol ester/DAG-binding domains (green) ofthe RasGRPs are highlighted.

Figure 15 shows comparative representations of 3D models of mRasGRP4. Fig. 15A shows a representation ofa 3D model of mRasGRP4 (residues 34-445) (grey) bound to H- Ras (blue). The two residues in mRasGRP4 predicted to be essential for its interaction with H-Ras are shown in ball and stick representation (yellow). The N and C termini ofthe modeled segment are indicated (orange). Fig. 15B and Fig. 15C show 3D models ofthe DAG-binding domain of nιRasGRP4 with (Fig. 15B) and without (Fig. 15C) the 5-residue insertion (green). The residues predicted to be most important for DAG-binding are indicated (red). All representations were rendered using Molscript (Kraulis, PJ. J. Appl. Cryst. 24:946-950, 1991).

Figure 16 shows digitized photomicrographs ofthe morphology of normal and

HiRasGRP4-expressing fibroblasts before and after exposure to phorbol- 12-m ristate 13- acetate (PMA). Fig. 16 A- Fig. 16D show normal mRasGRP4^" fibroblasts (Fib) and Fig. 16E- Fig. 16H show nιRasGRP4⁺ fibroblast transfectants that were evaluated before (Fig. 16 A, Fig. 16B, Fig. 16E, and Fig. 16F) and after a 15-min exposure at 37°C to 10 nM PMA (Fig. 16C, Fig. 16D, Fig. 16G, and Fig. 16H). At the light level (Fig. 16A, Fig. 16E), both populations of fibroblasts are extremely adherent to plastic culture dishes before PMA treatment and form prominent focal adhesions and extended membrane projections. mRasGRP4^" fibroblasts do not undergo noticeable morphologic changes when exposed to low levels of PMA for a brief period of time (Fig. 16C). However, many ofthe mRasGRP4⁺ fibroblasts (arrows in g) quickly round up when similarly treated. As assessed immunohistochemically (Fig. 16B-Fig. 16H), actin (red) redistributes in those mRasGRP4⁺ fibroblasts that have been exposed to PMA (Fig. 16H). Approximately 80% ofthe fibroblasts shown in panel G express mRasGRP4.

Figure 17 shows graphs and a blot of mRasGRP4 in COS-7 cells and fibroblasts, and then its guanine nucleotide exchange activity. Fig. 17A shows purified native (B) and heat- denatured (•) recombinant mRasGRP4 were evaluated for their ability to transfer radiolabeled GTP to GDP-loaded H-Ras in a kinetic manner at room temperature. Similar data were obtained in >5 other experiments. The level of immunoreactive mRasGRP4 in the conditioned media ofthe expressing cells always was below detection. The digitized image insert shows the SDS-PAGE/immunoblot analysis of lysates of COS-7 cells exposed to vector alone (-) or the mRasGRP4 construct (+); the depicted blot was probed with anti-V5 antibody. Molecular weight markers are shown on the left. Recombinant mRasGRP4 is ~10 kDa larger than the native protein (Fig. 13) due to the additional V5-His peptide tag attached to its C tenninus. Fig. 17 B- Fig. 17D show the ability of purified recombinant mRasGRP4 to transfer radiolabeled GTP to GDP-loaded recombinant H-Ras, RhoA, Cdc42, Racl, Rab3 A, and Ran, which was compared after a 20-min incubation at room temperature in the absence (Fig. 17B) or presence of anti-n RasGRP4 antibody (Fig. 17C), an inelevant control antibody (Fig. 17C), or CaCl₂ (Fig. 17D). As noted in Fig. 17D, calcium is able to dominantly inhibit the GRP transfer activity of recombinant mRasGRP4 even ifthe reaction is carried out in the presence of magnesium.

Figure 18 shows hRasGRP4 expression in the mast cell (MC) leukemia cell line HMC-1. Oligonucleotides conesponding to sequences in exon 2 (E2) and exon 6 (E6) ofthe hRasGRP4 gene were used in an RT-PCR approach to evaluate the extent of processing of the hRasGRP4 precursor transcript in the HMC-1 cell line (lane 2) and spleen of 22 pooled Caucasian fetuses (ages 20-33 weeks, Clontech) (lane 1). The normal 625-bp, properly processed portion ofthe transcript was detected in the pooled spleen sample, as well as the 742-bp variant 1 transcript that contains intron 5 (15). In contrast, the normal hRasGRP4 transcript and the variant 1 transcript were not detected in the HMC-1 cell line. Rather an 866-bp form (variant 3) was detected in the HMC-1 cell line whose nucleotide sequence revealed that it contained both intron 3 (13) and intron 5 (15). The failure to remove these introns causes a premature translation-termination codon (*).

Fig. 19 shows the granulation of HMC-1 cells and their mRasGRP4-transfectants. Because only non-functional forms of hRasGRP4 are present in HMC-1 cells, a transfection approach was used to force this immature human MC line to express a functional form ofthe mRasGRP4 GEF (Fig. 19A). As assessed immunohistochemically (Fig. 19C-Fig. 19H) and by SDS-PAGE-immunoblot analysis (Fig. 19B), the nιRasGRP4-expressing transfectants (+) contained substantially more tryptase (Fig. 19B, Fig. 19C, Fig.l9D), chymase (Fig. 19E, Fig. 19F), and carboxypeptidase A (CPA) (Fig. 19G, Fig. 19H) in their granules than the non- transfectants (-). While granules are rarely found in HMC-1 cells (Fig. 191), many granules that contain electron dense material are found in the transfectants (Fig. 19J). Shown in the insert in the lower right portion of/^' is a higher magnification of two typical granules in the transfectants. Although the transfectant depicted in the electron micrograph (EM) shown in / is more typical ofthe cells in the mRasGRP4-expressing cultures, -10% ofthe transfectants contain granules that are nearly completely filled with electron dense material. Fig. 20 shows PGD₂ synthase protein levels in RasGRP4^" and RasGRP4⁺ HMC-1 cells. Using a transfection approach, HMC-1 cells were induced to express a functional form of RasGRP4. The proteins present in the lysates of 10⁵ starting cells (middle lane) and 10⁵ transfectants (right lane) were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. The resulting blot was probed with anti-PGD₂ synthase antibody to detect the presence ofthe appropriately sized protein. Human recombinant PGD₂ synthase (left lane) was used as a positive control in this SDS/PAGE immunoblot analysis.

Fig. 21 shows calcium ionophore-induced expression of PGD₂ in RasGRP4^" and RasGRP4⁺ HMC-1. Washed HMC-1 cells and their RasGRP4 expressing transfectants were separately resuspended in Ca²⁺- and Mg²⁺-free phosphate buffered saline at a density of 10⁶ cells/ml. Calcium ionophore A23187 (0.5 μM) was added with or without 0.1 μM PMA. After a 30-min incubation at 37°C, the level ofthe arachonidate metabolite PGD₂ in the conditioned media ofthe resulting four populations of cells was determined using a commercial ELISA kit. The depicted data are the mean + range (p<0.05) of an experiment carried out in duplicate.

Brief Description of the Sequences

SEQ DD NO: 1 is the nucleotide sequence of hRasGRP4 GenBank Acc No.: AY048119. SEQ DD NO: 2 is the amino acid sequence of hRasGRP4. GenBank Acc No. : AAK85701.1 SEQ DD NO: 3 is the nucleotide sequence of Mutant hRasGRP4 (variant 1). GenBank Acc

No: AY048120. SEQ DD NO: 4 is the amino acid sequence of Mutant hRasGRP4 (variant 1). GenBank Acc

No.: AAK85702.1 SEQ DD NO: 5 is the nucleotide sequence of Mutant hRasGRP4 (variant 2). GenBank Acc

No: AY048121. SEQ ED NO: 6 is the amino acid sequence of Mutant hRasGRP4 (variant 2). GenBank Acc

No: AAK85703.1.

SEQ DD NO 7 is the nucleotide sequence of mRasGRP4. GenBank Acc No.: AF331457. SEQ ED NO 8 is the amino acid sequence of nιRasGRP4. GenBank Acc No.: AAK842991 SEQ ED NO 9 is the nucleotide sequence of Mutant mRasGRP4. GenBank Acc

No: AY040628 SEQ DD NO: 10 is the amino acid sequence of a Mutant mRasGRP4. GenBank Acc

No: AAK81694.1. SEQ ED NO: 11 is the 5'-RACE primer 5'-ACCTGTCGGGCTGTGCCTCA-3'. SEQ ED NO: 12 is the 3'-RACE primer 5'-CAGCACCAAGGCCCTCCTGGAGCT-3'. SEQ DD NO: 13 is 5'-AATGCACCGGAAAAACAGGA- 3'a sense primer for hRasGRP4. SEQ DD NO: 14 is 5'-TGAGTCTGGAGATGGCACTG-3', antisense primer for hRasGRP4. SEQ DD NO: 15 is 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' primer for hRasGRP4. SEQ DD NO: 16 is 5'-CATGTGGGCCATGAGGTCCACCAC-3' primer for hRasGRP4. SEQ DD NO: 17 is 5'-TGCAGATCTGTCACCTGGTC-3', sense primer for hRasGRP4. SEQ DD NO: 18 is 5'-CGGAACTCCAGGTAGGTGAG-3', antisense primer for hRasGRP4. SEQ ED NO: 19 is 5'-CTTCTGACCTCCCAGGCCTG-3', sense primer for hRasGRP4. SEQ DD NO: 20 is 5'-GTAGCGGGC GTAGTTGTTGT-3', antisense primer for hRasGRP4. SEQ DD NO: 21 is a 14-mer peptide Met-Asn-Arg-Lys-Asp-Ser-Lys-Arg-Lys-Ser-His-Gln-

Glu-Cys residing at residues 1 to 14 in hRasGRP4. SEQ DD NO: 22 is 5'-CACCATGAACAGAAAAGACAGTAAGAGG-3', sense primer primer for entire coding domain of an hRasGRP4. SEQ DD NO: 23 is 5'-GGAATCCGGCTTGGAGGATGCAGT-3', antisense primer for entire coding domain of an hRasGRP4. SEQ ED NO: 24 is a mRasGRP4-specific sense primer:

5'-GCUGAUGGCGAUGAAUCAACACUGCGUUUGCUGGCUUUGAUGAAA-3'. SEQ ED NO: 25 is an mRasGRP4-specific antisense primer:

5 '-CGGAACTCCCAGGTAGTGA-3 ' SEQ DD NO: 26: 5'-CGCGGATCCGACACTCGTTTGCTGGCTTTGATGAAA-3', an adapter primer. SEQ DD NO: 27: 5'-CAGGACTTAGCAGGCTGGAG-3', mRasGRP4-specific antisense primer.

SEQ ED NO: 28 5'-CATGAATCTGGGAGTGCTGA-3', inner sense nested primer SEQ ED NO: 29 5'-CAGGACTTAGCAGGCTGGAG-3', antisense primer SEQ DD NO: 30 5'-GCAAGACTAGAGGCCAAATCA-3', sense primer for mRasGRPl. SEQ DD NO: 31 5'-ATGGTGGGGTTCTCTTTTACG-3', antisense primer for mRasGRPl. SEQ DD NO: 32 5'-CACAGCTAGTGCGCATGTTT-3', sense primer for mRasGRP2. SEQ ED NO: 33 5'-ATGCTGAAAAGCTGCCTCAT-3', antisense primer for mRasGRP2. SEQ DD NO: 34: 5'-TGAAGGTCGGAGTCAACGGATTTGGT-3', sense primer for glyceraldehyde-3-phosphate dehydrogenase (G3PDH). SEQ DD NO: 35: 5'-CATGTGGGCCATGAGGTCCACCAC-3', antisense primer for G3PDH. SEQ DD NO: 36: 12-mer peptide Arg-Lys-Asp-Ile-Lys-Arg-Lys-Ser-His-Gln-Glu-Cys residing at residues 3 to 14 in mRasGRP4. SEQ DD NO: 37: 5'-CACCATGAACCGGAAAGACATCAAA-3', sense primer for mRasGRP4. SEQ DD NO: 38: 5'-CCAAAACCGGCTTATGACTG-3\ antisense primer for mRasGRP4. SEQ DD NO: 39: 5'-CATGAATCTGGGAGTGCTGA-3', sense primer for mRasGRP4. SEQ DD NO: 40: 5'-GACGCTGGGCTTCGAGGAAGC-3', antisense primer for mRasGRP4. SEQ DD NO: 41: 5'-CACCATGAACCGGAAAGACATCAAA-3', sense primer for mRasGRP4. SEQ DD NO: 42: 5'-GACGCTGGGCTTCGAGGAAGC-3', antisense primer for mRasGRp4. SEQ DD NO: 43 is the nucleotide sequence of mRasGRPl. GenBank Acc No: NM_011246. SEQ DD NO: 44 is the nucleotide sequence of rRasGRPl. GenBank Acc No: NM_019211. SEQ DD NO: 45 is the nucleotide sequence of hRasGRPl. GenBank Acc No: NM_005739. SEQ ED NO: 46 is the nucleotide sequence of mRasGRP2. GenBank Acc No: NM Ol 1242. SEQ ID NO: 47 is the nucleotide sequence of hRasGRP2. GenBank Acc No: NM_005825. SEQ DD NO: 48 is the nucleotide sequence of hRasGRP3. GenBank Acc No: NM_015376. SEQ ED NO: 49 is the amino acid sequence of mRasGRPl GenBank accession No: NP_035376.1. SEQ DD NO: 50 is the amino acid sequence of rRasGRPl GenBank accession No: NP_062084.1 SEQ ED NO: 51 is the amino acid sequence of hRasGRPl GenBank accession

No: : NP_005730.1. SEQ DD NO: 52 is mRasGRP2. GenBank accession No: NP_035372.1. SEQ DD NO: 53 is hRasGRP2. GenBank accession No: NP_005816.2 SEQ DD NO: 54 is hRasGRP3. GenBank accession No:NP_056191.1. SEQ ED NO: 55: is HX₁₂CX₂CX₁₃CX₂CX₄HX₂CX₇C, where H is His, C is Cys and X is any other amino acid residue.

SEQ DD NO: 56: is VSTGP (Val-Ser-Thr-Gly-Pro).

SEQ DD NO: 57: Leu-Ser-Pro-Gly-Gly-Pro-Gly-Pro-Pro-Leu-Pro-Met-Ser-Ser.

SEQ DD NO: 58: Nucleotide sequence of hRasGRP4 variant 3.

SEQ DD NO:59: Amino acid sequence of hRasGRP4 variant 3.

SEQ DD NO: 60: is HX₁₂CX₂CX₁₄CX₂CX₄HX₂CX₇C, where H is His, C is Cys and X is any other amino acid residue.

SEQ DD NO: 61: Ser-Ser-Asp-Leu-Pro-Gly-Leu-Gly-Lys.

Detailed Description of the Invention

The present invention, in one aspect, involves the cloning of cDNAs that encode a human RasGRP4 protein (hRasGRP4) or a mouse RasGRP4 protein (mRasGRP4, also refened to as: CalDAG-GEFDV). The sequence ofthe coding portion ofthe human gene is presented as SEQ DD NO: 1, and the predicted amino acid sequence of this gene's protein product is presented as SEQ ID NO: 2. The sequence ofthe coding portion ofthe mouse gene is presented as SEQ DD NO: 7, and the predicted amino acid sequence of this gene's protem product is presented as SEQ ED NO: 8. The invention also involves an alternate form ofthe mRasGRP4 transcript, which is presented as SEQ DD NO: 9, and the predicted amino acid sequence of this transcript's protein product is presented as SEQ ED NO: 10. Sequence analysis shows that the human and mouse RasGRP4 proteins are -85% identical.

The invention thus involves in one aspect human and mouse RasGRP4 proteins, nucleic acid molecules encoding those proteins, functional modifications and variants ofthe foregoing, useful fragments ofthe foregoing, as well as therapeutic and diagnostic products (including antibodies), non-human animal models, and methods relating thereto.

The invention also includes several identified single nucleotide polymorphisms in the hRasGRP4 transcript (noted in SEQ DD NO: 1) that result in single amino acid polymoφhisms in the protein (noted in SEQ DD NO: 2). In addition to the sequence ofthe human RasGRP4 transcript and protein and the above-mentioned allelic polymorphisms of the hRasGRP4 transcript and protein, the invention also involves the identification of more severe alterations in the hRasGRP4 transcript and protein due to differential splicing ofthe precursor hRasGRP4 transcript. Variant hRasGRP4 and mRasGRP4 nucleotide sequences presented here in include SEQ DD NOs: 3, 5, 58, and 9 and variant hRasGRP4 and mRasGRP4 amino acid sequences presented herein include 4, 6, 59, and 10.

As used herein, the term "hRasGRP4" means human RasGRP4, the term "mRasGRP4" means mouse RasGRP4, and the term "RasGRP4" means human and/or mouse RasGRP4.

As used herein, the term "hRasGRP4-associated disorder" or "RasGRP4-associated disorder" means any disorder or disease characterized by abenant expression of RasGRP4.

As used herein, the term "abenant" refers to decreased expression (including zero expression) or increased expression (depending upon the disorder) ofthe natural RasGRP4 molecule (nucleic acid or protein) as compared to its expression in a subject who does not have a RasGRP4-associated disorder. Abenant expression can also refer to increased expression of a Mutant RasGRP4 molecule (nucleic acid or protein) as compared to its expression in a subject who does not have a RasGRP4-associated disorder. For example, the abenant expression is expression that is not 100% ofthe level of RasGRP4 in a subject free of a RasGRP4-associated disorder. Abenant expression may be determined by comparing levels of RasGRP4 or a Mutant RasGRP4 to those levels in controls. The control(s) include positive and negative controls which may be a predetermined value that can take a variety of forms. The control(s) can be a single cut-off value, such as a median or mean, or can be established based upon comparative groups, such as in groups having normal amounts of RasGRP4 and groups having abnormal amounts of RasGRP4.

Another example of a comparative group is a group having a particular disease, condition and/or symptoms and a group without the disease, condition and/or symptoms. Another comparative group is a group with a family history of a particular disease and a group without such a family history ofthe particular disease. The predetermined control value can be ananged, for example, where a tested population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group or into quadrants or quintiles, the lowest quadrant or quintile being individuals with the lowest risk or expression levels of RasGRP4 and the highest quadrant or quintile being individuals with the highest risk or expression levels of RasGPR4. The predetermined value of a control will depend upon the particular population selected. For example, an apparently healthy population will have a different "normal" RasGRP4 expression level range than will a population which is known to have a condition characterized by abenant RasGRP4 expression. Accordingly, the predetermined value selected may take into account the category in which an individual falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art. By "abnormally high" it is meant high relative to a selected control. Typically the control will be based on apparently healthy individuals in an appropriate age bracket.

It will also be understood that the controls according to the invention may be, in addition to predetermined values, samples of materials tested in parallel with the experimental materials. Examples include samples from control populations or control samples generated through manufacture to be tested in parallel with the experimental samples.

Human RasGRP4 (hRasGRP4) Nucleic Acid Compositions and Utilities

According to one aspect ofthe invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the group consisting of:

(a) nucleic acid molecules which hybridize under high stringency conditions to a, nucleic acid molecule comprising a nucleotide sequence set forth as SEQ DD NO: 1 and which code for an hRasGRP4 protein,

(b) deletions, additions and substitutions ofthe nucleic acid molecules of (a), (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy ofthe genetic code, and (d) complements of (a), (b), or (c).

The prefened isolated nucleic acids ofthe invention are hRasGRP4 nucleic acid molecules which encode a hRasGRP4 protein. As used herein, a hRasGRP4 protein refers to a protein that is encoded by a nucleic acid having SEQ ID NO: 1 or a functional fragment thereof, or a functional equivalent thereof (e.g., a nucleic acid sequence encoding the same protein as encoded by SEQ DD NO: 1), provided that the functional fragment or equivalent encodes a protein which exhibits a hRasGRP4 functional activity. As used herein, a hRasGRP4 functional activity refers to the ability of a hRasGRP4 protein to modulate one or more ofthe following parameters: mast cell development, mast cell function, and/or cell-cell interactions. An exemplary hRasGRP4 functional activity is mast cell activation such as initiating and/or increasing mast cell development, or activity. Although not wishing to be bound to any particular theory or mechanism, it is believed that the hRasGRP4 protein may effect at least some ofthe above-noted cell functions by participating in the varied signal transduction pathways in mature human mast cells, and may also regulate one or more ofthe intracellular pathways that control the differentiation and/or maturation of committed progenitors (e.g. CD47CD87CD14⁺ mononuclear cells) as they develop into fully granulated human mast cells, and, thereby, modulate mast cell responses, such as allergic reactions and mast-cell associated disease. RasGRP4 functional activity can be determined, for example, by measuring the ability of hRasGRP4 to transfer GTP to H-Ras, and/or its family members (e.g., RhoA, Cdc42, Racl, Rab3A, and Ran). (See, for example, the assays described in the Examples.)

In the prefened embodiments, the isolated nucleic acid molecule is SEQ DD NO: 1 or the coding region thereof.

The invention provides isolated nucleic acid molecules which code for hRasGRP4 proteins and which hybridize under high stringency conditions to a nucleic acid molecule consisting ofthe nucleotide set forth in SEQ ED NO: 1. Such nucleic acids may be DNA, RNA, composed of mixed deoxyribonucleotides and ribonucleotides, or may incorporate synthetic non-natural nucleotides. Various methods for determining the expression of a nucleic acid and/or a polypeptide in cells are known to those of skill in the art and are described further below and in the Examples. As used herein, the term protein is meant to include large molecular weight proteins and polypeptides and low molecular weight polypeptides or fragments thereof.

The term "high stringency conditions" as used herein refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Cm-rent Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. More specifically, high stringency conditions, as used herein, refers, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyπolidone, 0.02% Bovine Serum Albumin (BSA), 2.5mM NaH₂PO₄ (pH 7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride/0.015M sodium citrate, pH 7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic acid. After hybridization, the membrane upon which the DNA is transfened is washed at 2 x SSC at room temperature, and then at 0.1 x SSC/0.1 x SDS at temperatures up to 68°C.

The foregoing set of hybridization conditions is but one example of high stringency hybridization conditions known to one of ordinary skill in the art. There are other conditions, reagents, and so forth which can be used, which result in a high stringency hybridization. The skilled artisan will be familiar with such conditions, and thus they are not given here. It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of hRasGRP4 nucleic acid molecules ofthe invention. The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation ofthe pertinent nucleic acid molecule and sequencing.

In general homologs and alleles typically will share at least 75% nucleotide identity and/or at least 90% amino acid identity to SEQ DD NO: 1 and SEQ DD NO: 2, respectively, in some instances will share at least 80% nucleotide identity and/or at least 95% amino acid identity and in still other instances will share at least 85%> nucleotide identity and/or at least 99% amino acid identity. Prefened homologs and alleles share nucleotide and amino acid identities with SEQ DD NO: 1 and SEQ DD NO: 2, respectively, and encode polypeptides of greater than 80%, more preferably greater than 90%, still more preferably greater than 95% and most preferably greater than 99% identity. The percent identity can be calculated using various publicly available software tools developed by NCBI (Bethesda, Maryland) that can be obtained through the internet (ftp :/ncbi. nlm.nih.gov/pub/). Exemplary tools include the BLAST system available at http://www.ncbi.nlm.nih.gov, which uses algorithms developed by Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). Pairwise and ClustalW alignments (BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysis can be obtained using the Mac Vector sequence analysis software (Oxford Molecular Group).

Watson-Crick complements ofthe foregoing nucleic acid molecules also are embraced by the invention.

In screening for hRasGRP4 genes, a Southern blot may be performed using the foregoing conditions, together with a detectably labeled probe (e.g. radioactive or chemiluminescent probes). After washing the membrane to which the DNA is finally transfened, the membrane can be placed against X-ray film or a phosphorimager to detect the radioactive or chemiluminescent signal. In screening for the expression of hRasGRP4 RNA, Northern blot hybridizations using the foregoing conditions can be performed on samples taken from subjects suspected of having a condition characterized by abenant expression of a hRasGRP4 molecule, e.g., abnormal mast cell function and/or abnormal hRasGRP4 protein expression. Amplification protocols such as real time RT-PCR and PCR using primers that hybridize to the presented nucleotided sequences, also can be used for detection ofthe RasGRP4 genes or expression thereof.

Identification of related sequences can be achieved using PCR and other amplification techniques suitable for cloning related nucleic acid sequences. Preferably, PCR primers are selected to amplify portions of a nucleic acid sequence believed to encode conserved regions(e.g., the guanine nucleotide exchange factor domain or the phorbol ester receptor domain). Again, nucleic acids are preferably amplified from a tissue-specific library (e.g., mast cells). One also can use expression cloning utilizing the antisera described herein to identify nucleic acids that encode related antigenic proteins in humans or other species using the SEREX procedure to screen the appropriate expression libraries. (See: Sahin et al. Proc. Natl. Acad. Sci. USA 92:11810-11813, 1995).

Nucleic acid sequences ofthe invention also include nucleic acids that have nucleotide deletions, additions, and/or substitutions. Such nucleotide changes may be single nucleotide changes in a molecule, or may be more than one nucleotide change in a molecule. For example, the nucleotide sequence of SEQ DD NO:l may be changed by a single nucleotide change or by multiple nucleotide changes, including deletions, additions, and/or substitutions to the original nucleic acid sequence provided herein. As used herein the terms: "deletion", "addition", and "substitution" mean deletion, addition, and substitution changes to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleic acids ofa sequence ofthe invention. The invention also includes degenerate nucleic acid molecules which include alternative codons to those present in the native materials. For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Each ofthe six codons is equivalent for the purposes of encoding a serine residue. Thus, it will be apparent to one of ordinary skill in the art that any ofthe serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating hRasGRP4 protein. Similarly, nucleotide sequence triplets that encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). Other amino acid residues may be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy ofthe genetic code.

According to another aspect ofthe invention, further isolated nucleic acid molecules that are based on the above-noted hRasGRP4 nucleic acid molecules are provided. In this aspect, the isolated nucleic acid molecules are selected from the group consisting of: (a) a unique fragment ofthe nucleotide sequence set forth as SEQ DD NO: 1 between

12 and 32 nucleotides in length or more and (b) complements of (a), wherein the unique fragments exclude nucleic acids including nucleotide sequences that are contained within SEQ DD NO: 1, and that are known as ofthe filing date of this application.

The invention also provides isolated unique fragments of SEQ DD NO: 1 or complements of SEQ DD NO: 1. A unique fragment is one that is a 'signature' for the larger nucleic acid. It, for example, is long enough to assure that its precise sequence is not found in molecules outside ofthe hRasGRP4 nucleic acid molecules defined above. Those of ordinary skill in the art may apply no more than routine procedures to determine if a fragment is unique within the human or mouse genome. Unique fragments of SEQ DD NO:l, however, exclude fragments completely composed of nucleotide sequences that were known as ofthe filing date of this application (See list in Table 1).

Unique fragments can be used as probes in Southern blot, Northern blot, and Gene Chip/microanay assays to identify such nucleic acid molecules, or can be used in amplification assays such as those employing PCR. As known to those skilled in the art, large probes such as 200 nucleotides or more are prefened for certain uses such as Southern blots, while smaller fragments will be prefened for uses such as in PCR and Gene Chip/microanay assays. Unique fragments also can be used to produce fusion proteins for generating antibodies or determining binding ofthe polypeptide fragments, or for generating immunoassay components. Likewise, unique fragments can be employed to produce nonfused fragments ofthe hRasGRP4 polypeptides that are useful, for example, in the preparation of antibodies in immunoassays. Unique fragments further can be used as antisense or RNAi oligonucleotides to inhibit the expression of hRasGRP4 nucleic acids and polypeptides, particularly for therapeutic purposes as described in greater detail below.

As will be recognized by those skilled in the art, the size ofthe unique fragment will depend upon its conservancy in the genetic code. Thus, some regions of SEQ DD NO: 1 and its complement will require longer segments to be unique while others will require only short segments, typically between 12 and 32 nucleotides or more in length (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32 or more), up to the entire length ofthe disclosed sequence. Many segments ofthe polynucleotide coding region or complements thereof that are 18 or more nucleotides in length will be unique. Those skilled in the art are well- versed in methods for selecting such sequences, typically on the basis of the ability ofthe unique fragment to selectively distinguish the sequence of interest from non- hRasGRP4 nucleic acid molecules. A comparison ofthe sequence ofthe fragment to those on known data bases typically is all that is necessary, although in vitro confinnatory hybridization and sequencing analysis may be performed.

A unique fragment can be a functional fragment. A functional fragment of a nucleic acid molecule ofthe invention is a fragment which retains some functional property ofthe larger nucleic acid molecule, such as coding for a functional polypeptide, binding to proteins, regulating transcription of operably linked nucleic acid molecules, and the like. For example, the CDC25-like catalytic domain in hRasGRP4 noted in Figure 4 can be used in the development of a low molecular weight synthetic compound that specifically inactivates this signal-transduction protein in human mast cells. One of ordinary skill in the art can readily determine using the assays described herein and those well known in the art to determine whether a fragment is a functional fragment of a nucleic acid molecule using no more than routine experimentation. hi yet another aspect ofthe invention, Mutant hRasGRP4 nucleic acid molecules are provided. Exemplary Mutant hRasGRP4 molecules include SEQ DD NO: 3, SEQ DD NO: 5, or SEQ DD NO:58, which do not encode fully functional hRasGRP4 proteins. Rather, these Mutant hRasGRP4 nucleic acid molecules encode Mutant hRasGRP4 proteins, i.e., proteins that do not exhibit 100% of hRasGRP4 protein functional activity. It is understood that some mutants will encode non-functional hRasGRP4 proteins, and other mutants will encode RasGRP4 proteins with reduced or enhanced function. For example, a mutant may encode a hRasGRP4 protein that has from 0 through 25% of hRasGRP4 protein functional activity, 0 through 50% of hRasGRP4 functional activity, 0 through 75% of hRasGRP4 protein functional activity, or 0 through 95% of hRasGRP4 protein functional activity, as assessed, for example, by the guanine nucleotide exchange assay described in the Examples. It will be understood by one of ordinary skill in the art, that some mutant RasGRP4 nucleic acids may encode proteins that have over 100% of hRasGRP4 protein functional activity. For example, a mutant may encode an hRasGRP4 protein that has from 101% through 125% of hRasGRP4 functional activity, 125% through 150% of hRasGRP4 functional activity, or 150% through 200% or more of hRasGRP4 functional activity as assessed for example, by the guanine nucleotide exchange assay described in the Examples. The level of function of a Mutant hRasGRP4 protein can be determined and compared to that of a normal hRasGRP4 protein using standard assays known to one of ordinary skill in the art. Such assays include, but are not limited to the guanine nucleotide exchange assay described herein. As used herein, the term "affect the functional activity" means to either inhibit or enhance the functional activity. Additional Mutant hRasGRP4 nucleic acid molecules ofthe invention contain a sequence which is identical to SEQ ID NO: 1 with the exception that the sequence includes one or more mutations, e.g., deletions, additions or substitutions, such that the Mutant hRasGRP4 nucleic acid molecule does not encode a fully functional hRasGRP4 protein or encodes a hRasGRP4 protein with greater than 100% RasGRP4 functional activity. Rather, the Mutant hRasGRP4 nucleic acid molecules encode a Mutant hRasGRP4 protein, i.e., a protein which exhibits reduced or enhanced hRasGRP4 protein functional activity. It is to be understood that some mutants will encode non-functional hRasGRP4 proteins, and other mutants will encode hRasGRP4 proteins with reduced or enhanced function. The level of function of a Mutant hRasGRP4 protein can be determined and compared to that of hRasGRP4 protein using standard assays known to one of ordinary skill in the art. Such assays include, but are not limited to the guanine nucleotide exchange assay described herein.

