JP2002523078A - Novel IRAP-BP polypeptides and nucleic acid molecules and uses thereof - Google Patents

Abstract

  (57) [Summary] Disclose novel IRAP-BP polypeptides, proteins, and nucleic acid molecules. In addition to an isolated full-length IRAP-BP polypeptide, the invention further provides an isolated IRAP-BP fusion protein, an antigenic peptide, and an anti-IRAP-BP antibody. The present invention further provides an IRAP-BP nucleic acid molecule, a recombinant expression vector containing the nucleic acid molecule of the present invention, a host cell into which the expression vector has been introduced, and a non-human non-human into which the IRAP-BP gene has been introduced or disrupted. It provides a transgenic animal. Diagnostic, screening and therapeutic methods utilizing the compositions of the present invention are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

GOVERNMENT RIGHTS This invention was granted by the National Institutes of Health under contract number DK30425,
It was made with at least partial government support. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION [0002] Insulin regulates blood glucose levels primarily by exclusively increasing glucose transport to fat and skeletal muscle tissue. De Fronzo et al. (1981
Diabetes 30: 1000-1007 and James et al. (1985) Am. J. Physiol. 248: E567
-E574. Only these two tissues express the specific isoform of the glucose transporter, GLUT4, which transmits the hormonal action of insulin. (Glucose transporter isoforms and their expression are described in Deveskar and Mueckler (1992) Pediatr. Res. 31: 1-13; Bell et al.
al. (1993) J. Biol. Chem. 268: 3352-3356; and Baldwin (1993) Biochim. Bio
phys. Acta 1154: 17-49). The mechanism of glucose transport activation by insulin depends on the cell type and the method of measurement, but GLUT4 is an intracellular storage location so that the transporter concentration on the cell surface increases 10 to 40-fold. The translocation rate from the vesicles to the cell membrane increases in a hormone-dependent manner. (Zorzono et al. (1989) J. Biol. Chem. 264: 12358-12363; Holman
et al. (1990) J. Biol. Chem. 265: 18172-18179; Slot et al. (1991) J. Biol.
Chem. 113: 123-135; Slot et al. (1991) Proc. Nat'l. Acad. Sci. USA 88: 7.
815-7819; and Smith et al. (1991) Proc. Nat'l. Acad. Sci. USA 88: 6893-68
97). Glucose uptake increases in proportion to the increment of GLUT4 molecules in the plasma membrane, suggesting that transporter redistribution is the main, if not the only, mechanism that explains this effect. ing. Kandror and Pilch (1994) Proc. Nat'l
Acad. Sci. USA 91: 8017-8021.

[0003] GLUT4 is thought to circulate in cells as a component of a tissue-specific secretory-like microsome structure known as "GLUT4-containing vesicles". In addition to GLUT4, these vesicles can further contain phosphatidylinositol 4-kinase (De
l Vecchio and Pilch (1991) J. Biol. Chem. 266: 13278-13283), vesicle-associated membrane protein ("VAMPS") (Cain et al. (1992) J. Biol. Chem. 267: 11681-1).
1684), secretory component-related membrane protein (“SCAMPS”) Thoidis et al. (1993) J. Bio
l. Chem. 268: 11691-11696; and Laurie et al. (1993) J. Biol. Chem. 268: 19
110-19117) and GTP-binding proteins of the low molecular weight Rab family (Cormont et al.
al. (1993) J. Biol. Chem. 268: 19491-19497). In addition to the proteins listed above, insulin-reactive aminopeptidase (
A novel zinc-dependent protease, termed "IRAP"), has also been identified and characterized as being an important component of GLUT4-containing vesicles. (Previously called gp160. Kandror and Pilch (1994) Proc. Nat'l Acad. Sca USA 91: 8017
-8021; Kandror et al. (1994) J. Biol. Chem. 269: 30777-30780; and vp165,
Keller et al. (1995) J. Biol. Chem. 270: 23612-23618). Structurally, IRAP
Contains the amino terminus of 109 amino acids protruding into the cytoplasm, a single 22 amino acid transmembrane domain, and a large catalytic domain within the vesicle lumen responsible for the enzymatic activity of this protein . Keller et al. (1995) J. Biol. Ch
em. 270: 23612-23618. In the basal state, IRAP, like GLUT4, is located primarily in cells, but translocates markedly to the cell surface in response to insulin. Mastick et al
. (1994) J. Biol. Chem. 269: 6089-6092; Kandror and Pilch (1994) Proc. Na.
t'l Acad. Sca USA 91: 8017-8021; Ross et al. (1996) J. Biol. Chem. 271: 33.
28-3332; and Ross et al. (1997) Biochem. Biophys. Res. Commun. (1997) 2
39: 247-251. Furthermore, it suggests that the amino terminus of IRAP, which contains two dileucine motifs and multiple acidic regions similar to those found in GLUT4, functions in regulating GLUT4 intracellular transport and storage Have been. Waters et al. (1997) J. Biol
Chem. 272: 23323-23327.