According to an aspect ofthe invention related to the mutant nucleic acid molecules, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the group consisting of: (a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ ED NO: 3, SEQ DD NO: 5, or SEQ DD NO:58 and which codes for a Mutant hRasGRP4 protein, (b) deletions, additions and substitutions ofthe nucleic acid molecules of (a),

(c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy ofthe genetic code, and

(d) complements of (a), (b), or (c). The prefened isolated nucleic acids of this aspect ofthe invention are Mutant hRasGRP4 nucleic acid molecules which encode a Mutant hRasGRP4 protein.

As used herein with respect to nucleic acid molecules, in general, the term "isolated" means: (i) amplified in vitro by, for example, PCR; (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis. An isolated nucleic acid molecule is one which is readily manipulable by recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known or for which PCR primer sequences have been disclosed is considered isolated but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid molecule may be substantially purified, but need not be. For example, a nucleic acid molecule that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage ofthe material in the cell in which it resides. Such a nucleic acid molecule is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art. An isolated nucleic acid molecule as used herein is not a naturally occurring chromosome.

As mentioned above, the invention embraces antisense or RNAi oligonucleotides that selectively bind to a Mutant lιRasGRP4 nucleic acid molecule encoding a Mutant hRasGRP4 protein. This is desirable in medical conditions wherein an abenant hRasGRP4 expression is not desirable, e.g., mastocytosis and/or asthma. As used herein, a "Mutant hRasGRP4 nucleic acid molecule" refers to a fιRasGRP4 nucleic acid molecule which includes a mutation (addition, deletion, or substitution) such that the Mutant hRasGRP4 nucleic acid molecule does not encode a fully functional hRasGRP4 protein. Rather, the Mutant hRasGRP4 nucleic acid molecule encodes a Mutant hRasGRP4 protein, i.e., a protein which does not exhibit the same functional activity as a hRasGRP4 protein. Thus, a "Mutant hRasGRP4 protein" refers to a gene product of a Mutant hRasGRP4 nucleic acid molecule. As used herein, the term "abenant" includes decreased expression (including zero expression) ofthe natural hRasGRP4 molecule (nucleic acid or protein) as compared to its expression in a subject who does not have a RasGRP4-associated disorder or increased expression of a Mutant hRasGRP4 molecule (nucleic acid or protein) as compared to its expression in a subject who does not have a RasGRP4-associated disorder.

As used herein, the terms "antisense oligonucleotide," "antisense molecule," or "antisense" describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to a transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense or RNAi oligonucleotide molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript. Those skilled in the art will recognize that the exact length ofthe antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence ofthe target and the particular bases which comprise that sequence. It is prefened that the antisense oligonucleotide be constructed and ananged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon SEQ DD NO: 1, SEQ DD NO: 3, SEQ DD NO: 5, and SEQ DD NO:58 or upon allelic or homologous genomic and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense and/or RNAi molecules for use in accordance with the present invention, hi order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides (Wagner et al., Nature Biotechnology 14: 840-844, 1996).

Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases. Although oligonucleotides may be chosen which are antisense to any region ofthe gene or its transcripts, in prefened embodiments the antisense oligonucleotides conespond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation, or promoter sites. In addition, 3 '-untranslated regions may be targeted. Targeting to mRNA splicing sites also has been used in the art but may be less prefened because alternative mRNA splicing ofthe hRasGRP4 transcript occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439-457, 1994) and at which proteins are not expected to bind. The present invention also provides for antisense oligonucleotides which are complementary to genomic DNA and/or cDNA conesponding to SEQ DD NO: 1, SEQ DD NO: 3, SEQ DD NO: 5, or SEQ DD NO:58. Antisense to allelic or homologous cDNAs and genomic DNAs are enabled without undue experimentation.

The invention also relates in part to the use of RNA interference or "RNAi." RNAi involves the use of double-stranded RNA (dsRNA) to block gene expression, (see: Sui, G, et al, Proc Natl. Acad. Sci U.S.A. 99:5515-5520,2002). Methods of applying RNAi strategies in embodiments ofthe invention would be understood by one of ordinary skill in the art.

Mouse RasGRP4 (mRas GRP4) Nucleic Acid Compositions and Utilities

According to one aspect ofthe invention, an isolated nucleic acid molecule is provided. The isolated nucleic acid molecule is selected from the group consisting of: (a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ DD NO: 7 and which code for a mouse RasGRP4 (mRasGRP4) protein,

(b) deletions, additions and substitutions ofthe nucleic acid molecules of (a),

(d) complements of (a), (b), or (c).

The prefened isolated mouse nucleic acids ofthe invention are mRasGRP4 nucleic acid molecules which encode an mRasGRP4 protein. As used herein, a mRasGRP4 protein refers to a protein that is encoded by a nucleic acid having SEQ DD NO: 7 or a functional fragment thereof, or a functional equivalent thereof (e.g., a nucleic acid sequence encoding the same protein as encoded by SEQ DD NO: 7), provided that the functional fragment or equivalent encodes a protein which exhibits a nιRasGRP4 functional activity. As used herein, a mRasGRP4 functional activity refers to the ability of a mRasGRP4 protein to modulate one or more ofthe following parameters: mast cell development, mast cell function, and/or cell-cell interactions. An exemplary mRasGRP4 functional activity is mast cell activation such as initiating and/or increasing mast cell development, or activity. Although not wishing to be bound to any particular theory or mechanism, it is believed that the mRasGRP4 protein may affect at least some ofthe above-noted cell functions by participating in the varied signal transduction pathways in mature mouse mast cells, and may also regulate one or more ofthe intracellular pathways that control the differentiation and maturation of committed progenitors (e.g. CD47CD87CD14⁺ mononuclear cells) as they develop into fully granulated mouse mast cells, and, thereby, modulating mast cell responses, such as allergic reactions and mast-cell-associated disease. mRasGRP4 functional activity can be determined, for example, by measuring mRasGRP4 ability to transfer GTP to H-Ras and its family members (e.g., RhoA, Cdc42, Racl, Rab3A, and Ran), using, for example, the assays described in the Examples. In the prefened embodiments of this aspect ofthe invention, the isolated nucleic acid molecule is SEQ DD NO: 7 or the coding region thereof. The invention also provides nucleic acid molecules which code for mRasGRP4 proteins and which hybridize under high stringency conditions to a nucleic acid molecule consisting ofthe nucleotide set forth in SEQ DD NO: 7. Such nucleic acids may be DNA, RNA, composed of mixed deoxyribonucleotides and ribonucleotides, or may also incoφorate synthetic non-natural nucleotides. Various methods for determining the expression ofa nucleic acid and/or a polypeptide in cells are known to those of skill in the art and are described further below and in the Examples. As used herein, the term protein is meant to include large molecular weight proteins and peptides and low molecular weight peptides or fragments thereof. The term "high stringency conditions" as used herein is defined above in reference to the hRasGRP4 nucleic acid molecules.

In general homologs and alleles ofthe mRasGRP4 typically will share at least 75% nucleotide identity and/or at least 90% amino acid identity to SEQ DD NO: 7 and SEQ DD NO: 8, respectively, in some instances will share at least 80% nucleotide identity and/or at least 95% amino acid identity and in still other instances will share at least 85% nucleotide identity and/or at least 99% amino acid identity. Prefened homologs and alleles share nucleotide and amino acid identities with SEQ DD NO: 7 and SEQ DD NO: 8, respectively, and encode polypeptides of greater than 80%, more preferably greater than 90%, still more preferably greater than 95% and most preferably greater than 99% identity. The percent identity can be calculated using various, publicly available software tools such as those described above in reference to hRasGRP4 molecules. In screening for mRasGRP4 genes, a Southern blot may be performed using the foregoing conditions, together with a detectably labeled probe (e.g. radioactive or chemiluminescent probes), such as using the conditions described above in reference to the hRasGRP4 nucleic acid molecules. Identification of related sequences also can be achieved using PCR and other amplification techniques suitable for cloning related nucleic acid sequences such as those described above in reference to the hRasGRP4 nucleic acid molecules.

The invention also includes nucleic acids that have nucleotide deletions, additions, and/or substitutions. Such nucleotide deletions, additions, and/or substitutions are described above in reference to the hRasGRP4 nucleic acid molecules.

The invention also includes degenerate nucleic acid molecules which include alternative codons to those present in the native materials. Such alternative codons are described above in reference to the hRasGRP4 nucleic acid molecules.

According to another aspect ofthe invention, further isolated nucleic acid molecules that are based on the above-noted mRasGRP4 nucleic acid molecules are provided, hi this aspect, the isolated nucleic acid molecules are selected from the group consisting of:

(a) a unique fragment ofthe nucleotide sequence set forth as SEQ DD NO: 7 between 12 and 32 nucleotides in length or more and

(b) complements of (a), wherein the unique fragments exclude nucleic acids including nucleotide sequences that are contained within SEQ DD NO: 7, and that are known as ofthe filing date of this application. (See, e.g., Table 1.)

The invention also provides isolated unique fragments of SEQ DD NO: 7 or complements of SEQ ED NO: 7. A unique fragment is one that is a 'signature' for the larger nucleic acid and is described above in reference to the hRasGRP4 nucleic acid molecules.

Unique fragments ofthe mRasGRP4 nucleic acid molecules, however, exclude fragments completely composed ofthe nucleotide sequences that are contained within SEQ ED NO: 7 and that are known as ofthe filing date of this application.

Uses of unique fragments are described above in reference to the hRasGRP4 nucleic acid molecules. Likewise, unique fragments can be used in any ofthe applications described above. As described above in reference to the hRasGRP4 nucleic acid molecules and as will be recognized by those skilled in the art, the size ofthe unique fragment will depend upon its conservancy in the genetic code. Thus, some regions of SEQ ED NO: 7 and its complement will require longer segments to be unique while others will require only short segments, typically between 12 and 32 nucleotides or more in length (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32 or more), up to the entire length ofthe disclosed sequence.

A unique fragment ofthe mRasGRP4 molecule can be a functional fragment. A functional fragment of a mouse nucleic acid molecule ofthe invention is a fragment which retains some functional property ofthe larger nucleic acid molecule, such as coding for a functional polypeptide, binding to proteins, regulating transcription of operably linked nucleic acid molecules, and the like. For example, the CDC25-like catalytic domain in mRasGRP4 noted in Figure 14 could be used in the development of a low molecular weight synthetic compound that specifically inactivates this signal-transduction protein in mouse mast cells. One of ordinary skill in the art can readily determine using the assays described herein and those well known in the art to determine whether a fragment is a functional fragment of a nucleic acid molecule using no more than routine experimentation.

In yet another aspect ofthe invention, Mutant mRasGRP4 nucleic acid molecules are provided. Because the Mutant mRasGRP4 molecule (SEQ ID NO: 9) possesses an altered DAG-binding domain, its ability to activate varied Ras family members in the context ofa living cell could be altered. It is to be understood that some mutants will encode nonfunctional mRasGRP4 proteins, and other mutants will encode mRasGRP4 proteins with reduced or enhanced function. The level of function of a Mutant mRasGRP4 protein can be determined and compared to that of mRasGRP4 protein using standard assays known to one of ordinary skill in the art. Such assays include, but not limited to the guanine nucleotide exchange assay described herein.

Additional Mutant mRasGRP4 nucleic acid molecules ofthe invention contain a sequence which is identical to SEQ DD NO: 7 with the exception that the sequence includes one or more mutations, e.g., deletions, additions or substitutions, such that the Mutant nιRasGRP4 nucleic acid molecule does not encode a functional mRasGRP4 protein. Rather, the Mutant mRasGRP4 nucleic acid molecules encode a Mutant nιRasGRP4 protein, i.e., a protein which does not exhibit the same functional activity as the mRasGRP4 protein. It is to be understood that some mutants will encode non-functional mRasGRP4 proteins, and other mutants will encode mRasGRP4 proteins with reduced or enhanced functional activity.

For example, a mutant may encode a mRasGRP4 protein that has from 0 through 25% of mRasGRP4 protein functional activity, 0 through 50% of mRasGRP4 functional activity, 0 through 75% of mRasGRP4 protein functional activity, or 0 through 95% of mRasGRP4 protein functional activity, as assessed for example, by the guanine nucleotide exchange assay described in the Examples. It will be understood by one of ordinary skill in the art, that some mutant n RasGRP4 nucleic acids may encode proteins that have over 100% of mRasGRP4 protein functional activity. For example, a mutant may encode an mRasGRP4 protein that has from 101% through 125% of mRasGRP4 functional activity, 125% through 150% of mRasGRP4 functional activity, or 150% through 200% or more of mRasGRP4 functional activity as assessed for example, by the guanine nucleotide exchange assay described in the Examples. The level of function of a Mutant mRasGRP4 protein can be determined and compared to that of mRasGRP4 protein using standard assays known to one of ordinary skill in the art. Such assays include, but are not limited to the guanine nucleotide exchange assay described herein.

The level of function of a Mutant nιRasGRP4 protein can be determined and compared to that of mRasGRP4 protein using standard assays known to one of ordinary skill in the art. Such assays include, but are not limited to the guanine nucleotide exchange assay described herein. As used herein, the term "affect the functional activity" means to either inhibit or enhance the functional activity.

Additional Mutant mRasGRP4 nucleic acid molecules ofthe invention contain a sequence which is identical to SEQ DD NO: 7 with the exception that the sequence includes one or more mutations, e.g., deletions, additions or substitutions, such that the Mutant mRasGRP4 nucleic acid molecule does not encode a fully functional mRasGRP4 protein or encodes a mRasGRP4 protein with greater than 100% RasGRP4 functional activity. Rather, the Mutant mRasGRP4 nucleic acid molecules encode a Mutant mRasGRP4 protein, i.e., a protein which exhibits reduced or enhanced mRasGRP4 protein functional activity. It is to be understood that some mutants will encode non-functional mRasGRP4 proteins, and other mutants will encode mRasGRP4 proteins with reduced or enhanced function. The level of function of a Mutant mRasGRP4 protein can be determined and compared to that of mRasGRP4 protein using standard assays known to one of ordinary skill in the art. Such assays include, but are not limited to the guanine nucleotide exchange assay described herein.

Thus, according to another aspect ofthe invention, an isolated nucleic acid molecule that codes for a mutant mRasGRP4 protein is provided. The isolated nucleic acid molecule is selected from the group consisting of: (a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule having a nucleotide sequence set forth as SEQ DD NO: 9 and which codes for a Mutant mRasGRP4 protein,

(b) deletions, additions and substitutions ofthe nucleic acid molecules of (a),

(d) complements of (a), (b), or (c).

The prefened isolated nucleic acids ofthe invention are Mutant n RasGRP4 nucleic acid molecules which encode a Mutant mRasGRP4 protein.

As used herein with respect to nucleic acid molecules, the term "isolated" is as defined above in reference to the hRasGRP4 nucleic acid molecules.

As mentioned above, the invention embraces antisense and RNAi oligonucleotides that selectively bind to a Mutant mRasGRP4 nucleic acid molecule encoding a Mutant nιRasGRP4 protein. This is desirable in modulating expression of mRasGRP4 in, e.g., mouse models of a human mast cell-dependent disorder such as asthma, systemic mastocytosis, allergic inflammation. As used herein, a "Mutant mRasGRP4 nucleic acid molecule" refers to a mRasGRP4 nucleic acid molecule which includes a mutation (addition, deletion, or substitution) such that the Mutant nιRasGRP4 nucleic acid molecule does not encode a fully functional mRasGRP4 protein. Rather, the Mutant nιRasGRP4 nucleic acid molecule encodes a Mutant mRasGRP4 protein, i.e., a protein which does not exhibit the same functional activity as a mRasGRP4 protein. A "Mutant mRasGRP4 protein" refers to a mRasGRP4 protein that is a gene product of a Mutant mRasGRP4 nucleic acid molecule which includes a mutation that affects the functional activity ofthe mRasGRP4 molecule. As used herein, the term "abenant" refers to decreased expression (including zero expression) or increased expression compared to the expression of a mRasGRP4 molecule (nucleic acid or protein) in a subject who does not have a RasGRP4-associated disorder or increased expression of a Mutant mRasGRP4 molecule (nucleic acid or protein) as compared to its expression in a subject who does not have a RasGRP4-associated disorder. Because mice express mRasGRP4, which is the ortholog of hRasGRP4, the mouse also can be utilized as a model system in which to screen compounds for their ability to regulate human mast cells to identify lead compounds for diagnostic and therapeutic applications.

As used herein, the terms "antisense oligonucleotide," "antisense molecule" or "antisense," are as defined above in reference to hRasGRP4 molecules. Based upon SEQ DD NO: 7 and SEQ DD NO: 9, or upon allelic or homologous genomic and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for inhibition, such oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides (Wagner et al., Nature Biotechnology 14: 840-844, 1996).

Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases. The present invention also provides for antisense oligonucleotides which are complementary to genomic DNA and/or cDNA conesponding to SEQ DD NO: 7 or SEQ ED NO: 9. Antisense to allelic or homologous cDNAs and genomic DNAs are enabled without undue experimentation.

The invention also relates in part to the use of RNA interference or "RNAi." RNAi involves the use of double-stranded RNA (dsRNA) to block gene expression (see: Sui, G, et al, Proc Natl. Acad. Sci U.S.A. 99:5515-5520,2002). Methods of applying RNAi strategies in embodiments ofthe invention would be understood by one of ordinary skill in the art.

Human and Mouse Nucleic Acid Expression Veetors/Antisense/Delivery In one set of embodiments ofthe aforementioned human and mouse compositions and utilities, the antisense oligonucleotides ofthe invention maybe composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These oligonucleotides may be prepared by art-recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors. In prefened embodiments, however, the antisense oligonucleotides ofthe invention also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways, which do not prevent them from hybridizing to their target but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.

The term "modified oligonucleotide" as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Prefened synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides. The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus, modified oligonucleotides may include a 2'-O- alkylated ribose group, hi addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acid molecules encoding RasGRP4 proteins, together with pharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. The compositions should be sterile and contain a therapeutically effective amount ofthe antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness ofthe biological activity ofthe active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics ofthe carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.

According to yet another aspect ofthe invention, an expression vector comprising any ofthe isolated nucleic acid molecules ofthe invention, preferably operably linked to a promoter is provided. In a related aspect, host cells transformed or transfected with such expression vectors also are provided.

As used herein, a "vector" may be any of a number of nucleic acid molecules into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids and virus genomes. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication ofthe desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). Prefened vectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which they are operably joined.

As used herein, a coding sequence and regulatory sequences are said to be "operably" joined when they are covalently linked in such a way as to place the expression or transcription ofthe coding sequence under the influence or control ofthe regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription ofthe coding sequence and ifthe nature of the linkage between the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability ofthe promoter region to direct the transcription ofthe coding sequences, or (3) interfere with the ability ofthe conesponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.

The precise nature ofthe regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.

Especially, such 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors ofthe invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

It will also be recognized that the invention embraces the use ofthe RasGRP4 cDNA sequences or Mutant RasGRP4 cDNA sequences in expression vectors, as well as to transfect host cells and cell lines, be these prokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO cells, COS cells, yeast expression systems and recombinant baculovirus expression in insect cells). Especially useful are mammalian cells such as human, mouse, hamster, pig, goat, primate, etc. They may be ofa wide variety of tissue types, including mast cells, fibroblasts, oocytes, monocytes, lymphocytes, and leukocytes, and they may be primary cells or cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone manow stem cells and embryonic stem cells. The expression vectors require that the pertinent sequence, i.e., those nucleic acids described supra, be operably linked to a promoter. The invention also permits the construction of RasGRP4 polypeptide gene "knockouts" or "knock-ins" in cells and in animals, providing materials for studying certain aspects of RasGRP4-associated diseases and immune system responses to RasGRP4-associated diseases by regulating the expression of RasGRP4 polypeptides. For example, a knock-in mouse may be constructed and examined for clinical parallels between the model and a RasGRP4-associated disease affected mouse with upregulated expression ofa normal or mutated RasGRP4 polypeptide. Such a cellular or animal model may be useful for assessing treatment strategies for RasGRP4-associated diseases. An example ofa "knock-in" mouse, although not intended to be limiting, involves introducing a hRasGRP4 molecule into the V3 mast cell line. The cells are then injected into the tail vein of a normal BADB/c mouse.

Approximately 7 days after the cells have been adoptively transfened, the function ofthe V3 mast cells that home to the spleen, liver, intestine, and lung are evaluated. This type of "knock-in" model provides a model with which to evaluate the effects of candidate pharmacological agents (e.g. inhibitory effects) on a living animal that expresses a hRasGRP4 molecule.

Alternative types of animal models for RasGRP4-associated diseases may be developed based on the invention and may provide a model in which to test treatments, and assess the etiology of RasGRP4-associated diseases. As used herein the term "variable level" means a level of expression of an RasGRP4 molecule in a cell or non-human animal model, that differs from the level of expression of that molecule normally found under identical conditions in that cell or animal type. For example, a variable level of expression in a cell following transfection with a RasGRP4 molecule ofthe invention, means the level of expression of RasGRP4 molecule of in the transfected cell differs from the normal level in the same cell type cell under identical conditions. The level may represent an increase or decrease in the normal level.

According to still a further aspect ofthe invention, a transgenic non-human animal comprising an expression vector ofthe invention is provided, including a transgenic non- human animal which has reduced expression of a RasGRP4 nucleic acid molecule or elevated expression of a hRasGRP4 or Mutant RasGRP4 nucleic acid molecule. As used herein, "transgenic non-human animals" includes non-human animals having one or more exogenous nucleic acid molecules incoφorated in germ line cells and/or somatic cells. Thus the transgenic animal include "knockout" animals having a homozygous or heterozygous gene disruption by homologous recombination, animals having episomal or chromosomally incoφorated expression vectors, etc. Knockout animals can be prepared by homologous recombination using embryonic stem cells as is well known in the art. The recombination can be facilitated by the cre/lox system or other recombinase systems known to one of ordinary skill in the art. In certain embodiments, the recombinase system itself is expressed conditionally, for example, in certain tissues or cell types, at certain embryonic or post-embryonic developmental stages, inducibly by the addition ofa compound which increases or decreases expression, and the like. In general, the conditional expression vectors used in such systems use a variety of promoters which confer the desired gene expression pattern (e.g., temporal or spatial). Conditional promoters also can be operably linked to

RasGRP4 nucleic acid molecules to increase or decrease expression of a RasGRP4 molecule in a regulated or conditional manner. Trans-acting negative or positive regulators of RasGRP4 activity or expression also can be operably linked to a conditional promoter as described above. Such trans-acting regulators include antisense RasGRP4 nucleic acid molecules, nucleic acid molecules which encode dominant negative RasGRP4 molecules, ribozyme molecules specific for RasGRP4 nucleic acid molecules, and the like. The transgenic non-human animals are useful in experiments directed toward testing biochemical or physiological effects of diagnostics or therapeutics for conditions characterized by increased or decreased RasGRP4 molecule expression. Other uses will be apparent to one of ordinary skill in the art. Thus, the invention also permits the construction of RasGRP4 gene "knock-outs" in cells and in animals, providing materials for studying certain aspects of mast cell disorders and/or RasGRP4-associated diseases.

Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA) encoding RasGRP4 protein, fragment, or variant thereof. The heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression ofthe heterologous DNA in the host cell. Prefened systems for mRNA expression in mammalian cells are those such as pRc/CMV (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences. Additionally, suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Ban virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element. Another expression vector is the pEF- BOS plasmid containing the promoter of polypeptide Elongation Factor lα, which stimulates efficiently transcription in vitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another prefened expression vector is an adenovirus, described by Stratford-Perricaudet, which is defective for El and E3 proteins (J. Clin. Invest. 90:626-630, 1992). The use ofthe adenovirus as an Adeno.PlA recombinant is disclosed by Warmer et al., in intradermal injection in mice for immunization against PI A (Int. J. Cancer, 67:303-310, 1996).

The invention also embraces so-called expression kits, which allow the artisan to prepare a desired expression vector or vectors. Such expression kits include at least separate portions of each ofthe previously discussed coding sequences. Other components may be added, as desired, as long as the previously mentioned sequences, which are required, are included.

Human RasGRP4 (hRasGRP4) Protein Compositions and Utilities According to another aspect ofthe invention, an isolated protein encoded by any of the foregoing isolated nucleic acid molecules ofthe invention is provided. Preferably, the isolated protein comprises SEQ ED NO: 2. The invention also embraces Mutant hRasGRP4 proteins, including SEQ DD NO: 4, SEQ DD NO: 6, and SEQ DD NO:59, and others such as those described in the Examples. The invention also provides isolated proteins, which include the protein SEQ DD NO:

2 and unique fragments of SEQ ED NO: 2. Such proteins are useful, for example, alone or as fusion proteins to generate antibodies for hRasGRP4 proteins.

As used herein, a hRasGRP4 protein refers to a protein which is encoded by a nucleic acid having SEQ ED NO: 1, a functional fragment thereof, or a functional equivalent thereof (e.g., a nucleic acid sequence encoding the same protein as encoded by SEQ DD NO: 1), provided that the functional fragment or equivalent encodes a lιRasGRP4 protein which exhibits a hRasGRP4 functional activity. As used herein, a hRasGRP4 functional activity refers to the ability of a hRasGRP4 protein to modulate one or more ofthe following parameters: mast cell development, mast cell function, and/or cell-cell interactions. An exemplary hRasGRP4 functional activity is mast cell activation such as initiating and/or increasing mast cell development, or activity. Although not wishing to be bound to any particular theory or mechanism, it is believed that the hRasGRP4 protein may affect at least some ofthe above-noted cell functions by participating in signal transduction pathways in mature human mast cells, and may also regulate one or more ofthe intracellular pathways that control the differentiation and maturation of committed progenitors (e.g. CD47CD8^" /CD14⁺ mononuclear cells) as they develop into fully granulated mast cells, and, thereby, modulate mast cell responses, such as allergic reactions and mast-cell associated disease. RasGRP4 functional activity can be detemiined by measuring its ability to transfer GTP to H-Ras and its family members (e.g., RhoA, Cdc42, Racl, Rab3A, and Ran), using, for example, the assays described in the Examples.

Additional Mutant hRasGRP4 protein molecules ofthe invention contain a sequence which is identical to SEQ ED NO: 2 with the exception that the sequence includes one or more mutations, e.g., deletions, additions or substitutions, such that the Mutant hRasGRP4 protein molecule is not a fully functional hRasGRP4 protein or is a hRasGRP4 protein with greater than 100% RasGRP4 functional activity.

Mouse RasGRP4 (mRasGRP4) Protein Compositions and Utilities

According to another aspect ofthe invention, an isolated protein encoded by any ofthe foregoing isolated mRasGRP4 nucleic acid molecules ofthe invention is provided. Preferably, the isolated protein comprises SEQ ED NO: 8. The invention also embraces Mutant mRasGRP4 proteins, including SEQ DD NO: 10, and others such as those described in the Examples. Additional Mutant nιRasGRP4 protein molecules ofthe invention contain a sequence which is identical to SEQ DD NO: 8 with the exception that the sequence includes one or more mutations, e.g., deletions, additions or substitutions, such that the Mutant hRasGRP4 protein molecule is not a fully functional mRasGRP4 protein or is a mRasGRP4 protein with greater than 100% RasGRP4 functional activity. The invention also provides isolated proteins, which include the protein SEQ DD NO:

8 and unique fragments of SEQ DD NO: 8. Such proteins are useful, for example, alone or as fusion proteins to generate antibodies, as a component(s) of an immunoassay or for determining the binding specificity of HLA molecules and/or CTL clones for n RasGRP4 proteins.

As used herein, an mRasGRP4 protein refers to a protein which is encoded by a nucleic acid having SEQ DD NO: 7, a functional fragment thereof, or a functional equivalent thereof (e.g., a nucleic acid sequence encoding the same protein as encoded by SEQ DD NO: 7), provided that the functional fragment or equivalent encodes an mRasGRP4 protein which exhibits an mRasGRP4 functional activity. As used herein, an mRasGRP4 functional activity refers to the ability of an mRasGRP4 protein to modulate one or more ofthe following parameters: mast cell development, mast cell function, and/or cell-cell interactions. An exemplary mRasGRP4 functional activity is mast cell activation such as initiating and/or increasing mast cell development, or activity. Although not wishing to be bound to any particular theory or mechanism, it is believed that the nιRasGRP4 protein may affect at least some ofthe above-noted cell functions by participating in signal transduction pathways in mature mouse mast cells, and may also regulate one or more ofthe intracellular pathways that control the differentiation and maturation of committed progenitors (e.g. CD47CD8^" /CD14⁺ mononuclear cells) as they develop into fully granulated mast cells, and, thereby, modulating mast cell responses, such as allergic reactions and mast-cell associated disease. mRasGRP4 functional activity can be determined by measuring its ability to transfer GTP to H-Ras and its family members (e.g., RhoA, Cdc42, Racl, Rab3A, and Ran), using, for example, the assays described in the Examples.

Human and Mouse RasGRP4 Protein (general)

Proteins can be isolated from biological samples including tissue or cell homogenates, and can also be expressed recombinantly in a variety of prokaryotic and eukaryotic expression systems by constructing an expression vector appropriate to the expression system, introducing the expression vector into the expression system, and isolating the recombinantly expressed protein. Short polypeptides, including antigenic peptides (such as those presented by MHC molecules on the surface ofa cell for immune recognition) also can be synthesized chemically using well-established methods of peptide synthesis. Thus, as used herein with respect to proteins, "isolated" means separated from its native environment and present in sufficient quantity to permit its identification or use. Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression of a recombinant nucleic acid or (ii) purified as by chromatography or electrophoresis. Isolated proteins or polypeptides may, but need not be, substantially pure. The term "substantially pure" means that the proteins or polypeptides are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use. Substantially pure proteins may be produced by techniques well known in the art. Because an isolated protein may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the protein may comprise only a small percentage by weight ofthe preparation. The protein is nonetheless isolated in that it has been separated from the substances with which it may be associated in living systems, e.g. isolated from other proteins.

A fragment of a RasGRP4 protein, for example, generally has the features and characteristics of fragments including unique fragments as discussed above in connection with nucleic acid molecules. As will be recognized by those skilled in the art, the size ofa fragment which is unique will depend upon factors such as whether the fragment constitutes a portion of a conserved protein domain. Thus, some regions of RasGRP4 protein (e.g., its large CDC25-like catalytic domain shown in Figures 4, 5, 14, and 15) will require longer segments to be unique. Others will require only short segments, typically between 5 and 14 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14 amino acids long) as used to generate mRasGRP4- and hRasGRP4-specific antibodies shown in Figures 3 and 12. Unique fragments of a protein preferably are those fragments which retain a distinct functional capability ofthe protein. Functional capabilities which can be retained in a fragment of a protein include interaction with antibodies, interaction with other proteins or fragments thereof, selective binding of nucleic acid molecules, and enzymatic activity. One important activity is the ability to act as a signature for identifying the polypeptide. Another is the ability to provoke in a human an immune response to a Mutant RasGRP4 molecule but not provoke an immune response to normal levels of a nonmutated RasGRP4 molecule.

Those skilled in the art are well versed in methods for selecting unique amino acid sequences, typically on the basis ofthe ability ofthe fragment to selectively distinguish the sequence of interest from non-family members. A comparison ofthe sequence ofthe fragment to those on known databases typically is all that is necessary.

The invention embraces variants ofthe RasGRP4 proteins described herein. As used herein, a "variant" of a RasGRP4 protein is a protein which contains one or more modifications to the primary amino acid sequence ofa RasGRP4 protein. Modifications which create a RasGRP4 protein variant can be made to a RasGRP4 protein 1) to produce, increase, reduce, or eliminate an activity ofthe RasGRP4 protein or Mutant RasGRP4 protein; 2) to enhance a property ofthe RasGRP4 protein, such as protein stability in an expression system or the stability of protein-protein binding; 3) to provide a novel activity or property to a RasGRP4 protein, such as addition of an antigenic epitope or addition ofa detectable moiety; or 4) to provide equivalent or better binding to a mast cell signaling molecule. Modifications to a RasGRP4 protein or to a Mutant RasGRP4 protein are typically made to the nucleic acid molecule which encodes the protein, and can include deletions, point mutations, truncations, amino acid substitutions, and additions of amino acids or non-amino acid moieties. Alternatively, modifications can be made directly to the protein, such as by cleavage, addition of a linker molecule, addition ofa detectable moiety, such as biotin, addition ofa fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part ofthe RasGRP4 amino acid sequences. One skilled in the art will be familiar with methods for predicting the effect on protein conformation of a change in protein sequence, and can thus "design" a variant RasGRP4 polypeptide according to known methods. One example of such a method is described by Dahiyat and Mayo in Science 278:82-87, 1997, whereby proteins can be designed de novo. The method can be applied to a known protein to vary only a portion ofthe protein sequence. By applying the computational methods of Dahiyat and Mayo, specific variants of a RasGRP4 protein can be proposed and tested to determine whether the variant retains a desired conformation.

In general, variants include RasGRP4 proteins which are modified specifically to alter a feature ofthe protein unrelated to its desired physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a RasGRP4 protein by eliminating proteolysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present).

Mutations of a nucleic acid molecule which encode a RasGRP4 protein preferably preserve the amino acid reading frame ofthe coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a haiφins or loops, which can be deleterious to expression ofthe variant protein. Mutations can be made by selecting an amino acid substitution, or by random mutagenesis ofa selected site in a nucleic acid which encodes the protein. Variant proteins are then expressed and tested for one or more activities to determine which mutation provides a variant protein with the desired properties. Further mutations can be made to variants (or to non- variant RasGRP4 proteins) which are silent as to the amino acid sequence ofthe protein, but which provide prefened codons for translation in a particular host. The prefened codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a RasGRP4 gene or cDNA clone to enhance expression ofthe protein. The activity of variants of RasGRP4 proteins can be tested by cloning the gene encoding the variant RasGRP4 protein into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the variant RasGRP4 protein, and testing for a functional capability ofthe RasGRP4 protein as disclosed herein. For example, the variant RasGRP4 protein can be tested for reaction with autologous or allogeneic sera. Preparation of other variant proteins may favor testing of other activities, as will be known to one of ordinary skill in the art.

The skilled artisan will also realize that conservative amino acid substitutions may be made in RasGRP4 proteins to provide functional variants ofthe foregoing proteins, i.e, the variants which the functional capabilities ofthe RasGRP4 proteins. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution which does not alter the relative charge or size characteristics ofthe protein in which the amino acid substitution is made. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) Ε, D.

For example, upon determining that a peptide derived from a RasGRP4 protein possesses modulator activity such as suppressing/reducing mast cell development and activity, one can make conservative amino acid substitutions to the amino acid sequence of the peptide. The substituted peptides can then be tested for one or more ofthe above-noted functions, in vivo or in vitro. These variants can be tested for improved stability and are useful, inter alia, in pharmaceutical compositions. Functional variants of RasGRP4 proteins, i.e., variants of proteins which retain the function ofthe RasGRP4 proteins, can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al, eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplary functional variants ofthe RasGRP4 proteins include conservative amino acid substitutions ofthe proteins encoded by SEQ DD NO: 1 and SEQ DD NO: 7. Conservative amino-acid substitutions in the amino acid sequence of RasGRP4 proteins to produce functional variants of RasGRP4 proteins typically are made by alteration ofthe nucleic acid molecule encoding a RasGRP4 protein (e.g., SEQ ID NO: 1 or SEQ DD NO: 7). Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR- directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492, 1985), or by chemical synthesis of a gene encoding a RasGRP4 protem. Where amino acid substitutions are made to a small unique fragment of a RasGRP4 protein, the substitutions can be made by directly synthesizing the peptide. The activity of functional variants or fragments of RasGRP4 protein can be tested by cloning the gene or transcript that encodes the altered RasGRP4 protein into a bacterial, mammalian, or insect cell expression vector, introducing the vector into an appropriate host cell, expressing the altered RasGRP4 protein, and testing for a functional capability ofthe RasGRP4 proteins as disclosed herein. The invention as described herein has a number of uses, some of which are described elsewhere herein. First, the invention permits isolation ofthe RasGRP4 protein molecules. A variety of methodologies well known to the skilled practitioner can be utilized to obtain isolated RasGRP4 molecules. The polypeptide may be purified from cells which naturally produce the protein by chromatographic means or immunological recognition. Alternatively, an expression vector may be introduced into cells to cause production ofthe protein. In another method, mRNA transcripts may be microinjected or otherwise introduced into cells to cause production ofthe encoded protein. Translation of mRNA in cell-free extracts such as the reticulocyte lysate system also may be used to produce polypeptide. Those skilled in the art also can readily follow known methods for isolating RasGRP4 proteins. These include, but are not limited to, HPDC, size-exclusion chromatography, ion-exchange chromatography, and immune-affinity chromatography. Those skilled in the art will also be able to utilize recombinant RasGRP4 to determine the crystal structure ofthe RasGRP4 protein and its variants.

In addition, RasGRP4 specific antibodies and/or nucleotide probes could be used to monitor the abenant expression of this signaling protein in cells at the protein and mRNA levels. RasGRP4 normally is restricted to mast cells and its gene resides on chromosome 19ql3.1. As noted at the "Mitelman Database of chromosome abenations in cancer" at the NCI web site (cgap.nci.nih.gov/Chromosome/Mitelman), breakpoint alterations at chromosome 19ql3.1 often lead to leukemia. Thus, the expression of RasGRP4 in a hematopoietic cell other than a mast cell probably is a contributing factor in development of certain leukemias. In situ hybridization and/or RT-PCR/nucleotide sequencing approaches could be used to identify and characterize the RasGRP4 transcripts that are abenantly expressed in leukemia patients. Anti-hRasGRP4 antibodies could be used to evaluate the presence of normal and defective hRasGRP4 proteins in a diagnostic approach to identify the early development of a suspected leukemia or to monitor the success of different pharmacological approaches to eliminate the RasGRP4-expressing leukemia cell from a patient with advanced disease.

The isolation and identification of RasGRP4 nucleic acid molecules also makes it possible for the artisan to diagnose a disorder characterized by abenant expression of a RasGRP4 nucleic acid molecule or protein. These methods involve determining the abenant expression of one or more RasGRP4 nucleic acid molecules and/or Mutant RasGRP4 nucleic acid molecules, and/or encoded RasGRP4 proteins and/or Mutant RasGRP4 proteins. In the former two situations, such determinations can be carried out via any standard nucleic acid determination assay, including the PCR, or assaying with labeled hybridization probes. In the latter two situations, such determinations can be carried out by screening patient antisera for recognition ofthe polypeptide or by assaying biological samples with binding partners (e.g., antibodies) for RasGRP4 proteins or Mutant RasGRP4 proteins.

The invention also provides, in certain embodiments, "dominant negative" polypeptides derived from RasGRP4 proteins. A dominant negative polypeptide is an inactive variant of a protein, which, by interacting with the cellular machinery, displaces an active protein from its interaction with the cellular machinery or competes with the active protein, thereby reducing the effect ofthe active protein. Dominant negative polypeptides are useful, or example, for preparing transgenic non-human animals to further characterize the functions ofthe RasGRP4 molecules and Mutant RasGRP4 molecules disclosed herein. For example, a dominant negative receptor which binds a ligand but does not transmit a signal in response to binding ofthe ligand can reduce the biological effect of expression ofthe ligand. Likewise, a dominant negative catalytically-inactive kinase which interacts normally with target proteins but does not phosphorylate the target proteins can reduce phosphorylation of the target proteins in response to a cellular signal. Similarly, a dominant negative transcription factor which binds to a promoter site in the control region of a gene but does not increase gene transcription can reduce the effect of a normal transcription factor by occupying promoter binding sites without increasing transcription. Another example of a dominant negative RasGRP4 is a mutant form of this protein lacking its calcium-binding domain or its DAG-binding domain. The 3D model of hRasGRP4 (Fig. 5 A) based on the crystal structure of hSos-1 predicts that the guanine nucleotide exchange factor activity of hRasGRP4 is dependent on Glu . If this prediction is conect, the conversion of residue 393 to Ala should result in a dominant negative form of hRasGRP4. The end result ofthe expression ofa dominant negative polypeptide in a cell is an altered function of active RasGRP4. One of ordinary skill in the art can assess the potential for a dominant negative variant of a protein, and using standard mutagenesis techniques to create one or more dominant negative variant polypeptides. For example, one of ordinary skill in the art can modify the sequence of RasGRP4 proteins by site-specific mutagenesis, scanning mutagenesis, partial gene deletion or truncation, and the like. See, e.g., U.S. Patent No. 5,580,723 and Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. The skilled artisan then can test the population of mutagenized proteins for diminution in a selected activity and/or for retention of such an activity. Other similar methods for creating and testing dominant negative variants of a protein will be apparent to one of ordinary skill in the art.

In yet a further aspect ofthe invention, binding polypeptides that selectively bind to a RasGRP4 molecule and/or to a Mutant RasGRP4 molecule are provided. According to this aspect, the binding polypeptides bind to an isolated nucleic acid or protein ofthe invention, including binding to unique fragments thereof. Preferably, the binding polypeptides bind to a RasGRP4 protein, a Mutant RasGRP4 protein, or a unique fragment thereof. In certain particularly prefened embodiments, the binding polypeptide binds to a Mutant RasGRP4 protein but does not bind to a RasGRP4 protein, i.e., the binding polypeptides are selective for binding to the Mutant RasGRP4 protein and can be used in various assays to detect the presence ofthe Mutant RasGRP4 protein without detecting RasGRP4 protein. For example, the variant 2 isoform of RasGRP4 identified in an asthma patient possesses a 14-residue deletion (Figs. 7b and 7c). Thus, a peptide antibody directed against the new sequence that is formed (e.g., Ser-Ser-Asp-Leu-Pro-Gly-Leu-Gly-Lys ;SEQ DD NO: 61) should selectively recognize variant 2. As noted in Figures 2 and 4, 10 single nucleotide polymoφhisms in the hRasGRP4 gene have been identified that result in 5 amino acid differences. In a similar manner, a peptide antibody directed against one or more of these variable sites (e.g., residues 18, 120, 145, 261, or 335) could be used to monitor the expression of different alleles ofthe hRasGRP4 gene in the human population. Mutant RasGRP4 protein binding polypeptides also can be used to selectively bind to a Mutant RasGRP4 molecule in a cell (in vivo or ex vivo) for imaging and therapeutic applications in which, for example, the binding polypeptide is tagged with a detectable label and/or a toxin for targeted delivery to the Mutant RasGRP4 molecule. In prefened embodiments, the binding polypeptide is an antibody or antibody fragment, more preferably, an Fab or F(ab)₂ fragment of an antibody. Typically, the fragment includes a complementarity-determining region (CDR) that is selective for the RasGRP4 protein or Mutant RasGRP4 protein. Any ofthe various types of antibodies can be used for this puφose, including monoclonal antibodies, anti-peptide antibodies, humanized antibodies, single chain antibodies, and chimeric antibodies.

Thus, the invention provides agents which bind to RasGRP4 proteins or Mutant RasGRP4 proteins encoded by RasGRP4 nucleic acid molecules or Mutant RasGRP4 nucleic acid molecules, respectively, and in certain embodiments preferably to unique fragments of the RasGRP4 proteins or Mutant RasGRP4 proteins. Such binding partners can be used in screening assays to detect the presence or absence of a RasGRP4 protein or a Mutant

RasGRP4 protein and in purification protocols to isolate such RasGRP4 proteins. Likewise, such binding partners can be used to selectively target drugs, toxins or other molecules to cells which express Mutant RasGRP4 proteins. In this manner, cells present in solid or non- solid tumors tissues which express Mutant RasGRP4 proteins can be treated with cytotoxic compounds. Such agents also can be used to inhibit the native activity ofthe RasGRP4 polypeptides, for example, by binding to such polypeptides, to further characterize the functions of these molecules The invention, therefore, provides antibodies or fragments of antibodies having the ability to selectively bind to Mutant RasGRP4 proteins, and preferably to unique fragments thereof. Antibodies include polyclonal, monoclonal, and chimeric antibodies, prepared, e.g., according to conventional methodology. The antibodies ofthe present invention thus are prepared by any of a variety of methods, including administering protein, fragments of protein, cells expressing the protein or fragments thereof and the like to an animal to induce polyclonal antibodies. The production of monoclonal antibodies is according to techniques well known in the art. As detailed herein, such antibodies may be used for example to identify tissues expressing protein or to purify protein. Antibodies also may be coupled to specific labeling agents for imaging or to cytotoxic agents, including, but not limited to, methotrexate, radioiodinated compounds, toxins such as ricin, other cytostatic or cytolytic drugs, and so forth. Additional suitable chemical toxins or chemotherapeutic agents that may be coupled to antibodies include members ofthe enediyne family of molecules, such as chalicheamicin and esperamicin. Cytotoxic radionuclides or radiotherapeutic isotopes may be alpha-emitting isotopes such as ²²⁵Ac, ²¹¹At, ²¹²Bi, or ²¹³Bi. Alternatively, the cytotoxic radionuclides may be beta-emitting isotopes such as ¹⁸⁶Rh, ¹⁸⁸Rh, ⁹⁰Y, ¹³¹I or ⁶⁷Cu. Further, the cytotoxic 10'ϊ '7*7 radionuclide may emit Auger and low energy electrons such as the isotopes I, I or Br. Other chemotherapeutic and radiotherapeutic agents are known to those skilled in the art. Significantly, as is well known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding ofthe antibody to its epitope (see, in general, Clark, W.R. (1986) The Experimental Foundations of Modern hnmunologv Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential hnmunolo v, 7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors ofthe complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')₂ fragment, retains both ofthe antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one ofthe antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion ofthe antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity-determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure ofthe paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FRl through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.

It is now well established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of nonspecific or heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody, (see, e.g., US. patents 4,816,567, 5,225,539, 5,585,089, 5,693,762, and 5,859,205). Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion ofthe murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often refened to as "chimeric" antibodies.

Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans.

Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')₂, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')₂ fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDRl and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDRl and/or CDR2 regions have been replaced by homologous human or non- human sequences. As noted in Figure 3 and 12, useful rabbit antibodies against the 12- to 14- mer unique sequences in hRasGRP4 and mRasGRP4, have been generated. Thus, the anti- peptide approach is another methodology that can be used to generate new antibodies.

The invention involves polypeptides of numerous size and type that bind specifically to Mutant RasGRP4 proteins and RasGRP4 proteins. These polypeptides may be derived also from sources other than antibody technology. For example, such polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptides and non-peptide synthetic moieties.

Phage display can be particularly effective in identifying binding peptides useful according to the invention. Briefly, one prepares a phage library (using e.g. ml3, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent a completely degenerate or biased array. One then can select phage-bearing inserts which bind to a RasGRP4 protein or a Mutant RasGRP4 protein. This process can be repeated through several cycles of reselection of phage that bind to a RasGRP4 protein or a Mutant RasGRP4 protein. Repeated rounds lead to enrichment of phage bearing particular sequences. DNA sequence analysis can be conducted to identify the sequences ofthe expressed polypeptides. The minimal linear portion ofthe sequence that binds to the RasGRP4 protein or the Mutant RasGRP4 protein can be determined. One can repeat the procedure using a biased library containing inserts containing part or all ofthe minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof. Thus, the RasGRP4 proteins ofthe invention can be used to screen peptide libraries, including phage display libraries, to identify and select peptide binding partners ofthe RasGRP4 proteins ofthe invention. Such molecules can be used, as described, for screening assays, for diagnostic assays, for purification protocols or for targeting drugs, toxins and/or labeling agents (e.g. radioisotopes, fluorescent molecules, etc.) to cells which express Mutant RasGRP4 genes such as leukocyte cells which have abenant RasGRP4 expression. Such binding agent molecules can also be prepared to bind complexes of an RasGRP4 protein and an HLA molecule by selecting the binding agent using such complexes. As detailed herein, the foregoing antibodies and other binding molecules may be used for example to identify tissues expressing mutant protein or to purify mutant protein. Antibodies also may be coupled to specific diagnostic labeling agents for imaging of cells and tissues with abenant RasGRP4 expression or to therapeutically useful agents according to standard coupling procedures. Diagnostic agents include, but are not limited to, barium sulfate, iocetamic acid, iopanoic acid, ipodate calcium, diatrizoate sodium, diatrizoate meglumine, metrizamide, tyropanoate sodium and radiodiagnostics including positron emitters such as fluorine- 18 and carbon-11, gamma emitters such as iodine- 123, technitium- 99m, iodine-131 and indium- 111, nuclides for nuclear magnetic resonance such as fluorine and gadolinium. Other diagnostic agents useful in the invention will be apparent to one of ordinary skill in the art. As used herein, "therapeutically useful agents" include any therapeutic molecule which desirably is targeted selectively to a cell or tissue selectively with an aberrant RasGRP4 expression, including radioiodinated compounds, toxins, other cytostatic or cytolytic drugs, and so forth. Toxins can be proteins such as, for example, pokeweed anti- viral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin, or Pseudomonas exotoxin. Toxin moieties can also be high energy-emitting radionuclides such as cobalt-60.

RasGRP4 Pharmaceutical Compositions (general)

According to a further aspect ofthe invention, pharmaceutical compositions containing the nucleic acid molecules, proteins, and binding polypeptides ofthe invention are provided. The pharmaceutical compositions contain any ofthe foregoing therapeutic agents in a pharmaceutically acceptable carrier. Thus, in a related aspect, the invention provides a method for forming a medicament that involves placing a therapeutically effective amount of the therapeutic agent in the pharmaceutically acceptable carrier to form one or more doses. When administered, the therapeutic compositions ofthe present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.

The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness ofthe biological activity ofthe active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics ofthe carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art. The therapeutics ofthe invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal. When antibodies are used therapeutically, a prefened route of administration is by pulmonary aerosol. Techniques for preparing aerosol delivery systems containing antibodies are well known to those of skill in the art. Generally, such systems should utilize components which will not significantly impair the biological properties ofthe antibodies, such as the paratope binding capacity (see, for example, Sciana and Cutie, "Aerosols," in Remington's Pharmaceutical Sciences. 18th edition, 1990, pp 1694-1712). Those of skill in the art can readily determine the various parameters and conditions for producing antibody aerosols without resort to undue experimentation. When using antisense preparations ofthe invention, slow intravenous administration is prefened.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. The preparations ofthe invention are administered in effective amounts. An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, stimulates the desired response. In the case of treating a RasGRP4-associated disorder, the desired response is inhibiting the progression ofthe RasGRP4-associated disorder. This may involve only slowing the progression ofthe disease temporarily, although more preferably, it involves halting the progression ofthe disease permanently. In the case of stimulating an immune response, the desired response is an increase in antibodies or T lymphocytes which are specific for the immunogen(s) employed. These responses can be monitored by routine methods or can be monitored according to diagnostic methods ofthe invention discussed herein.

The therapeutically effective amount ofthe RasGRP4 molecule is that amount effective to modulate RasGRP4 functional activity levels and reduce, prevent, or eliminate the RasGRP4-associated disorder. For example, using the assays described in the Example section, testing can be performed to determine the RasGRP functional activity in a subject's tissue and/or cells. Additional tests useful for monitoring the onset, progression, and/or remission, of RasGRP4-associated disorders such as bacterial infections, asthma, allergy, mastocytosis, and other RasGRP4-associated disorders, are well known to those of ordinary , skill in the art. As would be understood by one of ordinary skill, for some disorders (e.g. bacterial infection) an effective amount would be the amount of RasGRP4 molecules that increases RasGRP4 functional activity to a level that diminishes the disorder, as determined by the aforementioned tests. It is also understood that in other disorders (e.g. asthma, allergy, etc) an effective amount would be that amount of RasGRP4 molecules that decreases RasGRP4 functional activity to a level that diminishes the disorder, as determined by the aforementioned tests. The RasGRP4 molecule dosage may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 200 mg/kg, and most preferably from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days. Where it is desired to stimulate an immune response using a therapeutic composition ofthe invention (e.g., a Mutant RasGRP4 protein fragment which is a unique fragment ofthe Mutant RasGRP4 molecule), this may involve the stimulation of a humoral antibody response resulting in an increase in antibody titer in serum, a clonal expansion of cytotoxic lymphocytes, or some other desirable immunologic response. It is believed that doses of immunogens ranging from one nano gram/kilogram to 100 milligrams/kilogram, depending upon the mode of administration, would be effective. The prefened range is believed to be between 500 nanograms and 500 micrograms per kilogram. The absolute amount will depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual patient parameters including age, physical condition, size, weight, and the stage ofthe disease. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.

RasGRP4 Diagnostic methods (general)

According to another aspect ofthe invention, various diagnostic methods are provided. In general, the methods are for diagnosing "a disorder characterized by abenant expression of a RasGRP4 molecule". As used herein, "abenant expression" is dependent upon the particular disorder (i.e., characterized by increased or decreased RasGRP4 molecule expression). Thus "abenant expression" refers to increased expression of normal RasGRP4, decreased expression (including no expression) of a RasGRP4 molecule (nucleic acid or protein), or increased expression of a "Mutant RasGRP4 molecule". A Mutant RasGRP4 molecule refers to a RasGRP4 nucleic acid molecule which includes a mutation (deletion, addition, or substitution) or to a RasGRP4 protein molecule (e.g., gene product of Mutant

RasGRP4 nucleic acid molecule) which includes a mutation, provided that the mutation results in a Mutant RasGRP4 protein that has reduced or no RasGRP4 protein functional activity. As described above herein, some mutant RasGRP4 molecules ofthe invention may have enlianced RasGRP4 functional activity. The diagnostic methods ofthe invention can be used to detect the presence of a disorder associated with abenant expression ofa RasGRP4 molecule, as well as to assess the progression and/or regression ofthe disorder such as in response to treatment (e.g., chemical therapy). Expression of RasGRP4 can be evaluated using standard methods known to those of ordinary skill in the art. Such methods include, but are not limited to: PCR, RT-PCR and antibody methods, which can be used to evaluate defects in the expression of RasGRP4 at the gene, transcript, and protein levels, respectively. Expression of RasGRP4 can also be evaluated by determining the functional activity of RasGRP4 in cells and tissues with methods that include, but are not limited to the activity assay described in the Examples. According to this aspect ofthe invention, the method for diagnosing a disorder characterized by abenant expression of a RasGRP4 molecule involves: detecting in a first biological sample obtained from a subject, expression of a RasGRP4 molecule or a Mutant RasGRP4 molecule, wherein decreased or increased expression of a RasGRP4 molecule (depending upon the disorder as discussed below) or the increased expression of a Mutant RasGRP4 molecule compared to a control sample indicates that the subject has a disorder characterized by abenant expression of a RasGRP4 molecule. As used herein, a "disorder characterized by abenant expression of a RasGRP4 molecule" refers to a disorder in which there is a detectable difference in the expression levels of RasGRP4 molecule(s) and/or Mutant RasGRP4 molecule(s) in selected cells of a subject compared to the control levels of these molecules. Thus, a disorder characterized by abenant expression of a RasGRP4 molecule embraces overexpression of RasGRP4, underexpression (including no expression) of a RasGRP4 molecule compared to control levels of these molecules, as well as overexpression of a Mutant RasGRP4 nucleic acid molecule or Mutant RasGRP4 protein compared to control levels of these molecules. Such differences in expression levels can be determined in accordance with the diagnostic methods ofthe invention as disclosed herein. Disorders that are characterized by abenant expression of a RasGRP4 molecule include: disorders associated with abnormal mast cell development, function, and/or cell-cell interaction. Exemplary mast cell-dependent disorders include, but are not limited to: allergies, urticaria, systemic mastocytosis, cancer/leukemia, fibrosis, rheumatoid arthritis, neuron degeneration, and ADDS.

Mast cells play beneficial immunosurveillance and effector roles in the body, for example, for fighting bacterial infections, hi such situations, the reduction in RasGRP4 expression levels (e.g. functional activity) would indicate the presence ofa "reduced

RasGRP4-activity disorder" in a subject. In addition, the expression of a mutant form of RasGRP4 that is non-functional or has a reduced function as compared to RasGRP4, can be used as a diagnostic indicator for the presence of a disorder characterized by a decrease in RasGRP4 expression (e.g. functional activity) as described herein. In other diseases and disorders, including but not limited to: allergic inflammation, asthma, urticaria, systemic anaphylaxis, systemic mastocytosis, cancer/leukemia, fibrosis, rheumatoid arthritis, neuron degeneration, and ADDS, an increase in RasGRP4 expression levels and/or functional activity would indicate the presence of an "increased RasGRP4- activity disorder" in the subject. In addition, the expression of a mutant RasGRP4 that has RasGRP4-like functional activity, can also indicate the presence of a disorder, characterized by an increase in RasGRP4 expression (e.g. functional activity), as described herein. Thus, depending upon the nature ofthe disorder, i.e., whether it is attributable to reduced or elevated RasGRP4 molecule expression or elevated Mutant RasGRP4 molecular expression, one skilled in the art can select the appropriate type of treatment to modulate RasGRP4 or Mutant RasGRP4 expression to achieve levels of these molecules which are found in individuals who are not diagnosed with such disorders. In certain embodiments, the methods ofthe invention are to diagnose a RasGRP4- associated disorder including, but not limited to, systemic mastocytosis or common allergic inflammation disorders such as asthma.

In yet other embodiments, the diagnostic methods are useful for diagnosing the progression of a disorder and its treatment. According to these embodiments, the methods further involve: detecting in a second biological sample obtained from the subject, expression of a RasGRP4 molecule or a Mutant RasGRP4 molecule, and comparing the expression of the RasGRP4 molecule or the Mutant RasGRP4 molecule in the first biological sample and the second biological sample. In these embodiments, a decrease or an increase in the expression ofthe RasGRP4 molecule in the second biological sample compared to the first biological sample or an increase in the expression ofthe Mutant RasGRP4 molecule in the second biological sample compared to the first biological sample (depending upon the nature ofthe disorder) indicates progression ofthe disorder. One of ordinary skill will understand that the first and second biological samples can be taken at different times (e.g. days, weeks, months, or years apart), to assess changes in the subject's condition. In yet other embodiments, the diagnostic methods are useful for diagnosing the regression of a disorder. According to these embodiments, the methods further involve: detecting in a second biological sample obtained from the subject, expression of a RasGRP4 molecule or a Mutant RasGRP4 molecule, and comparing the expression ofthe RasGRP4 molecule or the Mutant RasGRP4 molecule in the first biological sample and the second biological sample. In these embodiments, an increase or a decrease in the expression ofthe RasGRP4 molecule in the second biological sample compared to the first biological sample or a decrease in the expression ofthe Mutant RasGRP4 molecule in the second biological sample compared to the first biological sample indicates regression ofthe disorder. As noted above, it is to be understood by one of ordinary skill that some disorders will be characterized by an increase in RasGRP4 functional activity and other disorders will be characterized by a decrease in RasGRP4 functional activity. One of ordinary skill will understand that the first and second biological samples can be taken at different times (e.g. days, weeks, months, or years apart), to assess changes in the subject's condition.

In certain embodiments, the diagnostic methods ofthe invention detect a RasGRP4 molecule that is a RasGRP4 nucleic acid molecule or a Mutant RasGRP4 nucleic acid molecule as described above. In yet other embodiments, the methods involve detecting a RasGRP4 protein or Mutant RasGRP4 protein as described above.

Various detection methods can be used to practice the diagnostic methods ofthe invention. For example, when the methods can involve contacting the biological sample with an agent that selectively binds to the RasGRP4 molecule or to the Mutant RasGRP4 molecule to detect these molecules. In certain embodiments, the RasGRP4 molecule is a nucleic acid and the method involves using an agent that selectively binds to the RasGRP4 molecule or to the Mutant RasGRP4 molecule, e.g., a nucleic acid that hybridizes to SEQ ED NO: 1 or to SEQ DD NO: 7 under high stringency conditions. In yet other embodiments, the RasGRP4 molecule is a protein and the method involves using an agent that selectively binds to the RasGRP4 molecule or to the Mutant RasGRP4 molecule, e.g., a binding polypeptide, such as an antibody, that selectively binds to SEQ ED NO: 2 or SEQ ED NO: 8.

According to still another aspect ofthe invention, kits for performing the diagnostic methods ofthe invention are provided. The kits include nucleic acid-based kits or protein- based kits. According to the former embodiment, the kits include: one or more nucleic acid molecules that hybridize to a RasGRP4 nucleic acid molecule or to a Mutant RasGRP4 nucleic acid molecule under high stringency conditions; one or more control agents; and instructions for the use ofthe nucleic acid molecules, and agents in the diagnosis of a disorder associated with abenant expression of a RasGRP4 molecule. Nucleic acid-based kits optionally further include a first primer and a second primer, wherein the first primer and the second primer are constructed and ananged to selectively amplify at least a portion of an isolated RasGRP4 nucleic acid molecule comprising SEQ DD NO: 1 and/or SEQ ED NO: 7. Alternatively, protein based-kits are provided. Such kits include: one or more binding polypeptides that selectively bind to a RasGRP4 protein or a Mutant RasGRP4 protein; one or more control agents; and instructions for the use ofthe binding polypeptides, and agents in the diagnosis of a disorder associated with abenant expression of a RasGRP4 molecule. In the prefened embodiments, the binding polypeptides are antibodies or antigen-binding fragments thereof, such as those described above. In these and other embodiments, certain of the binding polypeptides bind to the Mutant RasGRP4 protein but do not bind to the RasGRP4 protem to further distinguish the expression of these proteins in a biological sample.