Given that IRAP plays an important role in regulating the insulin responsive translocation of the glucose transporter GLUT4, we investigated the mechanism of action of IRAP and identified novel proteins with which IRAP interacts Thus, there is a need to identify modulators of such molecules that are used in modulating various insulin-sensitive cellular responses.

SUMMARY OF THE INVENTION [0005] The present invention relates, at least in part, to a novel family of proteins that bind and / or regulate insulin-reactive aminopeptidase ("IRAP"), referred to herein as IRAP binding protein. "IRAP-BP" family or "IRAP-BP" polypeptide "). IRAP-BP of the present invention
Molecules and IRAP-BP modulators are useful for modulating various cellular processes, especially insulin responsive processes. Thus, in one aspect, the present invention relates to IRAP-B
Provided is an isolated nucleic acid molecule encoding a P polypeptide or a biologically active portion thereof, or a nucleic acid fragment suitable as a primer or hybridization probe for detection of a nucleic acid encoding IRAP-BP. Things.

[0006] In one embodiment, the IRAP-BP nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 1.
Sequence, the nucleotide sequence shown in SEQ ID NO: 4, or their complement (original: compl
ement) is 60% homologous. In one preferred embodiment, the isolated IRAP-BP nucleus
The acid molecule has the nucleotide sequence set forth in SEQ ID NO: 3 or its complement. Another
In an example, the IRAP-BP nucleic acid molecule further comprises nucleotides 1-689 of SEQ ID NO: 1.
including. In another embodiment, the IRAP-BP nucleic acid molecule further comprises the nucleotide sequence of SEQ ID NO: 1.
Includes Leotide 2187-2947. In another preferred embodiment, the isolated IRAP-BP nucleic acid molecule
Has the nucleotide sequence shown in SEQ ID NO: 1. In another preferred embodiment
The isolated IRAP-BP nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO: 7, or
Has its complement.

[0007] In another preferred embodiment, the isolated IRAP-BP nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO: 6, or the complement thereof. In another embodiment, the IRAP-BP nucleic acid molecule further comprises nucleotides 1-183 of SEQ ID NO: 4. In another embodiment, the IRAP
-BP nucleic acid molecule further comprises nucleotides 1684-2064 of SEQ ID NO: 4. In another preferred embodiment, the isolated IRAP-BP nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 4.

[0008] In another embodiment, the IRAP-BP nucleic acid molecule is SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 5.
Includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of D NO: 8. In another preferred embodiment, the IRAP-BP nucleic acid molecule comprises SEQ ID NO:
It includes a nucleotide sequence that encodes a protein having an amino acid sequence at least 60% homologous to the amino acid sequence of D NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In yet another preferred embodiment, the IRAP-BP nucleic acid molecule comprises a nucleotide sequence that encodes a protein having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8.

[0009] In another embodiment, the isolated nucleic acid molecule according to the present invention comprises a helix-turn-
It encodes an IRAP-BP polypeptide containing a helix domain. In yet another embodiment, the IRAP-BP nucleic acid molecule encodes an IRAP-BP polypeptide and is a naturally occurring nucleotide sequence.