The foregoing kits can include instructions or other printed material on how to use the various components ofthe kits for diagnostic puφoses. The kits ofthe invention can, but are not required to, include control agents the can be used for detennining kit component quality, reagent quality, reaction parameters, background "noise" level, and reaction thresholds and success. The use of control agents in tests would be known to one of ordinary skill in the art to be a routine procedure in the art.

As used herein, a subject is a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent, including but not limited to: guinea pig, rat, and mouse. As used herein an animal is a non-human primate, cow, horse, pig, sheep, goat, dog, cat, or rodent, including, but not limited to: guinea pig, rat, and mouse, hi all embodiments, human and mouse RasGRP4 molecules and human subjects are prefened.

The biological sample can be located in vivo or in vitro. For example, the biological sample can be a tissue in vivo and the agent specific for the RasGRP4 nucleic acid molecule or polypeptide can be used to detect the presence of such molecules in the tissue (e.g., for imaging portions ofthe tissue that express the RasGRP gene products). Alternatively, the biological sample can be located in vitro (e.g., a blood sample, biopsy (e.g., tumor or tissue biopsy), tissue extract). In a particularly prefened embodiment, the biological sample can be a cell-containing sample, more preferably a sample containing leukocyte cells. Samples of tissue and/or cells for use in the various methods described herein can be obtained through standard methods. Samples can be surgical samples of any type of tissue or body fluid. Samples can be used directly or processed to facilitate analysis (e.g., paraffin embedding). Exemplary samples include a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract or other methods. Samples also can be cultured cells, tissues, or organs. RasGRP4 Therapeutics f General)

The invention also provides treatment methods. As used herein, "treatment" includes preventing, delaying, abating or anesting the clinical symptoms of a disorder characterized by abenant expression of a RasGRP4 molecule. Treatment also includes reducing or preventing RasGRP4-associated disorders.

Mast cells play beneficial immunosurveillance and effector roles in the body, for example, they are involved in fighting bacterial infections. In some disorders, such as bacterial infections, it is desirable to increase RasGRP4 expression (e.g. functional activity) in cells and tissues of a subject. Such an increase in activity can be brought about by, for example, increasing expression of RasGRP4, and/or increasing the level of a mutant

RasGRP4 that has an increased level of activity as compared to the activity level of normal RasGRP4.

In other diseases and disorders, which include but are not limited to: allergic inflammation, asthma, urticaria, systemic anaphylaxis, systemic mastocytosis, cancer/leukemia, fibrosis, rheumatoid arthritis, neuron degeneration, and ADDS, it is desirable to reduce the activity of RasGRP4 to inhibit mast cell development and the function of their exocytosed mediators. Mast cell activity can be reduced using methods such as: reducing expression of RasGRP4 and/or inhibiting RasGRP4 activity (e.g. competitive inhibition, binding inhibition). Increasing levels of a mutant RasGRP4 that is non-functional or reduced-function as compared to nonnal RasGRP4, can also be therapeutically desirable in diseases and disorders where a reduction in normal RasGRP4 activity is desirable.

In general, the treatment methods involve administering an agent to modulate expression of a RasGRP4 molecule and/or modulate expression of a Mutant RasGRP4 molecule. Thus, these methods include gene therapy applications. In certain embodiments, the method for treating a subject with a disorder characterized by abenant expression of a RasGRP4 molecule, involves administering to the subject an effective amount of a RasGRP4 nucleic acid molecule to treat the disorder. In yet other embodiments, the method for treatment involves administering to the subject an effective amount of an antisense or RNAi oligonucleotide to modulate expression of a Mutant RasGRP4 nucleic acid molecule and thereby, treat the disorder. An exemplary molecule for modulating expression of a Mutant RasGRP4 nucleic acid molecule is an antisense molecule that is selective for the Mutant nucleic acid and that does not modulate expression ofthe RasGRP4 nucleic acid molecule. Alternatively, the method for treating a subject with a disorder characterized by abenant expression of a RasGRP4 molecule involves administering to the subject an effective amount of a RasGRP4 protein to treat the disorder. In yet another embodiment, the treatment method involves administering to the subject an effective amount of a binding polypeptide to inhibit a Mutant RasGRP4 protein and, thereby, treat the disorder. In some embodiments, the treatment method involves administering to the subject an effective amount of a binding polypeptide to inhibit RasGRP4 to reduce or eliminate Mutant RasGRP4 activity. In certain prefened embodiments, the binding polypeptide is an antibody or an antigen-binding fragment thereof; more preferably, the antibodies or antigen-binding fragments are labeled with one or more cytotoxic agents.

The invention also contemplates gene therapy. The procedure for performing ex vivo gene therapy is outlined in U.S. Patent 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents. In general, it involves introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy ofthe gene, and returning the genetically engineered cell(s) to the subject. The functional copy ofthe gene is under operable control of regulatory elements which permit expression ofthe gene in the genetically engineered cell(s). Numerous transfection and transduction techniques as well as appropriate expression vectors are well known to those of ordinary skill in the art, some of which are described in PCT application WO95/00654. In vivo gene therapy using vectors such as adenovirus, retroviruses, heφes virus, and targeted liposomes also is contemplated according to the invention.

In prefened embodiments, a virus vector for delivering a nucleic acid molecule encoding a RasGRP4 protein is selected from the group consisting of adenoviruses, adeno- associated viruses, poxviruses including vaccinia viruses and attenuated poxviruses, Semliki Forest virus, Venezuelan equine encephalitis virus, retroviruses, Sindbis virus, and Ty viruslike particle. Examples of viruses and virus-like particles which have been used to deliver exogenous nucleic acids include: replication-defective adenoviruses (e.g., Xiang et al., Virology 219:220-227, 1996; Eloit et al., J Virol. 7:5375-5381, 1997; Chengalvala et al., Vaccine 15:335-339, 1997), a modified retrovirus (Townsend et al., J. Virol. 71:3365-3374, 1997), a nonreplicating retrovirus (Irwin et al., J Virol. 68:5036-5044, 1994), a replication defective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA 92:3009-3013, 1995), canarypox virus and highly attenuated vaccinia virus derivative (Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353, 1996), non-replicative vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA 93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol. Stand. 82:55-63, 1994), Venzuelan equine encephalitis virus (Davis et al., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et al., Virology 212:587-594, 1995), and Ty virus-like particle (AUsopp et al., Eur. J. Immunol 26:1951-1959, 1996). In prefened embodiments, the virus vector is an adenovirus.

Another prefened virus for certain applications is the adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus is capable of infecting a wide range of cell types and species and can be engineered to be replication-deficient. It further has advantages, such as heat and lipid solvent stability, high transduction frequencies in cells of diverse lineages, including hematopoietic cells, and lack of superinfection inhibition thus allowing multiple series of transductions. The adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression. In addition, wild-type . adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

In general, other prefened viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non- cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Adenoviruses and retroviruses have been approved for human gene therapy trials. In general, the retroviruses are replication-deficient (i.e., capable of directing synthesis ofthe desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incoφoration of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection ofthe target cells with viral particles) are provided in Kriegler, M., "Gene Transfer and Expression, A Laboratory Manual," W.H. Freeman Co., New York (1990) and Muny, E.J. Ed. "Methods in Molecular Biology," vol. 7, Humana Press, Inc., Cliffton, New Jersey (1991).

Preferably the foregoing nucleic acid delivery vectors: (1) contain exogenous genetic material that can be transcribed and translated in a mammalian cell and that can suppress RasGRP4-associated disease (e.g. mast cell associated disorders), and preferably (2) contain on a surface a ligand that selectively binds to a receptor on the surface of a target cell, such as a mammalian cell, and thereby gains entry to the target cell.

Various techniques may be employed for introducing nucleic acid molecules ofthe invention into cells, depending on whether the nucleic acid molecules are introduced in vitro or in vivo in a host. Such techniques include transfection of nucleic acid molecule-calcium phosphate precipitates, transfection of nucleic acid molecules associated with DEAE, transfection or infection with the foregoing viruses including the nucleic acid molecule of interest, liposome-mediated transfection, and the like. For certain uses, it is prefened to target the nucleic acid molecule to particular cells. In such instances, a vehicle used for delivering a nucleic acid molecule ofthe invention into a cell (e.g., a retrovirus, or other virus; a liposome) can have a targeting molecule attached thereto. For example, a molecule such as an antibody specific for a surface membrane protein on the target cell or a ligand for a receptor on the target cell can be bound to or incoφorated within the nucleic acid molecule delivery vehicle. Especially prefened are monoclonal antibodies. Where liposomes are employed to deliver the nucleic acid molecules ofthe invention, proteins that bind to a surface membrane protein associated with endocytosis may be incoφorated into the liposome formulation for targeting and/or to facilitate uptake. Such proteins include capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half life, and the like. Polymeric delivery systems also have been used successfully to deliver nucleic acid molecules into cells, as is known by those skilled in the art. Such systems even permit oral delivery of nucleic acid molecules. hi addition to delivery tlirough the use of vectors, RasGRP4 nucleic acids may be delivered to cells without vectors, e.g. as "naked" nucleic acid delivery using methods known to those of skill in the art.

Various forms ofthe RasGRP4 polypeptide or nucleic acid, as described herein, can be administered and delivered to a mammalian cell (e.g., by virus or liposomes, or by any other suitable methods known in the art or later developed). The method of delivery can be modified to target certain cells, and in particular, cell surface receptor molecules or antigens present on mast cells and/or tumor cells. Methods of targeting cells to deliver nucleic acid constructs are known in the art. The RasGRP4 polypeptide can also be delivered into cells by expressing a recombinant protein fused with peptide carrier molecules, examples of which, though not intended to be limiting, are tat or antennapedia. These delivery methods are known to those of skill in the art and are described in US patent 6,080,724, and US patent 5,783,662, the entire contents of which are hereby incoφorated by reference.

In addition to the methods described herein for delivering exogenous RasGRP4, expression of endogenous normal or mutant RasGRP4 can be induced (e.g., upregulated) in cells harboring the virus by the administration of chemicals or other molecules that specifically increase the level of RasGRP4 mRNA and/or protein. Such induction and/or upregulation of endogenous RasGRP4 may occur through methods that include, but not limited to: (a) activation ofthe RasGRP4 promoter, (b) stabilization of RasGRP4 mRNA, (c) increased translation of RasGRP polypeptide and (d) stabilization of RasGRP4 polypeptide. The invention provides various research methods and compositions. Thus, according to one aspect ofthe invention, a method for producing a RasGRP4 protein is provided. The method involves providing a RasGRP4 nucleic acid molecule operably linked to a promoter, wherein the RasGRP4 nucleic acid molecule encodes the RasGRP4 protein or a fragment thereof; expressing the RasGRP4 nucleic acid molecule in an expression system; and isolating the RasGRP4 protein or a fragment thereof from the expression system. Preferably, the RasGRP4 nucleic acid molecule has SEQ DD NO: 1 or SEQ DD NO: 7. According to yet another aspect ofthe invention, a method for producing a Mutant RasGRP4 protein is provided. This method involves: providing a Mutant RasGRP4 nucleic acid molecule operably linked to a promoter, wherein the Mutant RasGRP4 nucleic acid molecule encodes the Mutant RasGRP4 protein or a fragment thereof; expressing the Mutant RasGRP4 nucleic acid molecule in an expression system; and isolating the Mutant RasGRP4 protein or a fragment thereof from the expression system. Preferably, the Mutant RasGRP4 nucleic acid molecule has SEQ DD NO: 1 or SEQ DD NO: 7 with one or more deletions, additions, or substitutions to encode a Mutant RasGRP4 protein. As described above herein, the terms:

"deletion", "addition", and "substitution" mean deletion, addition, and substitution changes to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleic acids ofa sequence ofthe invention.

The invention further provides efficient methods of identifying pharmacological agents or lead compounds for agents which mimic the functional activity of a RasGRP4 molecule. Such RasGRP4 functional activities include tumor suppression, cytoskeletal organization, and cell migration. Generally, the screening methods involve assaying for compounds which modulate (up- or down-regulate) a RasGRP4 functional activity. As used herein: the term "regulation of mast cells" means the modulation of one or more ofthe following parameters: mast cell development, mast cell function, and/or mast cell interactions with other cells, and includes the modulation of RasGRP4 functional activity. RasGRP4 functional activity includes but is not limited to mast cell activation such as initiating and/or increasing mast cell development, or activity.

A wide variety of assays for pharmacological agents can be used in accordance with this aspect ofthe invention, including, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays, cell-based assays such as two- or three- hybrid screens, expression assays, etc. The assay mixture comprises a candidate pharmacological agent. Typically, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a different response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration of agent or at a concentration of agent below the limits of assay detection. Candidate agents encompass numerous chemical classes, although typically they are organic compounds. Preferably, the candidate pharmacological agents are small organic compounds, i.e., those having a molecular weight of more than 50 yet less than about 2500, preferably less than about 1000 and, more preferably, less than about 500. Candidate agents comprise functional chemical groups necessary for structural interactions with proteins and/or nucleic acid molecules, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two ofthe functional chemical groups and more preferably at least three of the functional chemical groups. The candidate agents can comprise cyclic carbon or heterocyclic structure and/or aromatic or polyaromatic structures substituted with one or more ofthe above-identified functional groups. Candidate agents also can be biomolecules such as peptides, saccharides, fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like. Where the agent is a nucleic acid molecule, the agent typically is a DNA or RNA molecule, although modified nucleic acid molecules as defined herein are also contemplated.

It is contemplated that cell-based assays as described herein can be performed using cell samples and/or cultured cells. Cells include cells that transformed to express RasGRP4 and cells treated using methods described herein to inhibit or enhance RasGRP4 expression or functional activity. Cell lines useful in the methods ofthe invention, including the cell- based assays include, but are not limited to CD14⁺ cells, HMC-1 cells, and RasGRP4⁺ HMC- 1 cells.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides, synthetic organic combinatorial libraries, phage display libraries of random peptides, and the like. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds can be readily be modified through conventional chemical, physical, and biochemical means. Further, known pharmacological agents may be subjected to directed or random chemical modifications such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs ofthe agents. A variety of other reagents also can be included in the mixture. These include reagents such as salts, buffers, neutral proteins (e.g., albumin), detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding. Such a reagent may also reduce non-specific or background interactions ofthe reaction components. Other reagents that improve the efficiency ofthe assay such as protease inhibitors, nuclease inhibitors, antimicrobial agents, and the like may also be used.

An exemplary binding assay is described herein. In general the mixture ofthe foregoing assay materials is incubated under conditions whereby, but for the presence ofthe candidate pharmacological agent, the RasGRP4 molecule or the Mutant RasGRP4 molecule specifically binds the binding agent (e.g., antibody, complementary nucleic acid). The order of addition of components, incubation temperature, time of incubation, and other parameters ofthe assay may be readily determined. Such experimentation merely involves optimization ofthe assay parameters, not the fundamental composition ofthe assay. Incubation temperatures typically are between 4°C and 40°C. Incubation times preferably are minimized to facilitate rapid, high throughput screening, and typically are between 0.1 and 10 hours.

After incubation, the presence or absence of specific binding between the RasGRP4 molecule or the Mutant RasGRP4 molecule and one or more binding agents is detected by any convenient method available to the user. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. The separation step may be accomplished in a variety of ways. Conveniently, at least one ofthe components is immobilized on a solid substrate, from which the unbound components may be easily separated. The solid substrate can be made of a wide variety of materials and in a wide variety of shapes, e.g., microtiter plate, microbead, dipstick, resin particle, etc. The substrate preferably is chosen to maximum signal to noise ratios, primarily to minimize background binding, as well as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting a reservoir such as a microtiter plate well, rinsing a bead, particle, chromotograpic column or filter with a wash solution or solvent. The separation step preferably includes multiple rinses or washes. For example, when the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components ofthe incubation mixture that do not participate in specific bindings such as salts, buffer, detergent, non-specific protein, etc. Where the solid substrate is a magnetic bead, the beads may be washed one or more times with a washing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assays such as two- or three-hybrid screens. For cell- free binding assays, one ofthe components usually comprises, or is coupled to, a detectable label. A wide variety of labels can be used, such as those that provide direct detection (e.g., radioactivity, luminescence, optical or electron density, etc.) or indirect detection (e.g., epitope tag such as the FLAG epitope, enzyme tag such as horseseradish peroxidase, etc.). The label may be bound to a RasGRP4 binding partner (e.g., polypeptide), or incoφorated into the structure ofthe binding partner.

A variety of methods may be used to detect the label, depending on the nature ofthe label and other assay components. For example, the label may be detected while bound to the solid substrate or subsequent to separation from the solid substrate. Labels may be directly detected through optical or electron density, radioactive emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, strepavidin-biotin conjugates, etc. Methods for detecting the labels are well known in the art.

The invention further includes nucleic acid or protein microanays with RasGRP4 peptides or nucleic acids encoding such polypeptides. In this aspect ofthe invention, standard techniques of microanay technology are utilized to assess expression ofthe

RasGRP4 polypeptides and/or identify biological constituents that bind such polypeptides. The constituents of biological samples include antibodies, leukocytes, and the like. Protein microanay technology, which is also known by other names including: protein chip technology and solid-phase protein anay technology, is well known to those of ordinary skill in the art and is based on, but not limited to, obtaining an anay of identified peptides or proteins on a fixed substrate, binding target molecules or biological constituents to the peptides, and evaluating such binding. See, e.g., G. MacBeath and S.L. Schreiber, "Printing Proteins as Microanays for High-Throughput Function Determination," Science 289(5485):1760-1763, 2000. Nucleic acid anays, particularly anays that bind RasGRP4 peptides, also can be used for diagnostic applications, such as for identifying subjects that have a condition characterized by RasGRP4 or Mutant RasGRP4 polypeptide expression. Microanay substrates include but are not limited to glass, silica, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, various clays, nitrocellulose, or nylon. The microanay substrates may be coated with a compound to enhance synthesis of a probe (peptide or nucleic acid) on the substrate. Coupling agents or groups on the substrate can be used to covalently link the first nucleotide or amino acid to the substrate. A variety of coupling agents or groups are known to those of skill in the art. Peptide or nucleic acid probes thus can be synthesized directly on the substrate in a predetermined grid. Alternatively, peptide or nucleic acid probes can be spotted on the substrate, and in such cases the substrate may be coated with a compound to enhance binding ofthe probe to the substrate. In these embodiments, presynthesized probes are applied to the substrate in a precise, predetermined volume and grid pattern, preferably utilizing a computer-controlled robot to apply probe to the substrate in a contact-printing manner or in a non-contact manner such as ink jet or piezo-electric delivery. Probes may be covalently linked to the substrate. Targets are peptides or proteins and may be natural or synthetic. The tissue may be obtained from a subject or may be grown in culture (e.g. from a cell line). In some embodiments ofthe invention, one or more control peptide or protein molecules are attached to the substrate. Preferably, control peptide or protein molecules allow determination of factors such as peptide or protein quality and binding characteristics, reagent quality and effectiveness, hybridization success, and analysis thresholds and success. Nucleic acid microanay technology, which is also known by other names including:

DNA chip technology, gene chip technology, and solid-phase nucleic acid array technology, is well known to those of ordinary skill in the art and is based on, but not limited to, obtaining an anay of identified nucleic acid probes on a fixed substrate, labeling target molecules with reporter molecules (e.g., radioactive, chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, or Cye5-dUTP), hybridizing target nucleic acids to the probes, and evaluating target-probe hybridization. A probe with a nucleic acid sequence that perfectly matches the target sequence will, in general, result in detection ofa stronger reporter- molecule signal than will probes with less perfect matches. Many components and techniques utilized in nucleic acid microanay technology are presented in Tlie Chipping Forecast, Nature Genetics, Vol.21, Jan 1999, the entire contents of which is incoφorated by reference herein.

According to the present invention, nucleic acid microanay substrates may include but are not limited to glass, silica, aluminosilicates, borosilicates, metal oxides such as alumina and nickel oxide, various clays, nitrocellulose, or nylon. In all embodiments, a glass substrate is prefened. According to the invention, probes are selected from the group of nucleic acids including, but not limited to: DNA, genomic DNA, cDNA, and oligonucleotides; and may be natural or synthetic. Oligonucleotide probes preferably are 20 to 25-mer oligonucleotides and DNA/cDNA probes preferably are 500 to 5000 bases in length, although other lengths may be used. Appropriate probe length may be determined by one of ordinary skill in the art by following art-known procedures. In one embodiment, prefened probes are sets of one or more ofthe RasGRP4 and/or Mutant RasGRP4 polypeptide nucleic acid molecules set forth herein. Probes may be purified to remove contaminants using standard methods known to those of ordinary skill in the art such as gel filtration or precipitation. In one embodiment, the microanay substrate may be coated with a compound to enhance synthesis ofthe probe on the substrate. Such compounds include, but are not limited to, oligoethylene glycols. h another embodiment, coupling agents or groups on the substrate can be used to covalently link the first nucleotide or olignucleotide to the substrate. These agents or groups may include, for example, amino, hydroxy, bromo, and carboxy groups.

These reactive groups are preferably attached to the substrate through a hydrocarbyl radical such as an alkylene or phenylene divalent radical, one valence position occupied by the chain bonding and the remaining attached to the reactive groups. These hydrocarbyl groups may contain up to about ten carbon atoms, preferably up to about six carbon atoms. Alkylene radicals are usually prefened containing two to four carbon atoms in the principal chain.

These and additional details ofthe process are disclosed, for example, in U.S. Patent

4,458,066, which is incoφorated by reference in its entirety. In one embodiment, probes are synthesized directly on the subsfrate in a predetermined grid pattern using methods such as light-directed chemical synthesis, photochemical deprotection, or delivery of nucleotide precursors to the substrate and subsequent probe production. hi another embodiment, the substrate may be coated with a compound to enhance binding ofthe probe to the substrate. Such compounds include, but are not limited to: polylysine, amino silanes, amino-reactive silanes (Chipping Forecast, 1999) or chromium. In this embodiment, presynthesized probes are applied to the substrate in a precise, predetermined volume and grid pattern, utilizing a computer-controlled robot to apply probe to the substrate in a contact-printing manner or in a non-contact manner such as ink jet or piezo-electric delivery. Probes may be covalently linked to the substrate with methods that include, but are not limited to, UV-iπadiation. In another embodiment probes are linked to . the substrate with heat.

Targets for microanays are nucleic acids selected from the group, including but not limited to: DNA, genomic DNA, cDNA, RNA, mRNA and may be natural or synthetic. In all embodiments, nucleic acid target molecules from human tissue are prefened. The tissue may be obtained from a subject or may be grown in culture (e.g. from a cell line). In some embodiments, targets for microanays are proteins/peptides. hi embodiments ofthe invention one or more control nucleic acid molecules are attached to the substrate. Preferably, control nucleic acid molecules allow determination of factors such as nucleic acid quality and binding characteristics, reagent quality and effectiveness, hybridization success, and analysis thresholds and success. Control nucleic acids may include but are not limited to expression products of genes such as housekeeping genes or fragments thereof.

The invention will be more fully understood by reference to the following examples. These examples, however, are merely intended to illustrate the embodiments ofthe invention and are not to be construed to limit the scope ofthe invention.

Examples

With respect to the Example section, the term "variant" means mutant forms ofthe nucleic acids and/or proteins.

Example 1

Methods

Evaluation of hRasGRP mRNA levels in varied fetal and adult tissues, and in different populations of peripheral blood mononuclear cells. A semi-quantitative RT-PCR approach was used to evaluate hRasGRP4 mRNA levels in the indicated fetal and adult human tissues and cells. The primers used in these transcript analyses conespond to sequences residing in exons 1 and 9 ofthe hRasGRP4 gene. Because the hRasGRP4 transcript was abundant in the leukocyte preparation, a second experiment was carried out in which pooled peripheral blood mononuclear cells from 4-36 individuals were sorted by Clontech based on their expression of CD4, CD8, CD14, and CD19. Samples ofthe resulting cell populations were evaluated for their expression of hRasGRP4 mRNA immediately after their isolation (resting) or after their subsequent exposure to phytohemaggultinin, pokeweed mitogen, and/or concanavalin A (lectin activated). hRasGRP4 mRNA was only detected in unfractionated and the "resting" CD14⁺ population of peripheral blood mononuclear cells. Glyceraldehyde-3 -phosphate dehydrogenase specific primers were used in a similar analysis to confirm the presence of intact RNA in all samples.

Cloning of the hRasGRP4 cDNA. The site where the hRasGRP4 gene resides on human chromosome 19ql3.1 had not been sequenced in its entirety before we released our mRasGRP4 and hRasGRP4 nucleotide sequence data to the public as GenBank entries:

AF331457, AY040628, AY048119, AY048120, and AY048121. Nevertheless, a genomic fragment was identified near the chromosome 19ql3.1 gap site that was homologous to the nucleotide sequence that conesponds to exons 1 to 9 ofthe nιRasGRP4 gene. Using primers that correspond to sequences in this human genomic fragment, a 900-base pair (bp) cDNA that we concluded encoded the conesponding portion ofthe mRasGRP4 transcript, was isolated from the mononucleoar cells in human peripheral blood. To deduce the nucleotide sequence ofa full-length hRasGRP4 transcript in this population of cells, a "rapid amplification of cDNA ends" (RACE) approach was carried out with an RLM-RACE kit from Ambion (Austin, Texas) and total RNA obtained from the leukocytes of normal individuals. After the mononuclear cells were enriched from peripheral blood using Ficoll- Paque (Pharmacia Biotech, Uppsala, Sweden), they were lysed and total RNA was extracted and purified using Trizol reagent (Life Technologies, Inc). Human leukocyte Marathon- Ready™ cDNAs (Clontech) also were used in the identification ofthe major hRasGRP4 expressed in the human population. The RACE was carried out using the 5'-RACE primer 5'-ACCTGTCGGGCTGTGCCTCA-3' (SEQ DD NO: 11) and the 3'-RACE primer 5'-CAGCACCAAGGCCCTCCTGGAGCT-3' (SEQ ED NO: 12). Each ofthe 30 cycles in the PCR consisted ofa 30-sec denaturing step at 94°C, a 30-sec annealing step at 55°C, and a 2.5-min extension step at 72°C. Multiple amplified products were subcloned into pcDNA3.1/Directional/V5-His-TOPO (Invitrogen, Carlsbad, CA) and their inserts were sequenced in both directions using an ABI-377 sequencer (Applied Biosystems, Foster City, CA).

Analysis of Tissue Distribution of the hRasGRP4 Transcript. Human cDNA libraries generated by Clontech (Palo Alto, CA) from adult heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, and leukocyte; from fetal heart, brain, lung, liver, kidney, spleen, thymus, and skeletal muscle; and from peripheral blood CD4⁺T lymphocytes, CD8⁺ T lymphocytes, CD 19⁺ B lymphocytes, and CD14⁺ mononuclear cells before and after lectin activation were used to • evaluate the relative distribution ofthe hRasGRP4 transcript in varied adult and fetal human tissues and varied circulating cells. Each library was created using pooled polyA^"1" RNA from large numbers of individuals. For example, the leukocyte cDNA library used to clone hRasGRP4 cDNAs was generated using mRNA pooled from 550 individuals. A blot

(Clontech) containing -2 μg of poly (A)⁺ RNA from varied human tissues also was probed under conditions of high stringency with a radiolabeled 303-pb probe that conesponds to residues 329 to 631 in a hRasGRP4 cDNA.

Four apparently normal individuals, a patient with asthma, a patient with systemic mastocytosis, and a mast cell line derived from a mast cell leukemia patient (Butterfield, et al., Leuk. Res. (1988) 12, 345-355) were evaluated individually for the presence of abnormal hRasGRP4 transcripts. The first patient used for this study was a 53-year-old male that had systemic mastocytosis for -25 years. He presented in 1997 with marked abdominal swelling and extensive lymphadenopathy on a background of long-term severe flushing and dianhea. Physical examination demonstrated extensive skin involvement with pigmentation and erythematous areas on his face trunk and limbs. He had marked ascites hepatomegaly down to the right iliac fossa and marked axillary, inguinal, cervical and epitrochlear lymphadenopathy. He also had a mild splenomegaly. Bone manow trephine carried out in July 1997 demonstrated extensive fibrosis and abnormal megakarocytes, moderate eosinophilia, and reduced numbers of erythrocytes and granulocytes. Approximately 3% of the cells in the patient's bone manow biopsy were mast cells that possessed an abnormal moφhology. The manow was hypercellular with moderate infiltration of abnormal primitive cells. These cells did not express CD3, CDl 5, CD20, CD34, CD45, CD45RO, and K or λ light chains; they also failed to stain when incubated with periodic acid schiff reagent. However, they expressed CD43 and CDl 17 (c-kit). Analysis ofthe cells in the patient's bone marrow on November 1997 revealed 30% blast cells consistent with the development of an acute myeloid leukemia. The patient was given chemotherapy. Although the initial response was good, he had a stormy course over the next 3 years and was given a number of different courses of chemotherapy. Eventually he died due to the complications of his leukemia. When the patient was in clinical remission, peripheral blood white cells were obtained and frozen down for future study. RNA was purified from these isolated cells in order to evaluate hRasGRP4 expression.

The asthma patient was a 32-year-old Caucasian male with a history of asthma, allergic rhinitis, conjunctivitis, and atopic dermatitis. The initial diagnosis of asthma was made at age 7. He used inhaled steroids from age 7 to 12; from age 18 to the present he uses inhaled β2 agonists and inhaled steroids twice per day. He has been hospitalized three times due to the acute exacerbations of his severe asthma. The patient has a history of allergic reactions to house dust mites and cats; he often gets asthmatic attacks during upper respiratory tract infections.

RNA, isolated from the HMC-1 mast cell line and the mononuclear cells of four normal individuals and the above two patients, was converted into cDNA by using Promega's reverse transcription system (Promega, Madison, WI) and then PCRs were carried out using 5-μl portions of each cDNA preparation (conesponding to -1 ng cDNA) and 0.4 μM ofthe sense oligonucleotide 5'-AATGCACCGGAAAAACAGGA- 3' (SEQ ED NO: 13) and 0.4 μM ofthe antisense oligonucleotide 5'-TGAGTCTGGAGATGGCACTG-3' (SEQ ID NO: 14) to generate the 900-bp product that conesponded to exons 1 to 9 in the hRasGRP4 transcript. In all instances, the 30 cycles of each PCR consisted of a 30-sec denaturing step at 94°C, a 30-sec annealing step at 55°C, and a 1-min extension step at 72°C. As an internal confrol, samples also were evaluated for the presence of glyceraldehyde-3 -phosphate dehydrogenase (G3PDH)with the sense and antisense oligonucleotides 5'- TGAAGGTCGGAGTCAACGGATTTGGT-3' (SEQ DD NO: 15) and 5'- CATGTGGGCCATGAGGTCCACCAC-3 ' (SEQ ED NO: 16). Additional PCRs were carried out with the sense oligonucleotide 5'-TGCAGATCTGTCACCTGGTC-3' (SEQ ED NO: 17) and the antisense oligonucleotide 5'-CGGAACTCCAGGTAGGTGAG-3' (SEQ DD NO: 18) and the sense oligonucleotide 5'-CTTCTGACCTCCCAGGCCTG-3' (SEQ ED NO: 19) and the antisense oligonucleotide 5'-GTAGCGGGCGTAGTTGTTGT-3' (SEQ ED NO: 20) to evaluate the expression ofthe variant 1 and 2 transcripts, respectively, in the region that conesponds to exons 5 to 6 ofthe normal hRasGRP4 transcript.

hRasGRP4 immunohistochemistry. Analysis ofthe primary amino acid sequences ofthe varied mouse and human RasGRP family members revealed that the N terminus is poorly conserved in this family of GEFs. A peptide antibody directed against the N-terminus of mRasGRP4 also specifically recognizes this mouse GEF. A routine computer search failed to reveal any amino acid sequence in varied protein databases that resembled the 14-mer peptide Met-Asn-Arg-Lys-Asp-Ser-Lys-Arg-Lys-Ser-His-Gln-Glu-Cys (SEQ DD NO: 21) residing at residues 1 to 14 in hRasGRP4. Thus, an anti-peptide approach was used to obtain rabbit antibodies that specifically recognize the N-terminus of lιRasGRP4. The synthetic peptide was generated by Affinity Bioreagents, Inc (Golden, CO) and then coupled to Keyhole Limpet hemocyanin through the SMCC linker with the thiol group ofthe C-terminal Cys in the peptide. A rabbit was immunized four times with the peptide conjugate (0.5 mg/immunization) over a 60-day period. The resulting anti-hRasGRP4 antibodies were then purified using a standard peptide-affinity chromatography approach.

Tryptases and chymases are stored in abundance in the secretory granules of human mast cells, and the levels of these granule proteins greatly exceed that of any intracellular signaling protein in a mast cell. While double-staining approaches often are used to identify human mast cells in tissues that coexpress these two families of serine proteases (Irani, A, et al., Proc. Natl, Acad. Sci. U.S.A., 83:4464-4468, 1986), such an immunohistochemical approach cannot be used effectively to identify mast cells that express tryptase and hRasGRP4 due to the substantial differences in the levels of these two proteins. Thus, a more reliable serial-section approach (Friend, D.S., et al., J. Cell Biol. 135:279-290, 1996) was used to identify those cells in human breast and stomach that express hRasGRP4.

For immunohistochemistry, 4% paraformaldehyde/PBS-fixed paraffin-embedded human breast and stomach serial tissue sections were obtained from Imgenex (San Diego, CA). The sections were deparaffinized, hydrated, and washed twice with phosphate buffered saline (PBS). They were then incubated overnight at 4°C in buffer containing affinity- purified rabbit anti-hRasGRP4 antibody or preimmune rabbit serum. The adjacent section was incubated with a mouse monoclonal anti-tryptase antibody (Chemicon, Temecula, CA). Samples were washed, incubated for 1 h at room temperature in buffer containing biotin- labeled goat biotin-labeled goat anti-rabbit or anti mouse lg anti-rabbit IgG, washed twice in 0.1% BSA and 0.05% Tween 20 in PBS, incubated for 1 h at room temperature in Vectastain ABC-AP reagent (Vector Laboratories, Burlingame, CA), and then incubated for 15 min at room temperature in the presence of an alkaline phosphatase substrate solution. The resulting cells were then counterstained with Gill's hematoxylin and covered with Immu-Mount (Shandon, Pittsburgh, PA). Cytospin slides containing tryptase^"1" HMC-1 cells¹⁹ also were evaluated immunohistochemically for hRasGRP4 protein.

Generation of recombinant hRasGRP4. Using a standard PCR approach with the sense oligonucleotide 5'-CACCATGAACAGAAAAGACAGTAAGAGG-3' (SEQ DD NO: 22) and the antisense oligonucleotide 5 '-GGAATCCGGCTTGGAGGATGCAGT-3 ' (SEQ DD NO: 23), the entire coding domain of a hRasGRP4 cDNA was generated from one ofthe 2.1-kb cDNAs isolated from the human leukocyte library. This PCR consisted of a 30-sec denaturing step at 94°C, a 30-sec annealing step at 55°C, and a 1-min extension step at 72°C. Only 25 cycles were carried out in these PCRs in order to minimize the generation of point mutations.

In order to detect the recombinant protein and to purify it from the lysates of transfected COS-7 cells and 3T3 fibroblasts, the final PCR product was placed into the mammalian expression vector pcDNA3.1/Directional/V5-His-TOPO (Invitrogen) upstream ofthe sequences that encode the V5 and 6xHis peptides. Vector lacking the hRasGRP4 insert was used as a negative control in these experiments. African green monkey, SV40- transformed kidney COS-7 cells (line CRD-1651, ATTC) and 3T3 fibroblasts were cultured in DMEM containing 10% fetal bovine serum. Transient fransfections were performed with SuperFect (Qiagen, Valencia, CA) according to manufacturer's instructions. Cells were plated at a density of 2 x 10⁵ cells per well in a 6-well plate 24 h prior to transfection. Cells were transfected for 2-3 h, trypsinized, and then replated into parallel plates for both immunofluorescence (24- well plates containing 11 -mm coverslips) and SDS- PAGE/immunoblot analysis (12-well plates). Conditioned media and cells were collected 24h post transfection. hRasGRP4-expressing fibroblasts also were obtained by transfecting Swiss Albino mouse 3T3 fibroblasts with the above expression plasmid. The resulting fibroblasts were cultured in enriched media supplemented with 200-500 ng/ml G418 to increase the percentage of hRasGRP4-expressing cells in the culture. The presence of recombinant protein was evaluated by SDS-PAGE/immunoblotting with anti-V5 antibody (Invitrogen) or the above hRasGRP4-specific antibody. For immunodetection ofthe recombinant protein, cell or tissue lysates were boiled in SDS sample buffer containing β-mercaptoethanol, as were samples ofthe conditioned media. The resulting soluble proteins were resolved on a 12% polyacrylamide gel (Bio-Rad, Hercules, CA) and blotted onto PVDF membranes (Bio-Rad). They were then exposed to Tris-buffer saline containing 0.1% Tween-20, 5% non-fat milk, and 0.5% goat serum to minimize nonspecific binding ofthe relevant antibody. Treated lysates were exposed to a 5000-fold dilution ofa stock solution of mouse anti-V5 antibody or rabbit anti-hRasGRP4 antibody in Tris-buffered saline and 0.1 % Tween-20, followed by horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibody (Bio-Rad). hnmunoreactive proteins were detected using BioMax MR film (Eastman Kodak) and chemiluminescence kits from Pierce (Rockford, IL). Guanine nucleotide exchange assay. ProBond™ nickel-chelating resin (Invitrogen) was used to purify 6xHis-tagged recombinant hRasGRP4 from the transfectants. As recommended by the manufacturer, -1 x 10⁷ transfectants were placed in 4 ml of 20 mM sodium phosphate, pH 7.4, buffer containing 500 mM sodium chloride and multiple protease inhibitors (Roche Diagnostics, Indianapolis, IN). Each cell suspension was lysed by two freeze-thaw cycles using liquid nitrogen and a 42°C water bath. Liberated nuclear DNA was sheared by passing the resulting preparation through an 18-gauge needle four times, and then the cellular debris was removed by a 5-min centrifugation step at 4°C and at -14,000 x g. The resulting hRasGRP4-enriched supernatant was incubated with nickel-charged agarose resin for 1 h at 4°C to ensure efficient binding ofthe 6xHis-tagged recombinant protein. After the equilibration step, the resin was centrifuged in a Spin column at 800 x g for 2 min. The non-bound material was discarded and the column was washed extensively with 500 mM sodium chloride and 20 mM sodium phosphate, pH 6.0, to remove weakly associated protein. Five ml of 50 mM imidazole elution buffer was applied, and the resulting hRasGRP4- enriched eluate was concentrated to -0.5 ml with a Centriplus-50 (Millipore, Bedford, MA) filtering device having a 50-kDa cut-off membrane.

A modification ofthe guanine nucleotide exchange assay described by Zheng and coworkers (Zheng, Y. et al., Methods Enzymol. 256:77-84, 1995) was used to evaluate the function of mRasGRP4. In this assay, 2 μg of recombinant human H-Ras (Oxford

Biomedical Research, Oxford, MI), RhoA, Racl, Ran, Rab3A, or Cdc42 (Calbiochem), was placed in 60 μl of 100 mM NaCl, 2 mM EDTA, 0.2 mM dithiothreitol, 20 mM Tris-HCI, pH 8.0, supplemented with 100 μM adenylyl-imidodiphosphate tetralithium salt (AMP-PNP; Roche Diagnostics) and 10 μM GDP (Sigma). After a 5-min incubation at room temperature, MgCl₂ was added to achieve a final concenfration of 5 mM. The solution was then incubated for another 15 min at room temperature to load H-Ras and its varied family members with non-radiolabeled GDP. Twenty μl ofthe resulting solution was added to 75 μl of reaction buffer [100 mM NaCl, 10 mM MgCl₂, 20 mM Tris-HCI, pH 8.0, supplemented with 100 μM AMP-PNP, 0.5 mg/ml bovine serum albumin, and 2 μM guanosine 5'-(γ-³⁵S)triphosphate (-11 ,000 cpm/pmol; Amersham Pharmacia Biotech, Piscataway, NJ)], followed by 5 μl of purified recombinant hRasGRP4 in the above buffer. The resulting samples were incubated at room temperature generally for 20 min. In the kinetic study with H-Ras, 15-μl aliquots were removed every 5 min and placed in 4 ml of ice-cold termination buffer (100 mM NaCl, 10 mM MgCl₂, and 20 mM Tris-HCI, pH 8.0). Non-bound radioactivity was removed by filtering each reaction mixture through a 25-mm cellulose nitrate membrane possessing 0.45- μm pores (Whatman, Maidstone, England). After each filter was washed with 10 ml of ice- cold termination buffer, it was placed in 5 ml of Filtron-X scintillation fluid (National Dignostics, Atlanta, GA) and the amount of bound radioactivity was quantitated using a Beckman LS5801 machine. For a negative control, purified recombinant hRasGRP4 was boiled and sonicated for 5 and 5 min, respectively; the enzymatic activity ofthe denatured protein was then assessed. The ability of CaCl₂ (0.2-10 mM) to inhibit the guanine nucleotide exchange activity of recombinant nιRasGRP4 was also evaluated. In this competition assays, recombinant H- Ras was preloaded with GDP. CaCl₂ was then added, followed by recombinant hRasGRP4 and radiolabeled GTP. The reaction mixtures were then incubated 20 min at room temperature.

Protein modeling. 3D models of residues 34-445 and 541-597 of normal and variant-2 hRasGRP4 were built using MODELLER. (Sali, A. et al., J. Mol. Biol. 234:779-815, 1993; Sali. A. et al., Protein Sci. 3:1582-1596). Residues 34-445 contain the REM and CDC25-like catalytic domains and conespond to the initial 61% ofthe translated protein. This portion of hRasGRP4 was modeled based on the crystallographic structure of hSosl (residues 568-1032 of GenBank Accession number A37488) complexed to H-Ras (Brookhaven Protein Data Bank code IBRD). (Boriak-Sjodin, P.A. et al., Nature 394:337-343, 1998). Residues 541- 597 in hRasGRP4 conespond to its putative DAG-binding domain. This portion ofthe GEF was modeled based on the NMR structure ofthe Cys2 domain in rat protein kinase C-γ (Bookhaven Protein Data Bank code ITBN). Xu, R.X. et al., Biochemistry 36:10709-10717, 1997). The regions are -20% and -33% identical to the conesponding regions in Sosl and protein kinase C-γ, respectively.

Normal Isoforms of RasGRP4 in the Differentiation and Granule Maturation of Human Mast Cells. As described in the results section, the hRasGRP4 gene is transcribed but the resulting transcript is not processed conectly in HMC-1 cells. Because a functional form of the signaling protein is not expressed in HMC-1 cells, this mast cell leukemia cell line was used in an attempt to deduce the function of RasGRP4 in mast cells. We identified ten single nucleotide polymoφhisms in the hRasGRP4 transcript in the human population that, in turn, result in five amino differences in the translated products. Four of these amino acid polymoφhisms are non-conservative changes. There is only one mRasGRP4 allele in the inbred BALB/c mouse. Because it was not obvious which hRasGRP4 allele should be used in our initial attempt to conect the developmental problem in the HMC-1 cell line, the BALB/c mouse-derived RasGRP4 described in RESULTS was used in the transfection experiments. Recombinant RasGRP4 possesses the V5 epitope at its C terminus. Thus, anti- V5 antibody was used to confirm that a functional, non-truncated version ofthe GEF was expressed in the transfectants. Immunohistochemistry and/or SDS-PAGE/immunoblot approaches were then used with anti-tryptase and anti-chymase antibodies (Chemicon International, Temecula, CA) to evaluate the levels of these neutral proteases in the two populations of cells. A pancreatic carboxypeptidase A (CPA) derived antibody (Sigma) that also recognizes MC CPA was used to evaluate the levels of this exopeptidase. Finally, standard electron microscopic methodologies were carried out to evaluate the ultrastructure ofthe HMC-1 cell line before and after transfection.

hRasGRP4 expression in the mast cell leukemia cell line HMC-1. Oligonucleotides conesponding to sequences in exon 2 (E2) and exon 6 (E6) ofthe hRasGRP4 gene were used in an RT-PCR approach to evaluate the extent of processing ofthe hRasGRP4 precursor transcript in the HMC-1 cell line (Fig. 18, lane 2) and in the spleen of 22 pooled Caucasian fetuses (ages 20-33 weeks, Clontech) (Fig. 18 lane 1). The normal 625-bp, properly processed portion ofthe transcript was detected in the pooled spleen sample, as well as the 742-bp variant 1 transcript that contains intron 5 (15). In contrast, the normal hRasGRP4 transcript and the variant 1 transcript were not detected in the HMC-1 cell line. Rather an 866-bp form (variant 3) was detected in the HMC-1 cell line whose nucleotide sequence revealed that it contained both intron 3 (13) and intron 5 (15). The failure to remove these introns causes a premature translation-termination codon.

Granulation of HMC-1 cells and their mRasGRP4-transfectants. Because only nonfunctional forms of hRasGRP4 are present in HMC-1 cells, a fransfection approach was used to force this immature human MC line to express a functional form ofthe GEF. As assessed immunohistochemically and by SDS-PAGE-immunoblot analysis, the mRasGRP4- expressing transfectants contained substantially more tryptase, chymase, and CPA in their granules than the non-transfectants. While granules are rarely found in HMC-1 cells, many granules that contain electron dense material are found in the transfectants. Although the transfectant depicted in the electron micrograph (EM) shown in Fig. 19J is more typical of the cells in the mRasGRP4-expressing cultures, -10% ofthe transfectants contain granules that are nearly completely filled with electron dense material. The data indicated that RasGRP4 plays an important role in the final stages of mast cell differentiation and maturation, including what neutral proteases these cells store in their secretory granules.

Generation of RasGRP4-expressing HMC-1 cells and evaluation of the consequences on granule protease expression. As noted in Figure 18, the hRasGRP4 gene is transcribed but the resulting transcript is not processed conectly in HMC-1 cells. Because a functional form ofthe signaling protein is not expressed in HMC-1 cells, this mast cell leukemia cell line was used in an attempt to deduce the function of RasGRP4 in mast cells. As also noted in Figure 2, single nucleotide polymoφhisms have been identified in the hRasGRP4 transcript in the human population that, in turn, result in five amino differences in the translated products. Four of these amino acid polymoφhisms are non-conservative changes. There is only one mRasGRP4 allele in the inbred BALB/c mouse. Because it is not obvious which hRasGRP4 allele should be used to conect the developmental problem in the HMC-1 cell line, the BALB/c mouse-derived RasGRP4 described in Figure 8 was used in the transfection experiments. HMC-1 cells were transfected with the same expression construct used above to induce COS-1 cells and fibroblasts to express mRasGRP4 (Figures 16 and 17). Recombinant mRasGRP4 possesses the V5 epitope at its C terminus. Thus, anti-V5 antibody was used to confirm that a functional, non-truncated version ofthe GEF was expressed in the transfectants. Immunohistochemistry and/or SDS-PAGE/immunoblot approaches were then used with anti-tryptase and anti-chymase antibodies (Chemicon International, Temecula, CA) to evaluate the levels of these human neutral proteases in the two populations of cells. A pancreatic CPA derived antibody (Sigma) that also recognizes mast cell CPA was used to evaluate the levels of this exopeptidase. Finally, standard electron microscopic methodologies were carried out to evaluate the ultrastructure ofthe HMC-1 cell line before and after transfection. Transcript analysis of RasGRP4^" and RasGRP4⁺ HMC-1 cells. RasGRP4^" and RasGRP4⁺ HMC-1 cells were cultured in enriched medium in the absence of human cytokines. Total RNA was isolated from both populations of cells using TRTZOL^® Reagent (Invitrogen), and comparative transcript profiling was carried out with HG-U95A GeneChips (Affymetrix, Santa Clara, CA). Each HG-U95A GeneChip contains -12,600 probe sets. In this transcript analysis, 8 μg of total RNA from mRasGRP4^" and mRasGRP4^"t" HMC-1 cells were reverse- transcribed with an oligo(dT) primer coupled to a T7 RNA polymerase binding site. Biotinylated complementary RNA (cRNA) was synthesized from the resulting cDNAs using T7 polymerase. Twenty-five μg of biotinylated cRNA were then randomly sheared to an approximate length of 50 nucleotides and hybridized for 16 h to each Affymetrix GeneChip. The hybridized GeneChips were incubated with phycoerythrin-streptavidin and washed. The obtained signal was then amplified by incubating each GeneChip with polyclonal anti- streptavidin antibody coupled to phycoerythrin. Hybridization to the anay was quantified with a Hewlett-Packard gene anay laser scanner. First-pass analysis ofthe obtained data was performed with Affymetrix GeneChip software (version 3.3). The multiples of change in gene expression reported by the Affymetrix software in the paired experiment was used to determine whether a statistically significant change in expression had occuned.

PGD₂ synthase expression at the protein level. A SDS-PAGE/immunoblot approach was used to determine whether or not increased levels ofthe PGD₂ synthase transcript in HMC-1 cells results in increased levels of PGD synthase protein. For these experiments, RasGRP4^" and RasGRP4⁺ HMC-1 cells were washed with phosphate-buffered saline and then boiled in SDS sample buffer with β-mercaptoethanol. After electrophoresis, the resolved proteins were blotted onto polyvinylidene difluoride membranes (Bio-Rad). Each protein blot was exposed to Tris-buffered saline containing 5% non-fat milk, 0.1% Tween-20, and 0.5% goat serum. Antibodies directed against cPLA₂ (Cell Signaling), PG endoperoxide H synthase 1, PG endoperoxide H synthase 2, 5-lipoxygenase, and PGD₂ synthase (Cayman Chemical, Ann Arbor, MI) were added at a dilutions ranging from 1:300 to 1:1000. The resulting blots were incubated overnight at 4°C. After each blot was washed 3 times with Tris-buffered saline containing 0.1% Tween-20, it was exposed to Tris-buffered saline containing 5% non-fat milk, 0.1%Tween-20, 0.5% goat serum, and a 1:1000 dilution ofa stock solution of horseradish peroxidase-conjugated goat anti-mouse IgG (Bio-Rad) for lh at room temperature. The immunoreactive proteins were then visualized using a chemiluminescence kit (Genotech, St. Louis, MO) and BioMax MR film.

Calcium ionophore activation of mRasGRP4^" and mRasGRP4⁺ HMC-1 cells in the presence and absence of PMA. Calcium ionophore activation experiments were carried out to determine whether or not the induced PGD₂ synthase protein in HMC-1 cells is functional. mRasGRP4^" and mRasGRP4⁺ HMC-1 cells were washed, suspended at a concenfration of 10⁶ cells/ml in calcium-free, magnesium-free PBS, and stimulated with 0.5 μM calcium ionophore A23187 (Sigma Chemical Co., St Louis, MO) in the presence or absence of 0.1 μM PMA (Calbiochem) at 37°C for 30 min. Supematants were collected from the centrifuged samples and stored at -80°C until analysis. The generated eicosanoids PGD₂, PGE₂, and LTC₄ were measured by separate ELISA kits, according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI). The plate was read at 450 nm in an ELISA plate reader (Molecular Device). Data are given as mean ± SD. For changes in level of lipid mediators, values were compared between mRasGRP4^" and mRasGRP4⁺ HMC-1 cells by Student's t test. Significance was defined as p < 0.05.

Results Cloning of the hRasGRP4 transcript, evaluation of its expression, and chromosomal location of its gene. A computer search of varied nucleotide and protein databases failed to reveal any complete human cDNA, gene, or protein that closely resembles that ofthe mRasGRP4 transcript, gene, and/or protein. Nevertheless, the 11.5-kb nucleotide sequence residing at one end ofthe human chromosome 19q 13.1 -derived BAC clone AC011469 closely resembled that ofthe 5' half (i.e, exons 1-9 and introns 1-8) ofthe nιRasGRP4 gene. The nucleotide sequence of an overlapping BAC genomic clone that conesponds to the missing 3' half of the hRasGRP4 gene had not been deposited in GenBank. However, based on the chromosomal assignment of BAC clone AC011469, we concluded that the hRasGRP4 gene resides -8 kb downstream ofthe ryanodine receptor 1 (RYR1) gene (GenBank's LocusLink accession number 6261) within a small region of human chromosome 19ql3 that had not been sequenced in its entirety by the Human Genome Project before the release of our nucleotide sequence data (e.g. GenBank accession number AY048119) to the public on August 19, 2001.

Primers conesponding to the nucleotide sequences residing in exons 1 and 9 ofthe putative hRasGRP4 gene were used in RT-PCR and PCR approaches to determine whether or not the gene was transcribed in vivo. Because the expected 900-bp product was detected in nearly every examined human fetal tissue, the novel human gene we identified on chromosome 19ql3.1 is transcribed in vivo and is expressed early in development. The level ofthe hRasGRP4 transcript was more abundant in fetal lung than in heart, brain, liver, kidney, spleen, thymus, or skeletal muscle (Fig. 1 A). Although the expected 900-bp PCR product was detected in cDNA libraries generated from a number of pooled adult human tissues, the amount of hRasGRP4 mRNA in these tissue samples was less than that in fetal lung (Fig. IB). Despite this finding with tissue samples, the CD14⁺ population of mononuclear cells in the peripheral blood of adult humans subsequently was found to contain substantial amounts of hRasGRP4 mRNA (Fig. IC). The transcript also was detected in the tryptase^"1" human mast cell line HMC-1. However, the hRasGRP4 transcript was not detected in resting or lectin-activated CD19^"1" B lymphocytes, CD4⁺ T lymphocytes, or CD8⁺ T lymphocytes. Thus, like mRasGRP4 in the mouse, the expression of hRasGRP4 is highly restricted to MCs and their progenitors.

Different sized RT-PCR fragments were occasionally seen in the tissue and blood samples pooled from many individuals. As noted herein, sequence analysis ofthe conesponding transcripts in the mononuclear cells ofa systemic mastocytosis patient and an asthma patient revealed that these larger and smaller RT-PCR products are the result of differential splicing ofthe precursor transcript. The levels of hRasGRP4 mRNA were below detection in resting or lectin-activated CD19⁺ B lymphocytes, CD4⁺ T lymphocytes, or CD8⁺ T lymphocytes (Fig IC). The levels of hRasGRP4 mRNA also were below detection in lectin-treated mononuclear cells. Nevertheless, the steady-state levels ofthe hRasGRP4 transcript were sufficiently high enough in the non-lectin treated mononuclear leukocytes to detect the transcript by routine blot analysis.

Using leukocyte mRNA from pooled individuals in varied 3' and 5' RACE approaches, we deduced the nucleotide sequence ofthe primary hRasGRP4 transcript in normal individuals (Fig. 2). The GenBank accession number for the hRasGRP4 cDNA is AY048119. Sequence analysis of multiple subcloned hRasGRP4 cDNAs revealed ten single nucleotide differences in the eight analyzed clones that ultimately resulted in five amino acid differences in the translated products. We presume that these differences are the result of minor allelic polymoφhisms ofa single hRasGRP4 gene in the human genome rather than distinct genes that are >99% identical. The primary hRasGRP4 transcript is -300 bp larger than the primary mRasGRP4 transcript due to a somewhat longer 3' untranslated region. However, the rest ofthe hRasGRP4 transcript is >80% identical to that ofthe mRasGRP4 transcript.

No genomic fragment was detected in GenBank's genome database that conesponded to the 3' end ofthe hRasGRP4 cDNA. Probing of a commercial human genome database with the full-length hRasGRP4 cDNA also failed to reveal an intact hRasGRP4 gene in this database. Nevertheless, a genomic fragment was identified that conesponded to the 3' end of exon 10 to exon 18 ofthe hRasGRP4 gene. Using a PCR approach, we were able to deduce that the hRasGRP4 gene is >15 kb in size and consists of 18 exons. Like that in the mouse, the coding portion ofthe transcript is derived from 17 exons. Exon 18 conesponds to the 3' untranslated region. Exons 1-18 conespond to residues 1-237, 238-422, 423-530, 531-591, 592-723, 724-877, 878-1051, 1052-1168, 1169-1444, 1445-1525, 1526-1630, 1631-1750, 1751-1894, 1895-1931, 1932-2066, 2067-2189, 2190-2272, and 2273-end, respectively, in the cDNA depicted in Figure 2. The release of our hRasGRP4 cDNA to the public domain eventually enabled the Human Genome Project to fill in the missing gap on chromosome 19ql3.1 in the Fall of2001.

Evaluation of the structure of hRasGRP4 protein

As assessed immunohistochemically using an antibody directed against the predicted N-terminus ofthe translated product, the hRasGRP4 transcript is converted into protein in the tryptase HMC-1 cell line and the in vzvo-differentiated tryptase^"1" MCs that reside in varied human tissues (Fig. 3). Every tryptase MC in the interlobular connective tissue of human breast and the mucosa layer of human stomach contained hRasGRP4. Similar immunohistochemical data were obtained in the mouse with a different antibody. Thus^ hRasGRP4 is highly restricted to mature MCs and their progenitors. The nucleotide sequences ofthe isolated cDNAs indicate that mouse and human

RasGRP4 exist as 673-678-residue, -75 kDa proteins. Mouse and human RasGRP4 have a <50% amino acid sequence identity with mouse, rat, and human RasGRP 1, RasGRP2, and RasGRP3 (Fig. 14). As found for other cytosolic proteins, RasGRP4 lacks a hydrophobic signal peptide at its N terminus. A Kyte-Doolittle hydropathy analysis also failed to reveal an extended hydrophobic domain in the protein's primary amino acid sequence. Immunoreactive mRasGRP4 and hRasGRP4 were not constitutively secreted from the COS-7 cell and fibroblast transfectants, and immunoreactive mRasGRP4 was not detected in extracellular matrices adjacent to tissue MCs. Thus, RasGRP4 is an intracellular protein.

A dendrogram comparison ofthe nine most related mouse proteins in this superfamily revealed that mRasGRP4 is slightly more similar to mRasGRP2 than to mRasGRPl (Fig. 14A). The amino acid sequence of mRasGRP3 has not yet been deposited in GenBank. Nevertheless, mRasGRP4 is only distantly related to hRasGRP3. mRasGRP4 also lacks the extended C-terminal domains present in mRasGRPl, rRasGRPl, hRasGRPl, and hRasGRP3 (Fig. 5B). The putative REM, CDC25 catalytic, Ca²⁺-binding EF hands, and phorbol ester/DAG-binding domains ofthe varied RasGRPs are highlighted in Figure 14B. All of these domains are present in mouse and human RasGRP4 at the expected locations in its primary amino acid sequence. The most conserved region is the CDC25-like catalytic domain. For example, residues 405-433 in the CDC25-like catalytic domains of mRasGRP4 and mRasGRPl differ only in two amino acid residues, and even these differences are minimal (i.e., Leu— »Val and Phe— »Tyr). The amino acid sequence of hRasGRP4 is -85% identical to that of mRasGRP4 (Fig. 4). Except for the C-terminal 21 amino acids where the degree of sequence identity drops to 52%, the high degree of conservation extends throughout the entire length ofthe protein.

Residues 34-445 of hRasGRP4 (Fig. 5 A) are predicted to resemble the two α helical domains of hSos-1 with 6 and 11 helices in the first and second domains, respectively. Mimicking hSos-1, the eighth α helix in the CDC25-like catalytic domain of hRasGRP4 is predicted to interact with H-Ras. The residues involved in H-Ras interaction are generally conserved between hRasGRP4 and hSosl. The 3D model of residues 541-597 of hRasGRP4 (Fig. 5B) also closely resembles that ofthe putative DAG-binding domain of protein kinase C γ despite the poor degree of primary amino acid sequence identity. The minor allelic differences identified so far in hRasGRP4 are not expected to grossly alter its 3D structure, nor are they likely to affect its interaction with H-Ras or DAG greatly. Guanine nucleotide exchange activity of recombinant RasGRP4, and increased sensitivity of RasGRP4-expressing fibroblasts to PMA. Bioengineered forms of mouse and human RasGRP4 were expressed in COS-7 cells and fibroblasts that contained the V5 and 6xHis peptides at their C termini. The latter epitope tag was added so that each recombinant protein could be identified and purified. Although substantial amounts of immunoreactive RasGRP4 were always recovered in the soluble cytosolic portion ofthe lysates ofthe transfectants, a portion ofthe recombinant protein was consistently recovered in the microsomal fractions. Thus, RasGRP4 does not reside in a single intracellular compartment in either transfected cell type. As the 3D model of residues 34-445 predicted, purified recombinant hRasGRP4 was able to transfer [γ-³⁵S]GTP to GDP-loaded H-Ras in a catalytic manner (Fig. 6A). Subsequent specificity studies revealed that hRasGRP4 also can activate human RhoA, Cdc42, Racl, Rab3A, and Ran in vitro (Fig. 6B). Fig. 6B is a histogram illustrating the ability of purified recombinant hRasGRP4 to transfer radioactive GTP to GDP-loaded recombinant H-Ras, RhoA, Cdc42, Racl, Rab3A, and Ran after a 20-min incubation at room temperature in the absence (Fig. 6B) or presence of CaCl₂ (Fig. 6C). hRasGRP4 has a putative Ca2+-binding domain analogous to the other three members of its family (Fig. 4). The first EF hand in rRasGRPl functions as this protein's primary Ca²⁺-binding site (Ebinu, , DO, et al., Science 280:1082-1086, 1998) and the critical residues that form the "regulatory EF hand" in rRasGRPl and other Ca²⁺-binding proteins (Rashidi, H.H., et al., J. Mol.

Microbiol. Biotechnol. 1:176-182, 1999) are present in mouse and human RasGRP4 at the expected locations. Whether or not Ca²⁺ binds specifically to this domain is determined using a site-directed mutagenesis approach. Nevertheless, 1 mM Ca²⁺ was sufficient to significantly inhibit the ability of recombinant hRasGRP4 to transfer GTP to GDP-loaded H- Ras even if 5 mM Mg²⁺ was present in the reaction buffer (Fig. 6C). The phorbol ester/DAG- binding, Cl domain in protein kinase C is -50 residues in length and possesses the motif of HX₁₂CX₂CX₁₃CX₂CX₄HX₂CX₇C (SEQ ED NO:55) or HX₁₂CX₂CX₁₄CX₂CX₄HX₂CX₇C (SEQ DD NO: 60), where H is His, C is Cys, and X is any other amino acid residue. Because these residues are present in mouse and human RasGRP4 and because the 3D model predicts that residues 537-590 resemble a Cl-like domain, the possibility that RasGRP4 is a phorbol ester/DAG receptor also was tested experimentally. As noted in Figure 8, RasGRP4- expressing fibroblasts underwent dramatic moφhologic changes when exposed to low levels of PMA for only 15 min.