[0010] Another embodiment of the present invention provides an IRA for specifically detecting a nucleic acid molecule encoding an IRAP-BP polypeptide relative to a nucleic acid molecule encoding a non-IRAP-BP polypeptide.
Characterized by a P-BP nucleic acid molecule. For example, in one example, the IRAP-BP nucleic acid molecule is at least 550 nucleotides in length and, under stringent conditions, the nucleotide sequence set forth in SEQ ID NO: 1, the nucleotide sequence set forth in SEQ ID NO: 4 Or a nucleic acid molecule containing the complement thereof. In another embodiment of the present invention, there is provided an isolated nucleic acid molecule that is antisense to a codon strand of an IRAP-BP nucleic acid.

[0011] Another aspect of the invention provides a vector comprising an IRAP-BP nucleic acid molecule.
In some embodiments, the vector is a recombinant expression vector. In another embodiment, the present invention provides a host cell containing the vector of the present invention. Further, the present invention provides a method for producing an IRAP-BP polypeptide by culturing a host cell of the present invention containing a recombinant expression vector in a suitable medium so that an IRAP-BP polypeptide is produced. It also provides.

[0012] Another aspect of the invention features an isolated or recombinant IRAP-BP polypeptide. In one embodiment, the isolated IRAP-BP polypeptide comprises at least one helix-turn-helix domain. In another embodiment, the isolated IRAP-BP polypeptide has an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In one preferred embodiment, IRAP-B
The P polypeptide has an amino acid sequence that is at least about 60% homologous to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In another embodiment, the IRAP-BP
The polypeptide has the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. In one preferred embodiment, the IRAP-BP polypeptide interacts with the IRAP polypeptide.

Another embodiment of the present invention relates to an isolated IRAP-BP polypeptide, wherein the nucleic acid molecule has a nucleotide sequence at least about 60% homologous to the nucleotide sequence of SEQ ID NO: 1, or a complement thereof. It is characterized by. Another embodiment of the present invention provides that SEQ ID NO: 4
An isolated IRAP-BP polypeptide encoded by a nucleic acid molecule having a nucleotide sequence at least about 60% homologous to the nucleotide sequence of SEQ ID NO: 1 or its complement. In addition, the present invention provides a method of SEQ ID NO: 1, SEQ ID NO:
An isolated IRAP-BP polypeptide characterized by a nucleic acid molecule having a nucleotide sequence that hybridizes to a nucleic acid molecule comprising the nucleotide sequence of ID NO: 4 or a complement thereof.

An IRAP-BP polypeptide according to the present invention, or a biologically active portion thereof, may be operatively linked to a non-IRAP-BP polypeptide to form an IRAP-BP fusion protein. Further, the present invention relates to IRAP, such as monoclonal or polyclonal antibodies.
-Characterized by an antibody that specifically binds to a BP polypeptide. In addition, the IRAP-BP polypeptides or biologically active portions thereof may be incorporated into a pharmaceutical composition, which may optionally include a pharmaceutically acceptable carrier. .

[0015] In another aspect, the invention is capable of detecting an IRAP-BP nucleic acid molecule, protein or polypeptide such that the presence of the IRAP-BP nucleic acid molecule, protein or polypeptide is detected in a biological sample. It is intended to provide a method for detecting the expression of IRAP-BP in a biological sample by bringing the biological sample into contact with an agonist.

[0016] In another aspect, the invention provides a method of contacting a biological sample with an agent capable of detecting IRAP-BP activity, such that the presence of the activity of IRAP-BP is detected in the biological sample. It is intended to provide a method for detecting the presence of IRAP-BP activity in a biological sample.

In another aspect, the invention modulates IRAP-BP activity, comprising contacting the cell with an agent that modulates IRAP-BP activity, such that IRAP-BP activity in the cell is modulated. It provides a method. In one embodiment, the agent inhibits IRAP-BP activity. In another embodiment, the agent stimulates IRAP-BP activity. In one embodiment, the agent is an antibody that specifically binds to an IRAP-BP polypeptide. In another embodiment, the agent is transcription of the IRAP-BP gene or
It modulates the expression of IRAP-BP by modulating the translation of RAP-BP mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the codon strand of IRAP-BP mRNA or IRAP-BP gene.

In one embodiment, a subject having a disorder characterized by abnormal expression or activity of an IRAP-BP polypeptide or nucleic acid is identified using the method of the present invention.
It is treated by administering an agent that is a modulator. In one embodiment,
The IRAP-BP modulator is an IRAP-BP polypeptide. In another embodiment,
An IRAP-BP modulator is an IRAP-BP nucleic acid molecule. In yet another embodiment, the IR
AP-BP modulators are peptides, peptidomimetics, or other small molecules. In one preferred embodiment, the disorder characterized by abnormal IRAP-BP polypeptide or nucleic acid expression is insulin resistance or diabetes.

Further, the present invention provides (i) an abnormal modification or mutation of a gene encoding an IRAP-BP polypeptide, (ii) a misregulation of said gene, and (iii) an abnormal post-translational translation of the IRAP-BP polypeptide. A diagnostic assay for determining the presence or absence of a genetic change characterized by at least one of the following modifications, wherein the wild-type of the gene is a protein having IRAP-BP activity: Is to code.

In another aspect, the invention provides a method for identifying a compound that binds to or modulates the activity of an IRAP-BP polypeptide. In one embodiment, the invention provides a method for identifying a compound that binds to an IRAP-BP polypeptide, the method comprising expressing the IRAP-BP polypeptide, or expressing the IRAP-BP polypeptide. Contacting the cell with the test compound and determining whether the IRAP-BP polypeptide binds to the test compound. In another embodiment, the present invention provides a method for identifying a compound that modulates the activity of an IRAP-BP polypeptide, comprising contacting the IRAP-BP polypeptide with a test compound, Determining the effect of the test compound on the activity of the polypeptide, thereby identifying a compound that modulates the activity of the IRAP-BP polypeptide.

[0021] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to the discovery of novel molecules, here referred to as IRAP-BP polypeptides and nucleic acid molecules, which include a family of molecules having particular conserved structural and functional characteristics. Is based on The term "family" when referring to the proteins and nucleic acid molecules of the present invention refers to two or more that have a common structural domain and have sufficient amino acid or nucleotide sequence homology as defined herein. It is intended to mean further proteins or nucleic acid molecules. Such family members may be naturally occurring and may be from the same or different species. For example, a family may include not only the first protein of human origin, but also other distinct proteins of human origin, or homologs of non-human origin. Members of a family may also have common functional characteristics.

In one embodiment, the IRAP-BP polypeptide of the invention comprises about 250 to 750, preferably about 300 to 700, more preferably about 350 to 650, and even more preferably about 400 to 750. It is a protein having an amino acid sequence of 600, and even more preferably 450 to 550 amino acids in length. In another embodiment, the IRAP-BP polypeptide of the invention comprises about 475 to 975, preferably about 525 to 925, more preferably about 575.
Is a protein having an amino acid sequence that is from about 875 to 875, even more preferably from about 625 to 825, and even more preferably from about 675 to 775 amino acid residues.
In one embodiment, an IRAP-BP molecule of the invention comprises at least one "helix-turn-helix" domain.

A preferred IRAP-BP molecule of the present invention is SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8
Has an amino acid sequence sufficiently homologous to the amino acid sequence of As used herein, the term "sufficiently homologous" refers to a second amino acid or second amino acid such that the first and second amino acid or nucleotide sequences have a common structural domain and / or a common functional activity. Or a first amino acid or nucleotide comprising a sufficient or minimal number of identical or equivalent (eg, amino acid residues having similar side chains) amino acid residues or nucleotides to the nucleotide sequence. For example, an amino acid or nucleotide sequence having a common structural domain has at least about 50%
Homology, preferably 60% homology, more preferably 70% to 80%
%, And even more preferably 90 to 95%, 98% or more homology over the amino acid sequence of the domain, and comprising at least one, and preferably two, structural domains. Are defined to be sufficiently homologous here. Furthermore, an amino acid or nucleotide sequence having at least 50%, preferably 60%, more preferably 70-80, 90-95% or 98% or more homology and having a common functional activity, It is defined here as sufficiently homologous.