Isolation of abnormal hRasGRP4 transcripts in an asthma patient and a mastocytosis patient. Primers conesponding to nucleotide sequences in exons 1 and 17 ofthe hRasGRP4 gene were used to generate cDNAs that conespond to the entire coding domains ofthe forms of hRasGRP4 that are expressed in the mast cell-committed progenitors residing in the peripheral blood of an asthma patient. Five ofthe eight arbitrarily subcloned cDNAs from this patient conesponded in principle to the normal hRasGRP4 cDNA depicted in Figure 2. However, two ofthe cDNAs (designated variant 1) suφrisingly were 117 nucleotides larger in size. Sequence analysis revealed that variant 1 was created by a failure ofthe hRasGRP4- expressing cell to remove intron 5 from the precursor transcript (Figs. 7A and 7D). The failure to remove this single intron results in the formation of a premature stop codon early in the expressed transcript. Assuming the normal translation-initiation site is used at residues 215-217, the translated protein only contains 170 amino acids and will not be able to activate H-Ras because it lacks -75% of its primary amino acid sequence including the entire CDC25-like catalytic domain.

One ofthe eight arbitrarily subcloned cDNAs from the asthma patient possessed a 42- residue deletion (Fig. 7B) rather than a 117-bp insertion. Interestingly, this isoform (e.g. altered transcript), which is designated variant 2, also was caused by a failure ofthe hRasGRP4-expressing cell in this patient to properly remove intron 5. Because the normal intron 5/exon 6 splice-site was not used in the maturation ofthe precursor transcript, a cryptic splice-site residing 42 nucleotides within exon 6 was employed to remove intron 5 during the maturation ofthe variant 2 transcript. The open reading frame ofthe truncated variant 2 hRasGRP4 remains intact. However, the resulting protein lacks the 14-mer sequence that links the two major helical domains within the N-terminal segment (Fig,. 5A). The GenBank accession numbers for the variant 1 and variant 2 hRasGRP4 transcripts are AY048120 and AY048121, respectively.

An RT-PCR approach was used to evaluate the prevalence of both abnormal hRasGRP4 transcripts in the general population and in an additional mastocytosis patient. As noted in Figure 7c using different primer sets, the levels ofthe variant 1 and 2 transcripts were below detection in the mast cell progenitors residing in the blood of four normal individuals. Nevertheless, both abenant transcripts were detected in the leukocyte preparation derived from 550 individuals. Variant 1 mRNA was found in both patients but the level ofthe variant 2 transcript was below detection in the mastocytosis patient.

None ofthe eight hRasGRP4 cDNAs isolated and sequenced from the asthma patient and none ofthe hRasGRP4 cDNAs isolated and sequenced from the leukocyte preparation generated from multiple individuals lacked an entire exon. These preliminary data suggest that differential splicing of one or more exons in hRasGRP4 is a rare event, if it ever occurs, in the mast cell-committed progenitors circulating in the blood of nonnal individuals. Nevertheless, removal of any combination of exons 7 to 16 except exons 14 and 15 will result in a truncated transcript that remains in the conect reading frame relative to the normal stop codon.

Evaluation of hRasGRP4 Expression and Function in the HMC-1 Cell Line. The immature HMC-1 cell line was derived by Butterfield and co workers in 1988 from a patient with a MC leukemia (Butterfield, H., et al., Leuk. Res. 12:345-355, 1988) The identification of defective variants of hRasGRP4 in a mastocytosis patient (Fig. 7C) raised the possibility that the HMC-1 cell line might also expresses abnormal variants of hRasGRP4. Thus, hRasGRP4 expression at the mRNA level was next evaluated in this cell line. Using various primers sets, we discovered that the hRasGRP4 gene is transcribed in HMC-1 cells (Fig. 18). However, only abnormal variants of hRasGRP4 are expressed in this fransformed cell line. As noted in Figure 18, a new isoform of hRasGRP4 (designated variant 3, SEQ DD NO: 58) that is closely related to the variant 1 isoform was isolated from HMC-1 cells. Sequence analysis of this RT-PCR product revealed that the larger-sized transcript was caused by a failure to remove intron 5 and additionally intron 3 in the precursor transcript. The failure to remove these two infrons results in a translation-termination codon that occurs earlier in variant 3 than that in variant 1. Thus, even if translated, variant 3 also would be unable to activate any Ras family member. A premature translation-termination codon at a similar location in a tryptase transcript results in its rapid degradation in C57BL/6 mouse mBMMCs by the nonsense-mediated pathway (Hunt, J.,E., et al., J Biol. Chem. 271 :2851-2855, 1996). Ifthe defective hRasGRP4 transcript is being catabolized nearly as fast as it is being generated as we suspect, this would account for its lower levels in the HMC-1 cell line. HMC-1 cells are poorly granulated, blast-like leukemia cells (Fig. 191) that fail to express detectable amounts of MC chymase (Fig. 19E) or CPA (Fig. 19G) protein. Because HMC-1 cells only express small amounts of β tryptase (Figs. 19B and 19C), this cell line became an attractive MC-committed progenitor to begin to address the function of RasGRP4 in a more natural setting than occurs in a transfected fibroblast line. Thus, using an expression/transfection approach, HMC-1 cells were induced to express a normal, biologically active form of mRasGRP4 (Fig. 19A). As noted in Figure 19, the resulting transfectants underwent dramatic moφhologic changes (Fig. 19J) and increased their levels of tryptase substantially (Figs. 19B and 19D). The transfectants also began expressing MC chymase (Fig. 19G) and CPA (Fig. 19H).

Example 2

Methods

Cloning of mRasGRP4 and chromosomal location of its gene. Greater than 2000 clones were arbitrarily isolated and sequenced from a previously constructed mBMMC cDNA library (Lam, B.K. et al., Eur. J. Biochem. 238:606-612, 1996) using standard molecular biology procedures. The BALB/c mBMMCs used to create this library had been cultured for 6 weeks to ensure that no contaminating cell types were present. As assessed by RNA blot analysis, one ofthe isolated clones conesponded to all but a few hundred nucleotides ofthe major mRasGRP4 transcript present in mBMMCs. Thus, a "rapid amplification of cDNA ends" (RACE) approach was carried out with an RLM-RACE kit from Ambion (Austen, Texas) to deduce the nucleotide sequence ofthe missing 5' portion ofthe mRasGRP4 transcript. BALB/c mBMMCs, generated as previously described, (Razin, E. et al., J. Immunol. 132:1479-1486, 1984), were the cellular source ofthe mRNA used in this 5' RACE reaction. The first PCR was carried out using the outer adapter

5 '-GCUGAUGGCGAUGAAUCAACACUGCGUUUGCUGGCUUUGAUGAAA-3 '(SEQ ED NO: 24) and the mRasGRP4-specific antisense oligonucleotide 5'-CGGAACTCCCAGGTAGTGA-3' (SEQ DD NO: 25). The second nested PCR was carried out using the inner adapter 5 '-CGCGGATCCGACACTCGTTTGCTGGCTTTGATGAAA-3 ' (SEQ ED NO: 26) and the mRasGRP4-specific antisense oligonucleotide 5'-CAGGACTTAGCAGGCTGGAG-3' (SEQ ED NO: 27). Each ofthe 30 cycles in the PCR consisted ofa 30-sec denaturing step at 94°C, a 30-sec annealing step at 55°C, and a 2.5-min extension step at 72°C. Multiple amplified products were subcloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA) and their inserts were sequenced in both directions using an ABI-377 sequencer and standard methods to deduce the nucleotide sequence ofthe full-length transcript in mBMMCs. A FISH technique was used by Incyte Genomics (St. Louis, MO) to determine the chromosomal location ofthe mRasGRP4 gene. Preliminary studies revealed that the mRasGRP4 gene resided in the company's BAC mouse genomic clone F1231. Slides containing normal metaphase chromosomes derived from mouse embryonic fibroblasts were therefore incubated with the digoxigenin dUTP-labeled clone F1231 in the presence of 50% formamide, 10% dexfran sulfate, 2x SSC, and sheared mouse DNA. After this hybridization step, the slides were incubated with fluoresceinated anti-digoxigenin antibody and counterstained with 4,6 diamidino-2-phenylindole.

Tissue distribution of nιRasGRP4 mRNA. A semi-quantitative RT-PCR approach was used to evaluate mRasGRPl, mRasGRP2, and mRasGRP4 transcript expression in BALB/c mBMMCs, C57BL/6 mBMMCs, and in BALB/c mouse brain. The mBMMCs used in these experiments were derived by culturing bone manow progenitors for 4 weeks in the presence of recombinant IL-3. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was used as an additional control to confirm that intact RNA is present in all three samples. Although it has been reported that the mRasGRP3 gene resides on chromosome 17, the nucleotide sequence ofthe mRasGRP3 transcript has not been reported. Thus, it was not possible to evaluate whether or not mBMMCs express mRasGRP3. A similar semi-quantitative PCR approach also was used to screen various cDNA libraries to identify those tissues (i.e. spleen and lung) that contain the highest levels of mRasGRP4 mRNA. mRasGRP4 mRNA expression was also evaluated in a peritoneal cavity cellular exudate. cDNA libraries generated by Clontech (Palo Alto, CA) from adult mouse heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis, and from day-7 to -17 mouse embryos were used to evaluate the relative distribution ofthe mRasGRP4 transcript. Each tissue library was created using pooled RNA from BALB/c mice whose ages ranged from 8 to 12 weeks. Each embryo library was derived using pooled RNA from Swiss Webster/NIH mouse embryos of a fixed age. PCRs were canied out using 5-μl portions of each library (conesponding to -1 ng cDNA) and 0.4 μM ofthe sense oligonucleotide 5'- CATGAATCTGGGAGTGCTGA-3' (SEQ ID NO: 28) and 0.4 μM ofthe antisense oligonucleotide 5'-CAGGACTTAGCAGGCTGG AG-3' (SEQ ED NO: 29) to generate the mRasGRP4 product. Similar approaches were used to determine if C57BL/6 mBMMCs express mRasGRP4, and if BALB/c and C57BL/6 mBMMCs express mRasGRPl and mRasGRP2. The sense and antisense oligonucleotides

5'-GCAAGACTAGAGGCCAAATCA-3' (SEQ DD NO: 30) and

5'-ATGGTGGGGTTCTCTTTTACG-3' (SEQ DD NO: 31) were used to evaluate mRasGRPl expression, whereas the sense and antisense oligonucleotides 5'-CACAGCTAGTGCGCATGTTT-3' (SEQ DD NO: 32) and 5 '-ATGCTGAAAAGCTGCCTCAT-3 ' (SEQ DD NO: 33) were used to evaluate mRasGRP2 expression. In all three instances, the 30 cycles of each PCR consisted ofa 30-sec denaturing step at 94°C, a 30-sec annealing step at 55°C, and a 1-min extension step at 72°C. The 661-, 911-, 387-bp products derived from the mRasGRPl, mRasGRP2, and mRasGRP4 transcripts, respectively, were resolved on 1.5% agarose gels. As an internal control, samples also were evaluated for the presence of glyceraldehyde-3-phosphate dehydrogenase with the sense and antisense oligonucleotides.5'-TGAAGGTCGGAGTCAACGGATTTGGT-3' (SEQ DD NO: 34) and 5'-CATGTGGGCCATGAGGTCCACCAC-3' (SEQ DD NO: 35).

Blot analysis was carried out using total RNA isolated from mBMMCs developed for 2-7 weeks with IL-3, Razin, E. et al., J. Immunol. 132:1479-1486, 1984, the two BALB/c mouse mast cell lines V3 (Gurish, M.F. et al., Immunity 3:175-186, 1995) and C1.MC/C57.1 (Young, DD. et al., Proc. Natl. Acad. Sci. U.S.A. 84:9175-9179, 1987), the rat mast cell line RBD-1 [line CRL-1378, American Type Culture Collection (ATCC), Rockville, MD], Eccleston, E. et al., Nat. New Biol. 244:73-76, 1973; Seldin, D.C. et al., Proc. Natl. Acad. Sci. U.S.A. 82:3871-3875, 1985), the two mouse myelomonocytic/macrophage cell lines RAW 264.7 (line CRL-2278, ATCC) and WEHI-3 (line TDB-68, ATCC), the mouse fibroblast cell line 3T3-Swiss albino (line CCL-92, ATCC); and the mouse T cell hybridoma MTC-1. (Kilshaw, PJ. et al., Eur. J. Immunol. 20:2201-2207, 1990). The resulting blot was probed under standard conditions of high stringency with a 197-bp probe that conesponds to residues 1978 to 2174 in the mRasGRP4 cDΝA. The V3, C1.MC/C57.1, and RBL-1 mast cell lines were examined because all three are well-described IL-3-independent mast cell lines that have been maintained in culture for years; the RBL cell line also is one ofthe major cell lines used by MC investigators to study FcεRI-mediated signaling events. The two myelomonocytic/macrophage lines were examined because macrophages and mast cells originate from a common progenitor in the bone manow; (Valent, P. et al., Blood 73 : 1778- 1785, 1989); the IL-3 -producing WEHI-3 cell line (Dhle, J.N. et al, J. Immunol. 129:2431- 2436, 1982) also resembles mBMMCs in its IL-3 dependence. The 3T3 cell line was examined because this fibroblast line was used in the transfection experiments to evaluate the role of mRasGRP4 in cellular adherence and activation of different Ras family members. Finally, the T cell hybridoma was examined because mRasGRPl was initially cloned from a T cell hybridoma (Tognon, CE. et al., Mol. Cell Biol. 18:6995-7008, 1998) and because this GEF is essential for mouse thymocyte differentiation and TCR signaling of varied populations of T cells. (Ebinu, DO. et al., Blood 95:3199-3203, 2000; Dower, N.A. et al., Nat. Immunol. 1:317-321, 2000).

mRasGRP4 immunohistochemistry. Analysis ofthe primary amino acid sequences of mRasGRPl, mRasGRP2, and mRasGRP4 revealed that the N terminus is poorly conserved in this family of GEFs. A computer search also failed to reveal any amino acid sequence in varied protein databases that resembled the 12-mer peptide Arg-Lys-Asp-De-Lys-Arg-Lys- Ser-His-Gln-Glu-Cys (SEQ ED NO: 36) residing at residues 3 to 14 in mRasGRP4. Thus, an anti-peptide approach was used to obtain rabbit antibodies that recognize this novel peptide sequence in mRasGRP4. The synthetic peptide was generated by Genemed Synthesis (San Francisco, CA) and then coupled to Keyhole Limpet hemocyanin through the SMCC linker with the thiol group ofthe C-terminal Cys in the peptide. A rabbit was immunized four times with the peptide conjugate (0.5 mg/immunization) over a 60-day period. The resulting anti- mRasGRP4 antibodies were then purified using a standard peptide-affinity chromatography approach. For immunohistochemistry at the light level, the peritoneal exudates, tongue, and skin biopsies from wild type mice, and spleen biopsies from V3-mastocytosis mice were fixed for 4 h at room temperature in 4% paraformaldehyde and 0.1 M sodium phosphate (pH 7.6), and then were washed twice with PBS containing 2% DMSO. The spleen ofthe V3-mastocytosis mouse was examined because this organ contains large numbers of v-αb/-immortalized mast cells two weeks after the adoptive transfer ofthe V3-mast cell line into the tail vein of BALB/c mice. (Gurish, M.F. et al., Immunity 3:175-186, 1995). Each specimen was dehydrated and embedded in plastic accordance with the JB-4 kit from Polysciences (Warrington, PA). Sections were cut on a Reichert-Jung Supracut microtome (Leica, Deerfield, IL) with glass knives and then picked up on glass slides. The slides were incubated sequentially for 15 min at 37°C in 2 mM CaCl₂ containing 0.025% trypsin, for 15 min at room temperature in PBS containing 0.05% Tween 20 and 0.1% BSA, for 30 min at 37°C in PBS containing 0.05% Tween 20 and 1% normal goat serum, and then overnight at 4°C in buffer containing purified rabbit anti-mRasGRP4 antibody. Samples were washed, incubated for 1 h at room temperature in buffer containing biotin-labeled goat anti-rabbit IgG, washed twice in 0.1% BSA and 0.05% Tween 20 in PBS, incubated for 1 hat room temperature in Vectastain ABC-AP reagent (Vector Laboratories, Burlingame, CA), and then incubated for 15 min at room temperature in the presence of an alkaline phosphatase substrate solution. The tissue sections were then counterstained with Gill's hematoxylin and covered with hnmu-Mount (Shandon, Pittsburgh, PA). To ensure that most ofthe mRasGRP4 epitope was not lost and/or destroyed during the plastic-embedding step and to minimize background staining, non-plastic embedded frozen sections of tongue also were stained with anti- mRasGRP4 antibody.

Ultra-thin frozen sections of a spleen from a V3 -mastocytosis mouse were processed for immunoelectron microscopy in order to determine where mRasGRP4 resides inside V3 mast cells. Tissue biopsies (2 mm ) were fixed overnight in PBS containing 4% paraformaldehyde, infiltrated for -30 min at room temperature in PBS containing 2.3 M sucrose and 0.15 M glycine, mounted, and frozen in liquid nifrogen. Ultrathin sections were cut at -120°C with a cryo-diamond knife and a Reichert Ultracut-S Microtome, picked up with a loop dipped in a 1:1 mixture of 2.3 M sucrose and 2%> methylcellulose, transfened to a formvar-coated copper grid, and incubated for 15 min in PBS containing 1% BSA to minimize non-specific binding ofthe rabbit antibody. The treated sections were then placed in PBS containing 1 % BSA and a 1 :50-fold dilution of affinity-purified rabbit anti- mRasGRP4 antibody. After washing 4 times with PBS, each section was exposed to 10 nm protein A-gold (Univ. Med. Ctr. Utrecht, The Netherlands) in PBS containing 1% BSA for 30 min. Treated sections were washed again, exposed to 1% uranyl acetate, and finally examined in a JEOL 1200EX electron microscope.

Generation of recombinant mRasGRP4. A full-length mRasGRP4 cDNA was constructed using the ~2.1-kb cDNA clone isolated from the mBMMC library and the subsequent 565-bp product generated using the 5' RACE approach. The latter product was liberated from its vector with EcoRL and then a PCR was carried out with the sense oligonucleotide 5'- CACCATGAACCGGAAAGACATCAAA-3' (SEQ ED NO: 37) and the antisense oligonucleotide 5'-CCAAAACCGGCTTATGACTG-3' (SEQ ED NO: 38) to create a product that conesponds to residues 141 to 590 in the mRasGRP4 transcript. The larger fragment was liberated with HindDI and Xhol. A PCR was carried out with the sense oligonucleotide 5'-CATGAATCTGGGAGTGCTGA-3' (SEQ DD NO: 39) and the antisense oligonucleotide 5'-GACGCTGGGCTTCGAGGAAGC-3' (SEQ DD NO: 40). The resulting two PCR products were mixed and then used as templates to generate a single product that conesponds to the entire coding domain (i.e., residues 141 to 2174) ofthe mRasGRP4 cDNA. Primers used in this final PCR were the sense oligonucleotide 5'-

CACCATGAACCGGAAAGACATCAAA-3' (SEQ DD NO: 41) and the antisense oligonucleotide 5'-GACGCTGGGCTTCGAGGAAGC-3' (SEQ ED NO: 42). Each PCR consisted of a 30-sec denaturing step at 94°C, a 30-sec annealing step at 55°C, and a 1-min extension step at 72°C. Only 25 cycles were carried out in these PCRs in order to minimize the generation of point mutations.

In order to detect the recombinant protein and to purify it from the lysates of transfected COS-7 cells and 3T3 fibroblasts, the final PCR product was placed into the mammalian expression vector pcDNA3.1/Directional/V5-His-TOPO (Invitrogen) upstream ofthe sequences that encode the V5 and 6xHis peptides. Vector lacking the mRasGRP4 insert was used as a negative control in these experiments. African green monkey, SV40- transformed kidney COS-7 cells (line CRL-1651, ATTC) and 3T3 fibroblasts were cultured in DMEM containing 10% fetal bovine serum. Transient fransfections were performed with SuperFect (Qiagen, Valencia, CA) according to manufacturer's instructions. Cells were plated at a density of 2 x 10⁵ cells per well in a 6-well plate 24 h prior to transfection. Cells were transfected for 2-3 h, frypsinized, and then replated into parallel plates for both immunofluorescence (24-well plates containing 11-mm coverslips) and SDS- PAGE/immunoblot analysis (12-well plates). Conditioned media and cells were collected 24 h post transfection. mRasGRP4-expressing fibroblasts also were obtained by transfecting Swiss Albino mouse 3T3 fibroblasts with the above expression plasmid. The resulting fibroblasts were cultured in enriched media supplemented with 200-500 ng/ml G418 to increase the percentage of mRasGRP4-expressing cells in the culture. The presence of recombinant protein was evaluated by SDS-PAGE/immunoblotting with anti-V5 antibody (Invitrogen) or the above mRasGRP4-specific antibody. For immunodetection ofthe recombinant protein, cell or tissue lysates were boiled in SDS sample buffer containing β-mercaptoethanol, as were samples ofthe conditioned media. The resulting soluble proteins were resolved on a 12% polyacrylamide gel (Bio-Rad, Hercules, CA) and blotted onto PVDF membranes (Bio-Rad). They were then exposed to Tris-buffer saline containing 0.1% Tween-20, 5% non-fat milk, and 0.5% goat serum to minimize nonspecific binding ofthe relevant antibody. Treated lysates were exposed a 5000-fold dilution ofa stock solution of mouse anti-V5 antibody or rabbit anti-mRasGRP4 antibody in Tris- buffered saline and 0.1% Tween-20, followed by horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibody (Bio-Rad). Immunoreactive proteins were. detected using BioMax MR film (Eastman Kodak) and chemiluminescence kits from Pierce (Rockford, IL).

Subcellular fractionation studies were carried out to locate where recombinant mRasGRP4 resides in the COS-7 cell transfectants. For these experiments, 1 x 10⁷ transfectants were washed three times with Hanks-buffered salt solution, suspended in 1.4 ml of MIC buffer (4 mM HEPES, pH 7.0, containing 50 mM sucrose, 0.4 mM EDTA, and 0.2 mM dithiothreitol), and sonicated on ice. The resulting lysates were centrifuged at 4°C for 10 min at -1,500 x g and then at -10,000 x g for another 10 min in order to remove nuclei and other organelles. The soluble fraction at this step was centrifuged for an additional 30 min at -100,000 x g and the resulting supernatant (cytosolic fraction) was subjected to SDS- PAGE/immunoblot analysis. The resulting plasma membrane-enriched, microsomal fraction was washed once with MIC buffer and then placed in 1 ml of MIC buffer supplemented with 10%) deoxycholic acid, 10% Triton X-100, and 30% glycerol. After a 30-min incubation at 4°C, the detergent exfracted membrane fraction was centrifuged at -100,000 x g for another 30 min. A sample of this supernatant (detergent-extracted microsomal fraction) also was subjected to SDS-PAGE/immunoblot analysis.

In order to evaluate its transforming activity, mRasGRP4^" and mRasGRP4^"1" fibroblasts were placed on top of 11-nιm glass coverslips in 24- well culture dishes. The attached cells were then exposed to 10 nM PMA (Calbiochem, La Jolla, CA) for 15-30 min at 37°C. After the coverslips were washed, they were incubated for 10 min in PBS supplemented with 4% paraformaldehyde. The fixative was removed, the treated cells were immersed in -20°C methanol for 10 min, rinsed in PBS, and incubated in 5% normal horse serum in PBS for 30 min before the addition of mouse anti-V5 antibody (Invifrogen, Carlsbad, CA) and rabbit anti-actin antibody (Sigma, St. Louis, MO). The latter antibody recognizes the common C-terminal domain ofthe varied isoforms of actin. Anti-V5 antibody was used to identify epitope tag mRasGRP4. Cells were incubated with a 100-500-fold dilution of each primary antibody for 2 h, washed several times in PBS, and then exposed for 1 h to the relevant secondary antibodies (a 200-fold dilution of Cy2-anti-mouse antibody and a 2,000-fold dilution of Cy3-anti-rabbit antibody; Jackson ImmunoResearch, West Grove, PA) in blocking solution supplemented with Hoechst dye 33258 at 50 ng/ml (Sigma). Stained cells were washed extensively with PBS and mounted. The resulting cells were viewed using a Nikon Eclipse 800 microscope; images were digitally captured using a CCD- SPOT RT digital camera and compiled using Adobe Photoshop^® software (v5.5).

Guanine nucleotide exchange assay. ProBond™ nickel-chelating resin (Invitrogen) was used to purify 6xHis-tagged recombinant mRasGRP4 from the transfectants. As recommended by the manufacturer, -1 x 10⁷ transfectants were placed in 4 ml of 20 mM sodium phosphate, pH 7.4, buffer containing 500 mM sodium chloride and multiple protease inhibitors (Roche Diagnostics, Indianapolis, IN). Each cell suspension was lysed by two freeze-thaw cycles using liquid nitrogen and a 42°C water bath. Liberated nuclear DNA was sheared by passing the resulting preparation through an 18-gauge needle four times, and then the cellular debris was removed by a 5-min centrifugation step at 4°C and at -14,000 x g. The resulting mRasGRP4-enriched supernatant was incubated with nickel-charged agarose resin for 1 h at 4°C to ensure efficient binding ofthe 6xHis-tagged recombinant protein. After the equilibration step, the resin was centrifuged in a Spin column at 800 x g for 2 min. The non-bound material was discarded and the column was washed extensively with 500 mM sodium chloride and 20 mM sodium phosphate, pH 6.0, to remove weakly associated protein. Five ml of 50 mM imidazole elution buffer was applied, and the resulting mRasGRP4- enriched eluate was concentrated to -0.5 ml with a Centriplus-50 (Millipore, Bedford, MA) filtering device having a 50-kDa cut-off membrane. A modification ofthe guanine nucleotide exchange assay described by Zheng and coworkers (Zheng, Y. et al., Methods Enzymol. 256:77-84, 1995) was used to evaluate the function of mRasGRP4. In this assay, 2 μg of recombinant human H-Ras (Oxford Biomedical Research, Oxford, MI), RhoA, Racl, Ran, Rab3A, or Cdc42 (Calbiochem), was placed in 60 μl of 100 mM NaCl, 2 mM EDTA, 0.2 mM dithiothreitol, 20 mM Tris-HCI, pH 8.0, supplemented with 100 μM adenylyl-imidodiphosphate tetralithium salt (AMP-PNP; Roche Diagnostics) and 10 μM GDP (Sigma). After a 5-min incubation at room temperature, MgCl₂ was added to achieve a final concentration of 5 mM. The solution was then incubated for another 15 min at room temperature to load H-Ras and its varied family members with non-radiolabeled GDP. Twenty μl ofthe resulting solution was added to 75 μl of reaction buffer [100 mM NaCl, 10 mM MgCl₂, 20 mM Tris-HCI, pH 8.0, supplemented with 100 μM AMP-PNP, 0.5 mg/ml bovine serum albumin, and 2 μM guanosine 5'-(γ-³⁵S)triphosphate (-11,000 cpm/pmol; Amersham Pharmacia Biotech, Piscataway, NJ)], followed by 5 μl of purified recombinant mRasGRP4 in the above buffer. The resulting samples were incubated at room temperature generally for 20 min. In the kinetic study with H-Ras, 15-μl aliquots were removed every 5 min and placed in 4 ml of ice-cold termination buffer (100 mM NaCl, 10 mM MgCl₂, and 20 mM Tris-HCI, pH 8.0). Non-bound radioactivity was removed by filtering each reaction mixture through a 25-mm cellulose nitrate membrane possessing 0.45- μm pores (Whatman, Maidstone, England). After each filter was washed with 10 ml of ice- cold termination buffer, it was placed in 5 ml of Filtron-X scintillation fluid (National Dignostics, Atlanta, GA) and the amount of bound radioactivity was quantitated using a Beckman LS5801 machine. For a negative confrol, purified recombinant mRasGRP4 was sequentially boiled for 5 min and then sonicated for 5 min; respectively; the enzymatic activity ofthe denatured protein was hen assessed.

The ability of CaCl₂ (0.2-10 mM), PMA (3-200 μM) (Calbiochem), and affinity- purified rabbit peptide antibodies directed against mRasGRP4 (10 μg) or an inelevant protein (human eosinophil serine protease 1) (10 μg) to inhibit the guanine nucleotide exchange activity of recombinant mRasGRP4 was also evaluated. In these competition assays, recombinant H-Ras was preloaded with GDP. CaCl₂, PMA, or antibody was then added, followed by recombinant mRasGRP4 and radiolabeled GTP. The reaction mixtures were then incubated 20 min at room temperature.

Comparative protein structure modeling. 3D models of residues 34-445 and 541-597 of mRasGRP4 were built using MODELLER. (Sali, A. et al., J. Mol. Biol. 234:779-815, 1993; Sali, A. et al., Protein Sci. 3:1582-1596, 1994). Residues 34-445 contain the REM and CDC25-like catalytic domains and conespond to the initial 61% ofthe translated protein; this portion of mRasGRP4 was modeled based on the crystallographic structure of hSosl (residues 568-1032 of GenBank accession number A37488) complexed to H-Ras (Protein Data Bank code 1BKD). (Boriak-Sjodin, P.A. et al., Nature 394:337-343, 1998). Residues 541-597 in mRasGRP4 conespond to its putative DAG-binding domain; this portion ofthe GEF was modeled based on the NMR structure ofthe Cys2 domain in rat protein kinase C-γ (Protein Data Bank code ITBN). Xu, R.X. et al., Biochemistry 36:10709-10717, 1997). The regions are -20% and -33% identical to the conesponding regions in Sosl and protein kinase C-γ, respectively.

Results

Cloning of the mRasGRP4 transcript and chromosomal location of its gene. Sequence analysis of >2000 clones from a BALB/c niBMMC cDNA library resulted in the identification of a clone that contained a 2.1-kb insert whose nucleotide sequence did not match any sequence in GenBank's genome and EST databases. A search ofa commercial mouse genome database with the resulting product also failed to reveal any intact, completely sequenced gene or transcript that conesponded to the new mBMMC cDNA. The fact that the unknown transcript had not been identified in another cell type, coupled with the finding that it was a low abundant transcript in mBMMCs, raised the possibility that it encoded a novel MC-restricted regulatory protein. Thus, a 5' RACE approach was carried out to deduce the nucleotide sequence ofthe missing 227-bp portion ofthe full-length, ~2.3-kb cDNA (Fig. 8). The GenBank accession number for mRasGRP4 is AF331457.