As used interchangeably herein, “IRAP-BP activity”, “biological activity of IRAP-BP” or “functional activity of IRAP-BP” is defined based on standard techniques. It refers to the activity of an IRAP-BP polypeptide or nucleic acid molecule as it affects IRAP-BP-responsive cells when examined in vivo or in vitro. In one example, IRAP-BP activity is a direct activity, such as association with an IRAP-BP target molecule. As used herein, a "target molecule" or "binding partner" is a partner to which an IRAP-BP polypeptide naturally binds or interacts, such that an IRAP-BP-mediated function is achieved. Molecule. The IRAP-BP target molecule can be a non-IRAP-BP molecule or an IRAP-BP polypeptide of the invention. In one example, the IRAP-BP target molecule is an IRAP protein or polypeptide (eg, a human IRAP protein, etc.). Depending on your choice, IRAP
-BP activity is an indirect activity, such as an intracellular activity (eg, regulation of vesicle transport) transmitted by the interaction of the IRAP-BP polypeptide with the IRAP polypeptide.

In a preferred embodiment, the IRAP-BP activity is at least one or more of the following activities: (I) interaction with non-IRAP-BP molecules in cells (eg, IRAP and / or GLUT4, etc.), eg, IRAP-BP target molecules; (ii) non-I
Binding to RAP-BP molecules and IRAP-BP target molecules (such as IRAP and / or GLUT4), (
iii) Non-IRAP-BP molecules in cells and IRAP-BP target molecules (eg IRAP and / or
Or GLUT4) and (iv) non-IRAP-BP in cells, for example.
Binding to transport motifs within molecules and IRAP-BP target molecules (such as IRAP and / or GLUT4).

[0027] In yet another preferred embodiment, the IRAP-BP activity is at least one or more of the following activities. That is, (1) modulation of translocation of IRAP-BP target molecule, (2)
), Modulation of GLUT4 transposition (eg, exocytosis), (3) modulation of IRAP transposition (eg, exocytosis), modulation of the classification of IRAP-BP target molecules, (5) IRAP
And / or modulation of GLUT4 classification, (6) modulation of retention of IRAP-BP target molecule, (7) IR
Modulation of retention of AP and / or GLUT4 (eg, retention in specialized intracellular compartments, such as circulating compartments of endocytosis), (8) IRAP-B into circulating vesicles
Modulation of P target molecule entry, (9) modulation of IRAP and / or GLUT4 entry into circulating vesicles, (10) regulation of intracellular transport and / or retention of IRAP-BP target molecule, (11)
) Regulation of intracellular transport and / or retention of IRAP and / or GLUT4, and (12) GLUT4
And / or regulation of intracellular transport of vesicles containing IRAP.

Thus, another embodiment of the invention features an isolated IRAP-BP polypeptide having IRAP-BP activity. Preferred IRAP-BP polypeptides are those that have at least one helix-turn-helix domain and one IRAP-BP activity. In another preferred embodiment, the IRAP-BP polypeptide comprises one helix-turn-helix domain, one IRAP-BP activity, SEQ ID NO: 2, SEQ ID NO: 5,
Or one amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO: 8.

[0029] Human IRAP-BP-1 cDNA, which is at least about 2064 nucleotides in length, encodes a protein about 500 amino acid residues in length. Mouse IRAP that is about 2947 nucleotides long
-BP-1 cDNA encodes about 499 amino acid residues of the mouse IRAP-BP-1 protein.

The following sections describe various aspects of the invention in further detail.

I. Isolated Nucleic Acid Molecules One embodiment of the present invention relates to isolated nucleic acid molecules that encode an IRAP-BP polypeptide or a biologically active portion thereof, and nucleic acids that encode IRAP-BP (eg, IRAP-BP mRNA )
Nucleic acid fragments sufficient to be used as hybridization probes to identify E. coli, and fragments used as PCR primers for amplification or mutation of IRAP-BP nucleic acid molecules. As used herein, the term “nucleic acid molecule” is DN
It is intended to include A molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA), as well as analogs of DNA or RNA made using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA
It is.

An “isolated” nucleic acid molecule is one that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. Preferably, an “isolated” nucleic acid is a sequence that is naturally present on both sides of the nucleic acid in the genomic DNA of the organism from which the nucleic acid was obtained (ie, 5% of the nucleic acid).
Sequence at the 'end and 3' end). For example, in various embodiments, an isolated IRAP-BP nucleic acid molecule is present in the genomic DNA of the cell from which the nucleic acid was obtained, approximately 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, on either side of the nucleic acid molecule. ,
It may comprise a nucleotide sequence of less than 0.5 kb or 0.1 kb. In addition, an `` isolated '' nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular materials or media when produced by recombinant techniques, or when chemically synthesized. May be substantially free of chemical precursors or other chemical components.

The nucleic acid molecules of the invention, such as, for example, nucleic acid molecules having the nucleotide sequence of SEQ ID NO: 1, or SEQ ID NO: 4, or portions thereof, can be prepared using standard molecular biology techniques and
Isolation may be performed using the sequence information provided herein. Of the nucleic acid sequence of SEQ ID NO: 1,
Alternatively, by using all or part of the nucleotide sequence of SEQ ID NO: 4 as a hybridization probe, the IRAP-BP nucleic acid molecule can be converted using standard hybridization and cloning techniques (eg, Sambrook, J., Fritsh). , E.
F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed.,
Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Col
d Spring Harbor, NY, 1989). Further, a nucleic acid molecule comprising all or a portion of SEQ ID NO: 1 or SEQ ID NO: 4 is converted to a nucleic acid molecule comprising SEQ ID NO: 1.
Alternatively, it can be isolated by polymerase chain reaction (PCR) using a synthetic oligonucleotide primer designed based on the sequence of SEQ ID NO: 4.

[0034] The nucleic acids of the invention can be amplified using cDNA, mRNA, or alternatively genomic DNA, as a template and suitable oligonucleotide primers based on standard PCR amplification techniques. The nucleic acid thus amplified may be cloned into a suitable vector and characterized by DNA sequence analysis. In addition, oligonucleotides corresponding to IRAP-BP nucleotide sequences can be made by standard synthetic techniques, such as using an automated DNA synthesizer.

[0035] In one preferred embodiment, the isolated nucleic acid molecule according to the present invention comprises SEQ ID NO: 1.
And the nucleotide sequence shown in Table 1. The sequence of SEQ ID NO: 1 is mouse IRAP-BP-1 cDNA
Is equivalent to This cDNA contains a sequence encoding the mouse IRAP-BP-1 protein (ie, the "codon region" at nucleotides 690-2186) and a 5 'untranslated sequence (nucleotides 1-68).
9) and the 3 'untranslated sequence (nucleotides 2187-2947). Depending on your choice,
The nucleic acid molecule may include only the codon region of SEQ ID NO: 1 (eg, nucleotides 690-2186 corresponding to SEQ ID NO: 3).

In another preferred embodiment, the isolated nucleic acid molecule according to the present invention has SEQ ID NO: 4.
And the nucleotide sequence shown in Table 1. The sequence of SEQ ID NO: 1 corresponds to the human IRAP-BP-1 cDNA. This cDNA has a human IRAP-BP-1 protein-encoding sequence (ie, the "codon region" at nucleotides 184-1683) and a 5 'untranslated sequence (nucleotides 1-183).
And the 3 'untranslated sequence (nucleotides 1684-2064). In some selections, the nucleic acid molecule may include only the codon region of SEQ ID NO: 1 (eg, nucleotides 184-1683 corresponding to SEQ ID NO: 6).

In another embodiment, an isolated nucleic acid molecule according to the present invention may comprise a sequence encoding the mouse IRAP-BP protein (ie, the “codon region” of nucleotides 2-2186) or an untranslated sequence at the 3 ′ end. (Nucleotides 2187-2947). In some selections, the nucleic acid molecule comprises only the codon region of SEQ ID NO: 1 (
For example, it may contain nucleotide 2-2186) corresponding to SEQ ID NO: 7.

In another preferred embodiment, the isolated nucleic acid molecule of the present invention comprises SEQ ID NO: 1 or SEQ ID NO:
Nucleic acid molecules that are the complement of the nucleotide sequence shown in ID NO: 4, or any portion of these nucleotide sequences. A nucleic acid molecule complementary to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4 is a stable duplex by hybridizing to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4. It is sufficiently complementary to the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 4 to form a chain.