An alternate form ofthe transcript also was identified in mBMMCs that lacks the 15- nucleotide sequence that encodes the "VSTGP" sequence (SEQ DD NO: 56) in the protein's DAG-binding domain at residues 561-565. The initial 163 nucleotides in the depicted cDNA conespond to the 5' untranslated region and the first eight amino acids in the translated product. Whether or not the 5' untranslated region is derived from multiple exons remains to be determined. However, preliminary analysis ofthe mRasGRP4 gene indicates that the 3' end ofthe first coding exon conesponds to residues 118-140 in the depicted cDNA. The subsequent 16 exons conespond to residues 164-348, 349-454, 455-517, 518-649, 650-803, 804-977, 978-1094, 1095-1370, 1371-1451, 1452-1556, 1557-1675, 1676-1835, 1836-1872, 1873-2007, 2008-2117, and 2118-2294, respectively. The mRasGRP4 cDNA lacks a classical "AAT AAA" or "ATT AAA" polyadenylation regulatory site 10-30 residues upstream of its 3' poly(A) tract. Nevertheless, because "AAAAAA" has weak polyadenylation promoting activity, (Wickens, M., Trends Biochem. Sci. 15:277-281, 1990), it is presumed that nucleotides 2272-2277 confrol the polyadenylation ofthe mRasGRP4 transcript.

Preliminary analysis of its gene revealed that the coding portion ofthe mRasGRP4 transcript is derived from 17 exons. The mRasGRPl, mRasGRP2, and mRasGRP3 genes have not been sequenced. Nevertheless, the finding that the exon/intron organizations ofthe hRasGRP2 and mRasGRP4 genes are somewhat similar supports the nucleotide sequence data (Fig. 8) indicating that mRasGRP4 is a new member ofthe RasGRP family of GEFs. The initial fluorescent in situ hybridization (FISH) analysis showed that the mRasGRP4 gene resides on the proximal region of a medium-sized chromosome believed to be chromosome 7. Based on this finding, a second experiment was conducted in which a probe specific for the telomeric region of mouse chromosome 7 was co-hybridized with the mRasGRP4-containing genomic clone F1231. This experiment resulted in the specific labeling ofthe telomere and the proximal portion of chromosome 7 with the two probes (Fig. 9). Measurements of 10 specifically labeled chromosomes 7 demonstrated that the mRasGRP4 gene is located at a position that is 17% ofthe distance from the heterochromatic-euchromatic boundary to the telomere ofthe chromosome. This area conesponds to the interface between bands 7B4-7B5. When 80 metaphase cells were analyzed, 71 exhibited specific labeling.

Expression of mRasGRP4 at the mRNA and protein levels. RT-PCR (Fig. 10), RNA blot (Fig. 11), and immunohistochemical (Fig. 12) data revealed that the expression of mRasGRP4 is highly restricted to mature mast cells and their immature progenitors. The mRasGRPl and mRasGRP2 transcripts are present in mouse brain (Fig. 10a). However, using a similar RT-PCR approach, the levels ofthe mRasGRPl and mRasGRP2 transcripts were found to be below detection in BADB/c and C57BL/6 mBMMCs (Fig. 10A). Based on these data, mRasGRP4 is not coordinately expressed with the other members of its family at the mRNA level. Although the mRasGRP4 transcript was initially cloned from BALB/c mBMMCs (Fig. 8), the strain-dependent expression of granule proteases in mouse mast cells raised the possibility that the transcript had not been cloned earlier because mRasGRP4 is selectively expressed in the BALB/c mouse strain. Thus, C57BL/6 mBMMCs and Swiss Webster mouse embryos were examined. The presence ofthe appropriate sized RT-PCR product in C57BL/6 mBMMCs (Fig. 10A) and in the day 11-17 Swiss Webster mouse embryos (Fig. 10B) now indicates that n RasGRP4 is not restricted to the BALB/c mouse. When a blot containing RNA isolated from varied tissues was probed under conditions of high stringency, the level of mRasGRP4 mRNA was below detection in all examined tissues. These data are consistent with the mBMMC cDNA library data that indicated the steady-state level ofthe mRasGRP4 transcript in mast cells is substantially lower than that ofthe transcripts that encode its granule proteases. The data also are consistent with the fact that no mRasGRP4-related EST had been deposited earlier in varied databases. Nevertheless, using a more sensitive RT-PCR approach, low levels ofthe mRasGRP4 transcript were detected in many tissues and in a peritoneal cavity exudate (Fig. 10B). While the levels ofthe mRasGRP4 transcript were almost below detection in day-7 mouse embryos, this transcript was readily detected in mouse embryos after day-11. The liver and kidney ofthe adult mouse contain considerably fewer mast cells than heart, lung, or spleen. Toluidine blue^"1" mast cells also are more prominent in day-11 to day- 14 mouse embryos than day-7 embryos. Thus, the presence ofthe mRasGRP4 transcript in both embryos and adult tissues is directly conelated with mast cell numbers.

At the cellular level, the mRasGRP4 transcript was detected in mBMMCs, two well- described mouse mast cell lines, and a well-described rat mast cell line (Fig. 11 A). In contrast, the levels ofthe ~2.3-kb transcript were below detection by blot analysis in two myelomonocytic/macrophage cell lines, a fibroblast line, and a T cell hybridoma. The fact that the steady-state level ofthe mRasGRP4 transcript was actually higher in primary mBMMCs than in the three less mature mast cell lines supported the above RT-PCR data which had indicated that mRasGRP4 is expressed at virtually all stages of mast cell development. Subsequent kinetic analysis ofthe mRasGRP4 transcript levels in the mBMMC cultures (Fig. IIB) and immunohistochemistry analysis of tissue-residing mature mast cells (Fig. 12) confirmed this conclusion. mRasGRP4 mRNA was readily detected at the 2-week time point before most ofthe mast cell-committed progenitors in the cultures had granulated (Fig. 1 IB). However, the steady-state levels ofthe mRasGRP4 transcript did not decrease during the subsequent 5 weeks of culture. While the mRasGRP4 cDNA was initially isolated from an IL-3-dependent population of non-transformed mast cells, the mRasGRP4 transcript was detected in three different mast cell lines that are not dependent on IL-3 for their viability. The level ofthe mRasGRP4 transcript also was below detection in the IL-3 -dependent WEHI-3 myelomonocytic cell line (Fig. 11 A).

At the protein level, the endogenous in vivo-differentiated mature mast cells that reside in the tongue (Figs. 12A and 12B), skin (Figs. 12C and 12D), and peritoneal cavity (Figs. 12E and 12F) ofthe BALB/c mouse contain much higher levels of mRasGRP4 protein than in vitro-differentiated BALB/c mBMMC. The exogenous v-αW-immortalized MCs that develop in the spleen ofthe 2-week V3-mastocytosis mouse also contain substantial amounts of nιRasGRP4 protein (Fig. 12G and 12H). In the latter cell population, mRasGRP4 resides either in the cytoplasm or plasma membrane (Fig. 121).

Evaluation of the structure of mRasGRP4. An analysis ofthe nucleotide sequence ofthe isolated mBMMC cDNA (Fig. 8) predicted that mRasGRP4 exists in vivo as a 678-residue, -75 kDa protein. SDS-PAGE/immunoblot analysis ofthe heart and peritoneal cavity cellular exudates (Fig. 13) confirmed that the native protein is -75 kDa. mRasGRP4 has a <50% amino acid sequence identity with mouse, rat, and human RasGRP 1 , RasGRP2, and

RasGRP3 (Fig. 14). As found for other cytosolic proteins, mRasGRP4 lacks a hydrophobic signal peptide at its N terminus. A Kyte-Doolittle hydropathy analysis also reveal no extended hydrophobic domain in the protein's primary amino acid sequence. Immunoreactive mRasGRP4 was not constitutively secreted from the COS-7 cell and fibroblast transfectants, and immunoreactive mRasGRP4 was not detected in extracellular matrices adjacent to tissue mast cells (Fig. 12). Thus, mRasGRP4 is not constitutively secreted in vivo.

A dendrogram comparison ofthe nine most related mouse proteins in this superfamily revealed that mRasGRP4 is slightly more similar to mRasGRP2 than to mRasGRPl (Fig. 14A). The amino acid sequence of mRasGRP3 has not been deposited in GenBank. Nevertheless, mRasGRP4 is only distantly related to hRasGRP3. mRasGRP4 also lacks the extended C-terminal domains present in mRasGRPl, rRasGRPl, hRasGRPl, and hRasGRP3 (Fig. 14B). The putative REM, CDC25 catalytic, Ca²⁺-binding EF hand, and phorbol ester/DAG-binding domains ofthe varied RasGRPs are highlighted in Figure 14B. All of these domains are present in mRasGRP4 at the expected locations in its primary amino acid sequence. The most conserved region is the CDC25-like catalytic domain. For example, residues 405-433 in the CDC25-like catalytic domains of mRasGRP4 and mRasGRPl differ only in two amino acid residues, and even these differences are minimal (i.e., Leu— Val and Phe— »Tyr). Shown in Figure 15A is a comparative 3D model of residues 34-445 of mRasGRP4 complexed to H-Ras. This model is based on the 3D structure of hSosl (residues 568-1032) complexed to H-Ras which was determined by X-ray crystallography (Tognon, CE. et al. Mol. Cell Biol. 18:6995-7008, 1998) (Protein Data Bank code 1BKD). Residues 34-445 of mRasGRP4 are predicted to be structured as two α helical domains containing 6 and 11 helices, respectively. Analogous to other GEFs, α helix 8 in the CDC25-like catalytic domain is predicted to physically interact with H-Ras. The residues involved in H-Ras interaction are generally conserved. For example, Leu⁹³⁸ and Glu⁹⁴² in hSosl (GenBank accession number A37488) are essential for the ability of this GEF to activate H-Ras. The conesponding residues in mRasGRP4 predicted to be necessary for its interaction with H-Ras are Val and Glu . The conservation ofthe hydrophobic and acidic amino acids in the model supports the biochemical data noted below documenting the activation of H-Ras by recombinant mRasGRP4.

The first EF hand in rRasGRPl functions as this protein's primary Ca²⁺-binding site (Ebinu, J.O. et al., Science 280:1082-1086, 1998) and the critical residues that form the

"regulatory EF hand" in rRasGRPl and other Ca²⁺-binding proteins (Rashidi, H.H. et al., J. Mol. Microbiol. Biotechnol. 1:175-182, 1999) are present in mRasGRP4 at the required locations. The phorbol ester/DAG-binding, Cl domain in protein kinase C is -50 residues in length and possesses the motif of HXι₂CX₂CX₁₃CX₂CX₄HX₂CX₇C (SEQ ED NO: 55) or HX₁₂CX₂CX₁₄CX₂CX₄HX₂CX₇C (SEQ ED NO:60), where H is His, C is Cys and X is any other amino acid residue. The putative DAG-binding domain encoded by the first mRasGRP4 transcript cloned from BALB/c mBMMCs is 55 residues long due to an additional 5 residues in the middle ofthe least conserved portion ofthe Cl-like domain. Analysis ofthe exon intron organization ofthe mRasGRP4 gene revealed that the nucleotides encoding the "VSTGP" peptide sequence (SEQ DD NO: 56) in this domain reside at the very end ofthe gene's 13^th coding exon (Fig. 8). Introns generally begin with the "GT" sequence. The finding of an additional "GT" sequence 15 nucleotides upstream ofthe 3' end of exon 13 raised the possibility that this region ofthe mRasGRP4 transcript is alternately spliced in mast cells. Using an RT-PCR/sequencing approach, a second form ofthe mRasGRP4 transcript was subsequently identified that encoded a 50-residue DAG-binding domain lacking the "VSTGP" sequence (SEQ DD NO: 56). Ifthe "VSTGP" sequence (SEQ DD NO: 56) is removed, the critical His and Cys residues are present in mRasGRP4 precisely at the expected locations for a typical DAG receptor. Moreover, these His and Cys residues are 100% conserved when comparing mRasGRP4 (SEQ DD NO: 7), mRasGRPl (SEQ DD NO: 49), rRasGRPl (SEQ DD NO: 50), hRasGRPl (SEQ DD NO: 51), mRasGRP2 (SEQ DD NO: 52), hRasGRP2 (SEQ DD NO: 53), and hRasGRP3 (SEQ DD NO: 54), (Fig. 14B). The predicted 3D structures ofthe DAG-binding domain of nιRasGRP4 with and without the 5- residue insertion are shown in Figures 15B and 15C, respectively. Because the DAG-binding motif has a similar structure in these models, the 5-residue peptide loop may not predicted to interfere with DAG-binding.

Guanine nucleotide releasing activity of recombinant mRasGRP4. Analysis of its cDNA predicted that mRasGRP4 contains a phorbol ester/DAG-binding domain. As noted in Figure 16, mRasGRP4-expressing fibroblasts undergo dramatic moφhologic changes when exposed to low levels of PMA for only 15 min. This finding indicates that mRasGRP4 has transforming-like activity in cells. Moreover, mRasGRP4 is biologically active inside mammalian cells that normally do not express this protein. Based on these data, mRasGRP4 also was expressed in COS-7 cells and the affinity-purified recombinant protein from both cell populations was evaluated for its ability to activate varied members ofthe Ras superfamily. A bioengineered form of mRasGRP4 was expressed in COS-7 cells and fibroblasts that contained the V5 and 6xHis peptides at its C terminus. Although substantial amounts of immunoreactive mRasGRP4 were always recovered in the soluble cytosolic portion ofthe lysates of COS-7 cells and fibroblasts, a portion ofthe recombinant protein was consistently recovered in the microsomal fractions. Thus, like in mast cells (Fig. 121), mRasGRP4 does not reside in a single intracellular compartment in either transfectant.

Because MCs express H-Ras (Graham, T.E. et al., J. Immunol. 161:6733-6744, 1998) and because the 3D model of residues 34-445 of mRasGRP4 predicts that this mast cell- restricted protein can bind H-Ras (Fig. 15 A), we evaluated whether or not recombinant mRasGRP4 could activate this Ras isoform in vitro. As noted in Figure 17 A, purified recombinant nιRasGRP4, generated in either COS-7 cells or fibroblasts, was able to transfer radiolabeled GTP to H-Ras in a kinetic manner. Subsequent specificity studies revealed that mRasGRP4 also can activate human RhoA, Cdc42, Racl, Rab3A, and Ran in vitro (Fig.

17B). The fact that the anti-mRasGRP4 antibody can inhibit the transfer of GTP to H-Ras in this functional assay (Fig. 17C) confirmed that the guanine nucleotide exchange activities of the varied protein preparations originate from recombinant mRasGRP4 rather than a GEF contaminant constitutively produced by COS-7 cells and fibroblasts. mRasGRP4 has a putative Ca²⁺-binding domain analogous to the other two members of its family (Fig. 14B) and mM concentrations of Ca²⁺ (n = 3) dominantly inhibited the ability of recombinant mRasGRP4 to transfer GTP to GDP-loaded H-Ras even if 5 mM Mg²⁺ was present in the reaction buffer (Fig. 17D).

Example 3

Methods and Results Comparative transcript analysis of RasGRP4^" and RasGRP4⁺ HMC-1 cells.

Comparative transcript analysis of RasGRP4^" and RasGRP4⁺ HMC01 cells was done using an Affymetrix GeneChip approach. The analysis revealed a dramatic difference in the steady-state levels ofthe transcript that encodes hematopoietic-type, prostaglandin (PG) D₂ synthase (Table 2). While the levels of thromboxane A synthase and 15-lipoxygenase increased 4.5- and 6.2-fold, respectively, in the RasGRP4⁺ HMC-1 cells, the levels of PGD synthase was >100 fold higher in these cells relative to HMC-1 cells that express nonfunctional forms ofthe signaling protein. The HG-U95A GeneChip used in this transcript analysis contains -12,600 probe sets. No human transcript was induced to a level comparable to that ofthe PGD synthase transcript. Table 2 shows the transcript data relating to varied proteins that participate in arachidonic acid metabolism.

Table 2. Comparative expression of 19 transcripts that encode a number of proteins that participate in arachidonic acid metabolism in cells

Gene and its GenBank accession number Affymetrix Fold

ID number Change*

PGD₂ synthase, hematopoietic (AFl 50241) 35523 at 107.0

PGD₂ synthase, brain (M98539) 216 at 1.1

PGE synthase 1 (AF010316) 38131 at 1.8

PGF synthase/AKRlC3 (D 17793) 37399 at 1.4

PGIHprostacylin synthase (D38145) 759 at 1.0

15-hydroxyprostaglandin dehydrogenase, NAD+-dependent (X82460) 37322 s at 1.4

PGH₂ synthase 1/COXl (M59979) 37969 at 1.3

PGH₂ synthase 2/COX2 (U04636) 1069 at 1.4

5-lipoxygenase (J03600) 307 at 1.2

5-lipoxygenase-activating protein/FLAP (AI806222) 37099 at 0.9

12-lipoxygenase, 12R type (AF038461) 33029 at 0.9 12-lipoxygenase (M62982) 35124 at 1.8

15-lipoxygenase, type 1 (M23892) 34636 at 6.2

15-lipoxygenase, type 2 (U78294) 37430 at 3.4

LTB₄ hydroxylase (D12620) 1305 s at 1.0

LTA_t hydrolase (J03459) 38081 at 0.8

LTC₄ synthase (U50136) 39968 at 1.2

ATP-binding cassette, subfamily C (CFTR/MRP), member 1 (L05628) 11889966_ ss_ aatt 0.9

Thromboxane A synthase 1 (D34625) 33777 at 4.5

*Fold change in mRasGRP4⁺ HMC-1 cells relative to mRasGRJ ^" HMC-1 cells. Data were normalized to the ubiquitously expressed transcripts that encode β-actin and glyceraldehyde-3-phosphate dehydrogenase.

The above-described Affymetrix GeneChip data revealed that RasGRP4 preferentially induces an immature mast cell line to dramatically increase its expression ofthe transcript that encodes PGD₂ synthase. The level of a transcript in a cell sometimes is not reflective of its protein level. Thus, a SDS-PAGE/immunoblot was used to determine whether or not increased levels ofthe PGD₂ synthase transcript in HMC-1 cells results in a comparable increase in the levels of its protein.

PGD₂ synthase protein levels in RasGRP4^" and RasGRP4⁺ HMC-1 cells. Using a transfection approach, HMC-1 cells were induced to express a functional form of RasGRP4. The proteins present in the lysates of 10⁵ starting cells (shown in Fig. 20, middle lane) and 10⁵ transfectants (Fig. 20, right lane) were separated by SDS-PAGE and blotted onto a nifrocellulose membrane. The resulting blot was probed with anti-PGD₂ synthase antibody to detect the presence ofthe appropriately sized protein. Human recombinant PGD₂ synthase (Fig. 20, left lane) was used as a positive control in this SDS/PAGE immunoblot analysis. As noted in Fig. 20, RasGRP4-expressing HMC-1 cells dramatically increase their intracellulatr levels of PGD₂ synthase. No change was noted in the intracellular levels of a number of other proteins (e.g. PGE₂ synthase, PG endoperoxide H synthase 1, and 5-lipoxygenase) that participate in arachidonic acid metabolism.

Calcium ionophore-induced expression of PGD₂ in RasGRP4^" and RasGRP4⁺ HMC-1.

Washed HMC-1 cells (kindly provided by Dr. J. Butterfield, Mayo Clinic, Rochester, MN) and their RasGRP4 expressing transfectants (refened to herein as RasGRP4^" HMC-1 cells and RasGrRP4⁺ HMC-1 cells) were separately resuspended in Ca²⁺- and Mg²⁺-free phosphate buffered saline at a density of 10⁶ cells/ml. Calcium ionophore A23187 (0.5 μM) was added with or without 0.1 μM PMA. After a 30-min incubation at 37°C, the level of arachonidate metabolite PGD₂ in the conditioned media ofthe resulting four populations of cells was determined using a commercial ELISA kit. Significantly more PGD₂ release was found in the RasGRP4⁺ HMC-1 cells and the HMC-1 cells. The results are shown in Fig. 21 and the depicted data are the mean + range (p<0.05) of an experiment carried out in duplicate. RasGRP4⁺ HMC-1 cells are also refened to herein as HMC-1 /RasGRP4 cells. As noted in Fig. 21, RasGRP4-expressing HMC-1 cells dramatically increase their release of PGD₂ when these cells are calcium ionophore activated. No change was noted in the calcium ionophore- mediated production of PGE₂ and LTC₄.

Table 1

HRasGRP4

G50885.1, G25114.1, G22135.1, L30455.1, G57107.1, AL117285.1, G06579.1, AU049456.1, AE003452.2, AE003159.1, AE003622.3, AE003620.2, AE003609.1, AE003451.2, AE003438.2, AE003496.2, AE003488.1, AE003548.2, AE003793.2, AE003828.2, AE003587.2, AE003790.1, AE003789.2, AE003457.1, AE003568.2, AE003732.1, AE003706.2, AE003694.2, lGDX, 1DBM, IIBL, IIBK, IHNZ, IHNX, IHNW, 1FJG, 1HR0, 1FJF, 1C04, 430D, AC011469.6, AE004665.1, Z25465.1, NM_011242.1, U78171.1, AF081193.1, XM_015549.1, AE005906.1, AC005840.2, AC007240.2, AD132825.35, AC006064.9, AC007011.1, X06997.1, XM _042166.1, AC078911.il, AD162742.9, AL354923.12, AL031297.4, Yl 1467.1, AK022784.1, AK022617.1, AP001629.1, AP001748.1, D17217.1, D31124.1, BG031991.1, BF076966.1, BI298749.1, BF559384.1, BE101201.1, AW921088.1, AI408886.1, AI129481.1, AI070599.1, AI012611.1, BG988798.1, BF463869.1, AU152195.1, AU148850.1, AU148698.1, AV691390.1, BE755604.1, BE750976.1, BB481529.1, BB387956.1, BB381062.1, BB361063.1, BB335279.1, BB333601.1, BE233334.1, BE233330.1, BB310152.1, BE295367.1, BB236315.1, BB233733.1, BE278604.1, BB075675.1, AW212607.1, AV326972.1, AV325152.1, AI839459.1, AI671194.1, AI655221.1, AI651150.1, AI636517.1, AI635558.1, AI482407.1, AI435483.1, AI420003.1, AI142742.1, AI034182.1, AA737376.1, AA268607.1, AA181767.1, W95057.1, H79509.1, H79496.1, H68087.1, H47238.1, R82671.1, R67046.1, R34308.1, AX061208.1, AX135128.1, A86031.1, E66049.1, AR091275.1, AX049445.1, AX049443.1, AX049441.1, AX041008.1, AX041006.1, AX019488.1, BI298749.1, AC055840.5, AC092741.2, AC023903.8, AD450322.16, XM_042166.1, AC022315.32, AC092250.1, AC092285.1, AC092411.1, AD589943.12, AL160289.10, AL360004.21, AL391863.20, BI180201.1, XM_007711.3, XMJB8422.1, XM_028795.1, XM_004481.4, XM_003266.4, XM_041505.1, XM_041506.1, AL590378.5

mRasGRP4

XM_045647.1, XM W5646.1, XM_006536.2, XM_045645.1, NM_005825.1, AF043723.1, AF043722.1, AC000134.14, U78170.1, AF081194.1, Y12336.1, AC005284.1, AE007255.1, AC013447.4, AC024730.7, AY003974.1, NM_011242.1, AC022517.1, AL355102.5, AL160192.3, Z21854.1, AL159156.15, U78171.1, AF081193.1, AL356473.il, X62479.1, J03174.1, AX059486.1, AX031123.1, 187082.1, A85775.1, 145588.1, E28979.1, E65793.1, E04514.1, BF052229.1, BI031621.1, BG980607.1, BF340308.1, BF338895.1, AA459793.1, H30343.1, BE099647.1, BE099358.1, AI407426.1, AA819180.1, AI136516.1, AI008461.1, AA891082.1, AW656756.1, AA956275.1, AI104438.1, lGDY, AE003772.1, AE003827.2, AE003678.2, AE003597.1, AE003106.2, AE003663.2, AE003636.2, AE003423.1, AE003508.2, AE003523.2, AE003549.2, AE003564.2, AE003839.2, AE003587.2, AE003477.2, AE003475.2, AE003745.2, AE003732.1, AE003713.2, AD592913.1, G56431.1, G52568.1, G30726.1, G35850.1, Z18552.1, BH116710.1, AC091082.2, XM 345647.1, XM_045646.1, XM_006536.2, XM_045645.1, AC068494.8, AL596125.4, AC015892.5, AC073151.7, AC087222.3, AC009532.8, AL513102.7, AL445070.14, AC079926.7, AL139406.14, AL136451.17 EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments ofthe invention described herein. Such equivalents are intended to be encompassed by the following claims.

All references, including patent documents, disclosed herein are incoφorated by reference in their entirety.

We claim:

Claims

1. An isolated nucleic acid molecule selected from the group consisting of:

(a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ DD NO: 1 and which code for a human RasGRP4 protein,

(b) deletions, additions and substitutions ofthe nucleic acid molecules of (a), which code for a human RasGRP4 protein,

2. The isolated nucleic acid molecule of claim 1, wherein the isolated nucleic acid molecule comprises SEQ DD NO: 1.

3. An isolated nucleic acid molecule selected from the group consisting of:

(a) a unique fragment ofthe nucleotide sequence set forth as nucleotides 1-2634 of SEQ DD NO: 1 between 12 and 2633 nucleotides in length, and

(b) complements of (a), wherein the unique fragments exclude nucleic acids consisting of nucleotide sequences that are contained within SEQ ED NO: 1, and that are known as ofthe filing date of this application.

4. An expression vector comprising the isolated nucleic acid molecule of any of claims 1-3 operably linked to a promoter.

5. A host cell transformed or transfected with the expression vector of claim 4.

6. The host cell of claim 5, wherein the host cell is a CD14⁺ cell.

7. The host cell of claim 5, wherein the host cell is a HMC-1 cell.

8. A transgenic non-human animal comprising the expression vector of claim 4.

9. A transgenic non-human animal of claim 8, that expresses a variable level of hRasGRP4.

10 The transgenic non-human animal of claim 8, wherein the animal is a transgenic V3- mastocytosis animal.

11. An isolated protein encoded by the isolated nucleic acid molecule of any of claims 1 - 3.

12. The isolated protein of claim 11 , wherein the isolated protein comprises the amino acid sequence of SEQ ID NO: 2 or SEQ DD NO: 21.

13. A binding polypeptide that selectively binds to the isolated protein of claim 11.

14. The binding polypeptide of claim 13, wherein the binding polypeptide is an antibody or an antigen-binding fragment thereof.

15. The binding polypeptide of claim 14, wherein the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated protein comprising an amino acid sequence of SEQ DD NO: 2 or SEQ DD NO:21.

16. The binding peptide of claim 14, wherein the binding polypeptide selectively binds to the polypeptide sequence set forth as SEQ DD NO: 21.

17. A composition comprising: an active agent selected from the group consisting of:

(a) the nucleic acid of any of claims 1-3,

(b) the protein encoded by the isolated nucleic acid molecule of any of claims 1-3, and

(c) the binding polypeptide of any of claims 13-16, and a pharmaceutically acceptable carrier.

18. A method for making a medicament, comprising: placing an active agent selected from the group consisting of: (a) the isolated nucleic acid molecules of any of claims 1-3, (b) the isolated protein of any of claims 11-12, and (c) the binding polypeptides of any of claims 13-16, in a pharmaceutically acceptable carrier.

19. The method of claim 18, wherein placing comprises placing a therapeutically effective amount ofthe active agent in the pharmaceutically acceptable carrier to form one or more doses.

20. A method of making a human mast cell line in vitro, comprising transfecting CD14⁺ cells with an isolated nucleic acid of claims 1-3, that encodes an hRasGRP4 protein.

21. The method of claim 20, wherein the isolated nucleic acid comprises the nucleic acid sequence set forth as SEQ DD NO: 1.

22. An isolated nucleic acid molecule selected from the group consisting of:

(a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ DD NO: 3, SEQ DD NO: 5, or SEQ ID NO:58 and which code for a Mutant human RasGRP4 protein, (b) deletions, additions and substitutions ofthe nucleic acid molecules of (a), which code for a Mutant human RasGRP4 protein,

(d) complements of (a), (b) or (c).

23. The isolated nucleic acid molecule of claim 22, wherein the isolated nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: SEQ ED NO: 3, SEQ DD NO: 5, and SEQ DD NO:58.

24. An isolated nucleic acid molecule selected from the group consisting of:

(a) a unique fragment ofthe nucleotide sequence set forth as nucleotides 1-2749 of SEQ DD NO: 3 between 12 and 2748 nucleotides in length, or set forth as nucleotides 1-2592 of SEQ ID NO: 5 between 12 and 2591 nucleotides in length, or set forth as nucleotides 1- 883 of SEQ DD NO:58 between 12 and 882 nucleotides in length, and (b) complements of (a), wherein the unique fragments exclude nucleic acids consisting of nucleotide sequences that are contained within SEQ DD NO: 3, SEQ ED NO: 5, or SEQ DD NO:58, and that are known as ofthe filing date of this application.

25. An expression vector comprising the isolated nucleic acid molecule of any of claims 22-24 operably linked to a promoter.

26. A host cell transformed or transfected with the expression vector of claim 25.

27. The host cell of claim 26, wherein the host cell is a CD14⁺ cell.

28. The host cell of claim 26, wherein the host cell is a HMC- 1 cell.

29. A transgenic non-human animal comprising the expression vector of claim 25.

30. A transgenic non-human animal of claim 29, that expresses a variable level of Mutant hRasGRP4.

31. The transgenic non-human animal of claim 29, wherein the animal is a transgenic V3- mastocytosis animal.

32. An isolated protein encoded by the isolated nucleic acid molecule of any of claims 22-

24.

33. The isolated protein of claim 32, wherein the isolated protein comprises an amino acid sequence selected from the group consisting of SEQ DD NO: 4, SEQ DD NO: 6, and SEQ DD NO:59.

34. A binding polypeptide that selectively binds to the isolated protein of claim 32.

35. The binding polypeptide of claim 34, wherein the binding polypeptide is an antibody or an antigen-binding fragment thereof.

36. The binding polypeptide of claim 35, wherein the antibody or antigen-binding fragment thereof specifically binds to the N-terminus ofthe isolated protein comprising an amino acid sequence of SEQ DD NO:4, SEQ DD NO:6, or SEQ DD NO:59.

37. A composition comprising: an active agent selected from the group consisting of: (a) the nucleic acid of any of claims 22-24,

(b) the protein encoded by the isolated nucleic acid molecule of any of claims 22-24, and

(c) the binding polypeptide of any of claims 34-36, and a pharmaceutically acceptable carrier.

38. A method for making a medicament, comprising: placing an active agent selected from the group consisting of: (a) the isolated nucleic acid molecules of any of claims 22-24, (b) the isolated protein of any of claims 32-33 , and (c) the binding polypeptides of any of claims 34-36, in a pharmaceutically acceptable carrier.

39. The method of claim 38, wherein placing comprises placing a therapeutically effective amount ofthe active agent in the pharmaceutically acceptable carrier to form one or more doses.