[0039] In yet another preferred embodiment, the isolated nucleic acid molecule of the invention comprises SEQ ID NO: 1.
At least about 60-65%, preferably at least about 70%, relative to the nucleotide sequence set forth in SEQ ID NO: 4, or to any portion of these nucleotide sequences.
-75%, more preferably at least about 80-85%, and even more preferably at least about 90-95%, 98% or higher, comprising nucleotide sequences that are homologous.

In addition, the nucleic acid molecules of the present invention may have the sequence of SEQ ID NO: 1 or SEQ ID NO: It may include a very small portion of the nucleic acid sequence of SEQ ID NO: 4. Given the nucleotide sequence determined from the cloning of the IRAP-BP-1 gene, IRAP-BP derived from other IRAP-BP family members and other species
This allows the generation of probes and primers designed to be used to identify and / or clone homologs. The probe / primer typically comprises a substantially purified oligonucleotide. The oligonucleotide is typically a sense sequence of SEQ ID NO: 1 or SEQ ID NO: 4, or SEQ ID NO: 4.
O: 1 or the antisense sequence of SEQ ID NO: 4, or naturally occurring mutant SEQ ID NO:
At least about 12, preferably about 25, more preferably about 1 of 1 or SEQ ID NO: 4
It comprises a region of the nucleotide sequence that hybridizes under stringent conditions to 40, 50, or 75 contiguous nucleotides. In one embodiment, the nucleic acid molecule of the invention comprises
More than 550 nucleotides in length, preferably 551-600, more preferably 601-650,
More preferably 651-700, and even more preferably 701-800, 801-900, 901-1
000, 1001-1500, or 1501-2000 nucleotides in length, and SEQ ID NO: 1
Alternatively, it comprises a nucleotide sequence that hybridizes under stringent hybridization conditions to the nucleic acid molecule of SEQ ID NO: 4.

[0041] Probes based on IRAP-BP nucleotide sequences may be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In a preferred embodiment, the probe may further include a label group attached thereto, which label group may be, for example, a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used as part of a diagnostic test kit for identifying cells or tissues that mis-express the IRAP-BP polypeptide, e.g., IRAP-BP in a cell sample taken from a subject. It can be used by measuring the amount of the encoding nucleic acid, for example, detecting the amount of IRAP-BP mRNA, or checking whether the genomic IRAP-BP gene has been mutated or deleted.

The nucleic acid fragment encoding the “biologically active portion of an IRAP-BP polypeptide” can be used to generate IRAP-BP biological activity (eg, by recombinant expression in vitro). Isolating the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, which encodes a polypeptide having the peptide's biological activity as previously described, and encoding the encoded portion of the IRAP-BP polypeptide By expressing and evaluating the activity of the encoded portion of the IRAP-BP polypeptide.

The present invention further relates to SEQ ID NO: 1 or SEQ ID NO: 4, which differs from the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 4, but due to the degeneracy of the genetic code. As well as nucleic acid molecules that encode the same IRAP-BP polypeptide as the nucleotide sequence set forth in SEQ. In another embodiment, an isolated nucleic acid molecule according to the present invention has a nucleotide sequence that encodes a protein having the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 5.

[0044] In addition to the IRAP-BP nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 4, those skilled in the art will appreciate that DNA that results in a change in the amino acid sequence of the IRAP-BP polypeptide
It will also be appreciated that sequence polymorphisms may also be present in a population (eg, a human population). Such genetic polymorphisms in the IRAP-BP gene may exist between individuals in a population due to natural allelic differences. The terms "gene" and "recombinant gene" as used herein refer to a nucleic acid molecule comprising an open reading frame encoding an IRAP-BP polypeptide, preferably a mammalian IRAP-BP polypeptide. Because of these natural allelic differences, typically one
There may be 1-5% variance in the nucleotide sequence of the IRAP-BP gene. IRAP-BP
Such nucleotide differences in genes, and consequent amino acid polymorphisms, that result from natural allelic differences and that do not alter the functional activity of the IRAP-BP polypeptide All such are intended to be within the scope of the present invention.