40. A method for diagnosing a disorder characterized by abenant expression of an hRasGRP4 molecule, comprising: detecting in a first biological sample obtained from a subject, expression of an hRasGRP4 molecule or a Mutant hRasGRP4 molecule, wherein a difference in expression level ofthe hRasGRP4 molecule compared to an hRasGRP4 control or an increase in expression level ofthe Mutant hRasGRP4 molecule compared to a Mutant hRasGRP4 control indicates that the subject has a disorder characterized by abenant expression of an hRasGRP4 molecule.

41. The method of claim 40, further comprising the steps of: detecting in a second biological sample obtained from the subject, expression of an hRasGRP4 molecule or a Mutant hRasGRP4 molecule, and comparing the expression ofthe hRasGRP4 molecule or the Mutant hRasGRP4 molecule in the first biological sample and the second biological sample.

42. The method of claim 41, wherein the disorder is a bacterial infection, wherein an increase in expression level ofthe hRasGRP4 in the second biological sample compared to the expression level in the hRasGRP4 in the first biological sample indicates progression of the disorder characterized by abenant expression of hRasGRP4.

43. The method of claim 41, wherein the disorder is a bacterial infection, wherein a decrease in expression level ofthe hRasGRP4 in the second biological sample compared to the expression level in the hRasGRP4 in the first biological sample indicates regression of the disorder characterized by abenant expression of hRasGRP4.

44. The method of claim 41, wherein the disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS, and wherein an increase in expression level ofthe hRasGRP4 in the second biological sample compared to the expression level in the hRasGRP4 in the first biological sample indicates progression of the disorder characterized by abenant expression of hRasGRP4.

45. The method of claim 41 , wherein the disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS, and wherein a decrease in expression level ofthe hRasGRP4 in the second biological sample compared to the expression level in the hRasGRP4 in the first biological sample indicates regression ofthe disorder characterized by abenant expression of hRasGRP4.

46. The method of claim 41 , wherein the disorder is a bacterial infection, wherein an increase in expression level ofthe Mutant hRasGRP4 in the second biological sample compared to the expression level ofthe Mutant hRasGRP4 in the first biological sample indicates progression ofthe disorder characterized by abenant expression of hRasGRP4.

47. The method of claim 41 , wherein the disorder is a bacterial infection, wherein a decrease in expression level ofthe Mutant hRasGRP4 in the second biological sample compared to the expression level ofthe Mutant hRasGRP4 in the first biological sample indicates regression ofthe disorder characterized by abenant expression of hRasGRP4.

48. The method of claim 41 , wherein the disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS, and wherein an increase in expression level ofthe Mutant hRasGRP4 in the second biological sample compared to the expression level ofthe Mutant hRasGRP4 in the first biological sample indicates progression ofthe disorder characterized by abenant expression of hRasGRP4.

49. The method of claim 41, wherein the disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS, and wherein a decrease in expression level ofthe Mutant hRasGRP4 in the second biological sample compared to the expression level ofthe Mutant hRasGRP4 in the first biological sample indicates regression ofthe disorder characterized by abenant expression of hRasGRP4.

50. The method of claim 40, wherein the disorder characterized by abenant expression of an hRasGRP4 molecule is a bacterial infection.

51. The method of claim 40, wherein the mast cell disorder characterized by abenant expression of an hRasGRP4 molecule is a mast cell disorder.

52. The method of claim 51 , wherein the mast cell disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS.

53. The method of claim 51 , wherein the mast cell disorder is asthma.

54. The method of claim 51 , wherein the mast cell disorder is mastocytosis.

55. The method of claim 40, comprising detecting expression of an hRasGRP4 nucleic acid molecule.

56. The method of claim 55, wherein the hRasGRP4 nucleic acid molecule comprises the nucleotide sequence set forth as SEQ ED NO: 1.

57. The method of claim 40, comprising detecting expression of a Mutant hRasGRP4 nucleic acid molecule.

58. The method of claim 57, wherein the Mutant hRasGRP4 molecule is a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ DD NO: 3, SEQ ED NO: 5, and SEQ ED NO:58.

59. The method of claim 57, wherein the Mutant hRasGRP4 molecule is a nucleic acid comprising a nucleotide sequence set forth as SEQ ED NO: 1 with one or more nucleic acid additions, deletions, or substitutions which affect the functional activity ofthe hRasGRP4 nucleic acid molecule.

60. The method of claim 40, comprising detecting expression of an hRasGRP4 protein.

61. The method of claim 60, wherein the hRasGRP4 protein comprises a nucleotide sequence set forth as SEQ DD NO: 2.

62. The method of claim 40, comprising detecting expression of a Mutant hRasGRP4 protein.

63. The method of claim 62, wherein the Mutant hRasGRP4 protein comprises an amino acid sequence selected from the group consisting of SEQ DD NO: 4, SEQ DD NO: 6, and SEQ

DD NO:59.

64. The method of claim 62, wherein the Mutant hRasGRP4 protein comprises an amino acid sequence selected from the group consisting of SEQ DD NO: 2, SEQ ED NO: 4, SEQ ED NO: 6, and SEQ DD NO:59 with one or more amino acid additions, deletions, or substitutions which affect the functional activity of a hRasGRP4 protein.

65. The method of claim 40, wherein detecting comprises contacting the biological sample with an agent that selectively binds to the hRasGRP4 molecule or to the Mutant hRasGRP4 molecule.

66. The method of claim 65, wherein the hRasGRP4 molecule or the Mutant hRasGRP4 molecule is a nucleic acid and wherein the agent that selectively binds to the hRasGRP4 molecule or to the Mutant hRasGRP4 molecule is a nucleic acid that hybridizes to SEQ DD NO: 1 , SEQ DD NO: 3, SEQ DD NO: 5, or SEQ DD NO:58 under high stringency conditions.

67. The method of claim 65, wherein the hRasGRP4 molecule or the Mutant hRasGRP4 molecule is a protein and wherein the agent that selectively binds to the hRasGRP4 molecule or to the Mutant hRasGRP4 molecule comprises a binding polypeptide that selectively binds to an amino acid sequence selected from the group consisting of SEQ ED NO: 2, SEQ ED NO: 4., SEQ ED NO: 6, and SEQ ED NO:59.

68. The method of claim 40, wherein the biological sample is selected from the group consisting of: a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract.

69. A method for evaluating the effect of candidate pharmacological agents on the regulation of mast cells, comprising: administering a candidate pharmacological agent to a subject; determining the effect ofthe candidate phannacological agent on the expression level of hRasGRP4 relative to the expression level of hRasGRP4 in a subject to which no candidate pharmacological agent is administered, wherein a relative increase or relative decrease in the expression level of hRasGRE indicates regulation of mast cells by the candidate pharmacological agent.

70. A method for evaluating the effect of candidate pharmacological agents on the regulation of mast cells, comprising: contacting a candidate pharmacological agent with a mast cell sample; determining the effect ofthe candidate pharmacological agent on the expression level of hRasGRP4 relative to the expression level of hRasGRP4 in a mast cell sample not contacted with the candidate pharmacological agent, wherein a relative increase or relative decrease in the expression level of hRasGRP4 indicates regulation of mast cells by the candidate pharmacological agent.

71. The method of claim 70, wherein the mast cell sample comprises RasGRP4⁺ HMC- 1 cells.

72. A kit for diagnosing a disorder associated with abenant expression of an hRasGRP4 molecule, comprising: one or more nucleic acid molecules that hybridize to an hRasGRP4 nucleic acid molecule or to a Mutant hRasGRP4 nucleic acid molecule under high stringency conditions, and instructions for the use ofthe nucleic acid molecules, and control agents in the diagnosis of a disorder associated with abenant expression of an hRasGRP4 molecule.

73. The kit of claim 72, wherein the one or more nucleic acid molecules consist of a first primer and a second primer, wherein the first primer and the second primer are constructed and ananged to selectively amplify at least a portion of an isolated hRasGRP4 nucleic acid molecule comprising SEQ DD NO: 1 or an isolated Mutant hRasGRP4 nucleic acid molecule comprising SEQ DD NO: 3, SEQ DD NO: 5, or SEQ DD NO:58.

74. The kit of claim 72, wherein the nucleic acids and the one or more control agents are bound to a substrate.

75. A kit for diagnosing an hRasGRP4-associated disorder in a subject comprising: one or more binding polypeptides that selectively bind to an hRasGRP4 protein or a

Mutant hRasGRP4 protein, and instructions for the use ofthe binding polypeptides, and control agents in the diagnosis of a disorder associated with abenant expression of an hRasGRP4 molecule.

76. The kit of claim 75, wherein the one or more binding polypeptides are antibodies or antigen-binding fragments thereof.

77. The kit of claim 76, wherein the antibodies or antigen-binding fragments thereof and the one or more control agents are bound to a substrate.

78. The kit of claim 75, wherein the binding polypeptides bind to the Mutant hRasGRP4 protein but do not bind to the hRasGRP4 protein.

79. A method for treating a subject with a disorder characterized by decreased expression of an hRasGRP4 nucleic acid, comprising administering to the subject an effective amount of an hRasGRP4 nucleic acid molecule to treat the disorder.

80. A method for treating a subject with a disorder characterized by increased expression of an hRasGRP4 nucleic acid molecule, comprising administering to the subject an effective amount of an antisense or RNAi molecule to an hRasGRP4 nucleic acid molecule to treat the disorder.

81. A method for treating a subject with a disorder characterized by decreased expression of an hRasGRP4 protein, comprising administering to the subject an effective amount of an hRasGRP4 protein to freat the disorder.

82. A method for treating a subject with a disorder characterized by increased expression of an hRasGRP4 protein, comprising: administering to the subject an effective amount of a binding polypeptide to an hRasGRP4 protein to treat the disorder.

83. The method of claim 82, wherein the binding polypeptide agent is an antibody or an antigen-binding fragment thereof.

84. The method of claim 83, wherein the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated hRasGRP4 protein or Mutant hRasGRP4 protein.

85. The method of claim 83, wherein the antibodies or antigen-binding fragments are labeled with one or more cytotoxic agents.

86. A method for producing an hRasGRP4 protein comprising providing an isolated hRasGRP4 nucleic acid molecule operably linked to a promoter, wherein the hRasGRP4 nucleic acid molecule encodes the hRasGRP4 protein or a fragment thereof, and expressing the hRasGRP4 nucleic acid molecule in an expression system.

87. The method of claim 86, further comprising: isolating the hRasGRP4 protein or a fragment thereof from the expression system.

88. The method of claim 86, wherein the hRasGRP4 nucleic acid molecule comprises the nucleotide sequence set forth as SEQ DD NO: 1.

89. A method for producing a Mutant hRasGRP4 protein comprising providing an isolated Mutant hRasGRP4 nucleic acid molecule operably linked to a promoter, wherein the Mutant hRasGRP4 nucleic acid molecule encodes the Mutant hRasGRP4 protein or a fragment thereof, and expressing the Mutant hRasGRP4 nucleic acid molecule in an expression system.

90. The method of claim 89, further comprising isolating the Mutant hRasGRP4 protein or a fragment thereof from the expression system.

91. The method of claim 89, wherein the Mutant hRasGRP4 nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ DD NO: 3, SEQ ED NO: 5, and SEQ ED NO:58.

92. The method of claim 89, wherein the Mutant hRasGRP4 nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ ED NO: 1, SEQ DD NO: 3, SEQ DD NO: 5, and SEQ DD NO:58 with one or more point mutations or deletions to encode a Mutant hRasGRP4 protein.

93. An isolated nucleic acid molecule selected from the group consisting of: (a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ DD NO: 7 and SEQ ED NO: 9, and which codes for an mRasGRP4 protein,

(b) deletions, additions and substitutions ofthe nucleic acid molecules of (a), which code for an mRasGRP4 protein (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy ofthe genetic code, and (d) complements of (a), (b) or (c).

94. The isolated nucleic acid molecule of claim 93, wherein the isolated nucleic acid molecule comprises a nucleotide set forth as SEQ ED NO: 7.

95. An isolated nucleic acid molecule selected from the group consisting of: (a) a unique fragment ofthe nucleotide sequence set forth as nucleotides 1-2327 of SEQ ED NO: 7 between 12 and 2326 nucleotides in length, and

(b) complements of (a), wherein the unique fragments exclude nucleic acids consisting of nucleotide sequences that are contained within SEQ ED NO: 7 and that are known as ofthe filing date of this application.

96. An expression vector comprising the isolated nucleic acid molecule of any claims 93- 95 operably linked to a promoter.

97. A host cell transformed or transfected with the expression vector of claim 96.

98. The host cell of claim 97, wherein the host cell is a CD14⁺ cell.

99. The host cell of claim 97, wherein the host cell is a HMC-1 cell.

100. A transgenic non-human animal comprising the expression vector of claim 96.

101. A transgenic non-human animal of claim 100, that expresses a variable level of mRasGRP4 or Mutant mRasGRP4.

102. The transgenic non-human animal of claim 100, wherein the transgenic non-human animal is a transgenic V3 -mastocytosis animal.

103. An isolated protein encoded by the isolated nucleic acid molecule of any of claims 93- 95.

104. The isolated protein of claim 103, wherein the isolated protein comprises the amino acid sequence set forth as SEQ ED NO: 8.

105. A binding polypeptide that selectively binds to the isolated protein of claim 104.

106. The binding polypeptide of claim 105, wherein the binding polypeptide is an antibody or an antigen-binding fragment thereof.

107. The binding polypeptide of claim 106, wherein the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated protein comprising the amino acid sequence of SEQ DD NO:8.

108. An antigen used to generate the antibodies or antibody-binding fragments of claim 105, wherein the antigen comprises the amino acid sequence set forth as SEQ ED NO: 36.

109. An isolated nucleic acid molecule selected from the group consisting of:

(a) nucleic acid molecules which hybridize under high stringency conditions to a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ED NO: 9 and which code for a Mutant mRasGRP4 protein, (b) deletions, additions and substitutions ofthe nucleic acid molecules of (a), which code for a Mutant mRasGRP4 protein

(d) complements of (a), (b) or (c).

110. The isolated nucleic acid molecule of claim 109, wherein the isolated nucleic acid molecule comprises a nucleotide sequence set forth as SEQ ED NO: 9.

111. An isolated nucleic acid molecule selected from the group consisting of: (a) a unique fragment ofthe nucleotide sequence set forth as nucleotides 1-2312 of

SEQ BD NO: 9 between 12 and 2311 nucleotides in length, and

(b) complements of (a), wherein the unique fragments exclude nucleic acids consisting of nucleotide sequences that are contained within SEQ DD NO: 9, and that are known as ofthe filing date of this application.

112. An expression vector comprising the isolated nucleic acid molecule of any ofthe foregoing claims 109-111 operably linked to a promoter.

113. A host cell transformed or transfected with the expression vector of claim 112.

114. The host cell of claim 113, wherein the host cell is a CD14⁺ cell.'

115. The host cell of claim 113, wherein the host cell is a HMC-1 cell.

116. A transgenic non-human animal comprising the expression vector of claim 113.

117. A transgenic non-human animal of claim 116, that expresses a variable level of Mutant mRasGRP4.

118. The transgenic non-human animal of claim 116, wherein the transgenic non-human animal is a transgenic V3-mastocytosis animal.

119. An isolated protein encoded by the isolated nucleic acid molecule of any of claims 109-111.

120. The isolated protein of claim 119, wherein the isolated protein comprises the amino acid sequence set forth as SEQ ED NO: 10.

121. A binding polypeptide that selectively binds to the isolated protein of claim 119.

122. The binding polypeptide of claim 121, wherein the binding polypeptide is an antibody or an antigen-binding fragment thereof.

123. The binding polypeptide of claim 122, wherein the antibody or antigen-binding fragment thereof specifically binds to the N-terminus of the isolated protein comprising the amino acid sequence of SEQ DD NO: 10.

124. A method for preparing an animal model of a disorder characterized by abenant expression of an hRasGRP4 molecule, comprising:

(a) introducing into a non-human animal, a nιRasGRP4 molecule or a Mutant mRasGRP4 molecule; and

(b) detecting in a first biological sample obtained from the non-human animal, expression ofthe mRasGRP4 molecule or a Mutant n RasGRP4 molecule.

125. The method of claim 124, wherein the animal model is of a disorder that is a bacterial infection.

126. The method of claim 124, wherein the animal model is of a disorder selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS.

127. The method of claim 124, wherein the mRasGRP4 molecule is an mRasGRP4 nucleic acid molecule or a Mutant mRasGRP4 nucleic acid molecule.

128. The method of claim 124, wherein the mRasGRP4 molecule is an mRasGRP4 protein or a Mutant mRasGRP4 protein.

129. A method for evaluating the effect of candidate pharmacological agents on the regulation of mast cells, comprising: administering a candidate pharmacological agent to a subject; determining the effect ofthe candidate pharmacological agent on the expression level of mRasGRP4 relative to the expression level of mRasGRP4 in a subject to which no candidate pharmacological agent is administered, wherein a relative increase or relative decrease in the expression level of mRasGRP4 indicates regulation of mast cells by the candidate pharmacological agent.

130. A method for evaluating the effect of candidate pharmacological agents on the regulation of mast cells, comprising: contacting a candidate pharmacological agent with a mast cell sample; determining the effect ofthe candidate pharmacological agent on the expression level of mRasGRP4 relative to the expression level of nιRasGRP4 in a mast cell sample not contacted with a pharmacological agent, wherein a relative increase or relative decrease in the expression level of mRasGRP4 indicates regulation of cells by the candidate pharmacological agent.

131. A kit for preparing a non-human animal model of a disorder associated with abenant expression of an hRasGRP4 molecule, comprising: one or more nucleic acid molecules that hybridize to an mRasGRP4 nucleic acid molecule or to a Mutant mRasGRP4 nucleic acid molecule under high stringency conditions, and instructions for the use ofthe nucleic acid molecules, and control agents in the preparation of a non-human animal model of a disorder associated with abenant expression of an hRasGRP4 molecule.

132. The kit of claim 131, wherein the one or more nucleic acid molecules consist of a first primer and a second primer, wherein the first primer and the second primer are constructed and ananged to selectively amplify at least a portion of an isolated mRasGRP4 nucleic acid molecule comprising a nucleotide selected from the group consisting of SEQ DD NO: 7 and SEQ ED NO: 9.

133. The kit of claim 131, wherein the one or more nucleic acids or control agents are bound to a substrate.

134. A kit for preparing a non-human animal model of a disorder of a hRasGRP4- associated disorder in a subject comprising: one or more binding polypeptides that selectively bind to an mRasGRP4 protein or a Mutant n RasGRP4 protein, and instructions for the use ofthe binding polypeptides, and control agents in the preparation of a non-human animal model of a disorder associated with abenant expression of an hRasGRP4 molecule.

135. The kit of claim 134, wherein the one or more binding polypeptides are antibodies or antigen-binding fragments thereof.

136. The kit of claim 135, wherein the antibody or antigen-binding fragment specifically binds to the N-terminus of the isolated protein of an mRasGRP4 protein or a Mutant mRasGRP4 protein.

137. The kit of claim 135, wherein the antibodies or antigen-binding fragments thereof, or one or more control agents are bound to a subsfrate.

138. The kit of claim 134, wherein the binding polypeptides bind to the Mutant mRasGRP4 protein but do not bind to the mRasGRP4 protein.

139. A method for preparing a non-human animal model of a disorder characterized by abenant expression of an hRasGRP4 molecule, comprising administering to a non-human animal an effective amount of an antisense or RNAi molecule to a Mutant mRasGRP4 nucleic acid molecule or an anti-sense or RNAi molecule to a mRasGRP4 nucleic acid molecule to reduce expression ofthe Mutant mRasGRP4 nucleic acid molecule or the mRasGRP4 nucleic molecule in the non-human animal.

140. A method for preparing a non-human animal model of a disorder characterized by abenant expression of a RasGRP4 molecule, comprising administering to a non-human animal an effective amount of a binding polypeptide to a Mutant nιRasGRP4 protein or to a mRasGRP4 protein to reduce expression ofthe Mutant mRasGRP4 protein or ofthe mRasGRP4 protein in the non-human animal.

141. The method of claim 140, wherein the binding polypeptide agent is an antibody or an antigen-binding fragment thereof.

142. The method of claim 141, wherein the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated mRasGRP4 protein or Mutant mRasGRP4 protein.

143. The method of claim 141, wherein the antibodies or antigen-binding fragments are labeled with one or more cytotoxic agents.

144. A method for producing an mRasGRP4 protein comprising providing an isolated mRasGRP4 nucleic acid molecule operably linked to a promoter, wherein the mRasGRP4 nucleic acid molecule encodes the nιRasGRP4 protein or a fragment thereof, and expressing the mRasGRP4 nucleic acid molecule in an expression system.

145. The method of claim 144, further comprising; isolating the mRasGRP4 protein or a fragment thereof from the expression system.

146. The method of claim 144, wherein the mRasGRP4 nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of SEQ DD NO: 7 and SEQ ED NO: 9.

147. A method for producing a Mutant mRasGRP4 protein comprising providing an isolated Mutant nιRasGRP4 nucleic acid molecule operably linked to a promoter, wherein the Mutant mRasGRP4 nucleic acid molecule encodes the Mutant mRasGRP4 protein or a fragment thereof, and expressing the Mutant nιRasGRP4 nucleic acid molecule in an expression system.

148. The method of claim 147, further comprising: isolating the Mutant nιRasGRP4 protein or a fragment thereof from the expression system.

149. The method of claim 147, wherein the Mutant mRasGRP4 nucleic acid molecule comprises a nucleotide sequence set forth as SEQ DD NO: 9.

150. The method of claim 147, wherein the Mutant mRasGRP4 nucleic acid molecule comprises a nucleotide sequence set forth as SEQ DD NO: 7 with one or more point mutations or deletions to encode a Mutant mRasGRP4 protein.

151. A method for treating a subject with a disorder characterized by a decreased amount of a prostaglandin D₂ (PGD₂) molecule and/or its synthase, comprising admimstering to the subject an effective amount of a RasGRP4 molecule to treat the disorder.

152. The method of claim 151, wherein the RasGRP4 molecule is a RasGRP4 nucleic acid molecule.

153. The method of claim 151, wherein the RasGRP4 molecule is a RasGRP4 protein.

154. A method for treating a subject with a disorder characterized by an increased amount of a prostaglandin D₂ (PGD₂) molecule and/or its synthase, comprising administering to the subject an effective amount of an inhibitor of a RasGRP4 molecule to freat the disorder.

155. The method of claim 154, wherein the inhibitor of a RasGRP4 molecule is an antisense or RNAi molecule to a RasGRP4 nucleic acid molecule.

156. The method of claim 154, wherein the inhibitor of a RasGRP4 molecule is a binding polypeptide to a RasGRP4 protein.

157. The method of claim 156, wherein the binding polypeptide agent is an antibody or an antigen-binding fragment thereof.

158. The method of claim 157, wherein the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated RasGRP4 protein or the N terminus of a Mutant RasGRP4 protein.

159. The method of claim 158, wherein the antibodies or antigen-binding fragments are labeled with one or more cytotoxic agents.

160. A method for increasing the amount of a prostaglandin D₂ (PGD₂) molecule and/or its synthase in a tissue or cell, comprising increasing the amount of RasGRP4 in the tissue or cell.

161. A method for decreasing the amount of a prostaglandin D₂ (PGD₂) molecule and/or its synthase in a tissue or cell comprising inhibiting RasGRP4 activity in the tissue or cell.

162. The method of claim 161, wherein inhibiting RasGRP4 activity is interfering with RasGRP4 activity.

163. The method of claim 161, wherein inhibiting RasGRP4 activity is reducing the level of expression of RasGRP4.

164. A method for diagnosing a disorder characterized by abenant amounts ofa prostaglandin D₂ (PGD₂) molecule and/or its synthase, comprising: determining in a first biological sample obtained from a subject, an amount ofa RasGRP4 molecule, wherein a difference in the amount ofthe RasGRP4 molecule compared to a RasGRP4 control indicates that the subject has a disorder characterized by abenant amounts of a PGD₂ molecule and/or its synthase.

165. The method of claim 164, further comprising the steps of: detecting in a second biological sample obtained from the subject, an amount ofa RasGRP4 molecule, and comparing the amount ofthe RasGRP4 molecule in the first biological sample and the second biological sample.

166. The method of claim 164, wherein the disorder characterized by abenant expression of a prostaglandin D₂ (PGD₂) molecule and/or its synthase is a bacterial infection.

167. The method of claim 164, wherein the disorder characterized by abenant expression of a PGD₂ molecule and/or its synthase is a mast cell disorder.

168. The method of claim 167, wherein the mast cell disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS.

169. The method of claim 167, wherein the mast cell disorder is asthma.

170. The method of claim 167, wherein the mast cell disorder is mastocytosis.

171. The method of claim 164, wherein the biological sample is selected from the group consisting of: a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract.

172. A method for diagnosing a disorder characterized by abenant expression of a RasGRP4 molecule, comprising: determining in a first biological sample obtained from a subject, an amount of a prostaglandin D₂ (PGD₂) molecule and/or its synthase, wherein a difference in the amount ofthe PGD₂ molecule and/or its synthase compared to a PGD₂ and/or its synthase control indicates that the subject has a disorder characterized by abenant expression of a RasGRP4 molecule.

173. The method of claim 172, further comprising the steps of: detecting in a second biological sample obtained from the subject, an amount ofa prostaglandin D₂ (PGD₂) molecule and/or its synthase, and comparing the amount ofthe PGD₂ molecule and/or its synthase in the first biological sample and the second biological sample.

174. The method of claim 172, wherein the disorder characterized by abenant expression of a RasGRP4 molecule is a bacterial infection.

175. The method of claim 172, wherein the disorder characterized by abenant expression of a RasGRP4 molecule is a mast cell disorder.

176. The method of claim 175, wherein the mast cell disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS.

177. The method of claim 175, wherein the mast cell disorder is asthma.

178. The method of claim 175, wherein the mast cell disorder is mastocytosis.

179. The method of claim 172, wherein the biological sample is selected from the group consisting of: a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract.

180. A method for treating a subject with a disorder characterized by a decreased amount of a granule neutral protease, comprising administering to the subject an effective amount of a RasGRP4 molecule to treat the disorder.

181. The method of claim 180, wherein the RasGRP4 molecule is a RasGRP4 nucleic acid molecule.

182. The method of claim 180, wherein the RasGRP4 molecule is a RasGRP4 protein.

183. A method for treating a subj ect with a disorder characterized by increased amount ofa granule neutral protease, comprising administering to the subject an effective amount of an inhibitor of a RasGRP4 molecule to treat the disorder.

184. The method of claim 183, wherein the inhibitor of a RasGRP4 molecule is an antisense or RNAi molecule to a RasGRP4 nucleic acid molecule.

185. The method of claim 183, wherein the inhibitor of a RasGRP4 molecule is a binding polypeptide to a RasGRP4 protein.

186. The method of claim 185, wherein the binding polypeptide agent is an antibody or an antigen-binding fragment thereof.

187. The method of claim 186, wherein the antibody or antigen-binding fragment specifically binds to the N-terminus ofthe isolated RasGRP4 protein or the N terminus ofa Mutant RasGRP4 protein.

188. The method of claim 187, wherein the antibodies or antigen-binding fragments are labeled with one or more cytotoxic agents.

189. A method for increasing the amount of a granule neutral protease in a tissue or cell, comprising increasing the amount of RasGRP4 in the tissue or cell.

190. A method for decreasing the amount ofa granule neutral protease in a tissue or cell comprising inhibiting RasGRP4 activity in the tissue or cell.

191. The method of claim 190, wherein inhibiting RasGRP4 activity is interfering with RasGRP4 activity.

192. The method of claim 190, wherein inl ibiting RasGRP4 activity is reducing the level of expression of RasGRP4.

193. A method for diagnosing a disorder characterized by abenant amounts of a granule neutral protease molecule, comprising: determining in a first biological sample obtained from a subject, an amount ofa RasGRP4 molecule, wherein a difference in the amount ofthe RasGRP4 molecule compared to a RasGRP4 control indicates that the subject has a disorder characterized by abenant amounts of a granule neutral protease.

194. The method of claim 193, further comprising the steps of: detecting in a second biological sample obtained from the subject, an amount ofa RasGRP4 molecule, and comparing the amount ofthe RasGRP4 molecule in the first biological sample and the second biological sample.

195. The method of claim 193 , wherein the disorder characterized by abenant expression of a granule neutral protease is a bacterial infection.

196. The method of claim 193, wherein the disorder characterized by abenant expression of a granule neutral protease is a mast cell disorder.

197. The method of claim 196, wherein the mast cell disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS.

198. The method of claim 196, wherein the mast cell disorder is asthma.

199. The method of claim 196, wherein the mast cell disorder is mastocytosis.

200. The method of claim 193, wherein the biological sample is selected from the group consisting of: a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract.

201. A method for diagnosing a disorder characterized by abenant expression of a RasGRP4 molecule, comprising: determining in a first biological sample obtained from a subject, an amount ofa granule neutral protease, wherein a difference in the amount ofthe granule neutral protease compared to a granule neutral protease confrol indicates that the subject has a disorder characterized by abenant expression of a RasGRP4 molecule.

202. The method of claim 201, further comprising the steps of: detecting in a second biological sample obtained from the subject, an amount ofa granule neutral protease, and comparing the amount ofthe granule neutral protease in the first biological sample and the second biological sample.

203. The method of claim 201, wherein the disorder characterized by abenant expression of a RasGRP4 molecule is a bacterial infection.

204. The method of claim 201, wherein the disorder characterized by abenant expression of a RasGRP4 molecule is a mast cell disorder.

205. The method of claim 204, wherein the mast cell disorder is selected from the group consisting of: allergy (allergic inflammation), urticaria, systemic mastocytosis, cancer, fibrosis, rheumatoid arthritis, mast cell leukemia, neuron degeneration, and ADDS.

206. The method of claim 204, wherein the mast cell disorder is asthma.

207. The method of claim 204, wherein the mast cell disorder is mastocytosis.

208 The method of claim 201, wherein the biological sample is selected from the group consisting of: a cell, a cell scraping, a cell extract, a blood sample, a leukocyte sample, a tissue biopsy, including punch biopsy, a tumor biopsy, a bodily fluid, a tissue, or a tissue extract.

209. A method for evaluating the effect of candidate pharmacological agents on the regulation of mast cells, comprising: administering a candidate pharmacological agent to a subject; determining the effect ofthe candidate pharmacological agent on the amount of prostaglandin D₂ (PGD₂) relative to the amount of PGD₂ in a subject to which no candidate pharmacological agent is administered, wherein a relative increase or relative decrease in the amount of PGD₂ indicates regulation of mast cells by the candidate pharmacological agent.

210. A method for evaluating the effect of candidate pharmacological agents on the regulation of mast cells, comprising: contacting a candidate pharmacological agent with a mast cell sample; determining the effect ofthe candidate pharmacological agent on the amount of prostaglandin D₂ (PGD₂) and/or its synthase relative to the amount of PGD₂ and/or its synthase in a mast cell sample not contacted with the candidate pharmacological agent, wherein a relative increase or relative decrease in the amount of PGD₂ and/or its synthase indicates regulation of mast cells by the candidate pharmacological agent.

211. The method of claim 210 wherein the mast cells are RasGRP4⁺ HMC-1 cells.