In addition, it encodes other IRAP-BP family members and thus has the SEQ ID NO
Such nucleic acid molecules having a nucleotide sequence that differs from the IRAP-BP-1 sequence of SEQ ID NO: 1 or SEQ ID NO: 4 are intended to be within the scope of the invention. For example, IRAP-BP-2 cDNA
May be identified based on the nucleotide sequence of human IRAP-BP-1 or mouse IRAP-BP-1. Further, nucleic acid molecules that encode an IRAP-BP polypeptide from a different species, and thus have a nucleotide sequence that differs from the IRAP-BP sequence of SEQ ID NO: 1 or SEQ ID NO: 4, are also included in the invention. It is intended to be within range. For example, rat IRAP-BP cDNA can be identified based on the nucleotide sequence of human or mouse IRAP-BP.

Nucleic acid molecules corresponding to the naturally occurring allelic variants and homologues of the IRAP-BP cDNA of the present invention can be used to convert the cDNAs or portions thereof disclosed herein under stringent hybridization conditions using standard hybridization techniques. When used as a hybridization probe, they can be isolated based on their homology to the IRAP-BP nucleic acids disclosed herein.

Thus, in another embodiment, the isolated nucleic acid molecule according to the invention comprises at least 15
It is nucleotide long and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4. In another embodiment, the nucleic acids are at least 30, 50, 100, 250 or 500 nucleotides in length. As used herein, the term "hybridize under stringent conditions" refers to at least one other
It is intended to describe the hybridization and washing conditions under which nucleotide sequences that are 60% homologous often remain hybridized to each other. Preferably, the conditions are such that sequences that are at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% homologous to each other often remain hybridized to each other. It is a condition that can be put. Such stringent conditions are known to those of skill in the art and are described in Current Protocols in Molecular Biolo
gy, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. One example of suitable, but non-limiting, stringent hybridization conditions is hybridization in about 45 C 6-fold sodium chloride / sodium citrate, followed by 50-65 ° C. 0.2-fold SSC, 0 One or more washes in 1% SDS. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 or SEQ ID NO: 4 may correspond to a naturally occurring nucleic acid molecule. A "naturally occurring" nucleic acid molecule, as used herein, is
An RNA or DNA molecule having a naturally occurring nucleotide sequence (eg, encoding a naturally occurring protein).

[0048] In addition to naturally occurring allelic variants of the IRAP-BP sequence that may be present in the population, those skilled in the art will appreciate that mutations may result in nucleotides of SEQ ID NO: 1 or SEQ ID NO: 4. It will be appreciated that changes in the sequence may change the amino acid sequence of the encoded IRAP-BP polypeptide without altering the functional ability of the IRAP-BP polypeptide. For example, nucleotide substitutions may occur in the sequence of SEQ ID NO: 1 or SEQ ID NO: 4 that result in amino acid substitutions at "unimportant" amino acid residues. “Insignificant” amino acid residues are those residues that may be changed from the wild-type sequence of IRAP-BP (eg, such as the sequence of SEQ ID NO: 2) without changing its biological activity, "Important" amino acid residues are those required for biological activity. For example, conserved amino acid residues among the IRAP-BP polypeptides of the present invention are not expected to be particularly amenable to change. Furthermore, the amino acid residues defined by the helix-turn-helix domain are not particularly amenable to change.

Accordingly, one further aspect of the present invention relates to nucleic acid molecules encoding IRAP-BP polypeptides that include changes in amino acid residues that are not essential for activity. Such an IRAP-BP polypeptide can have SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 5.
Although it differs in amino acid sequence from D NO: 8, it retains its biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein is SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 5. : 8 containing an amino acid sequence at least about 60% homologous to the amino acid sequence. Preferably, the protein encoded by the nucleic acid molecule is SEQ ID NO: 2, SEQ ID NO:
: 5, or at least about 65-70% homologous to SEQ ID NO: 8, more preferably SEQ ID NO: 8
NO: 2, SEQ ID NO: 5, or at least about 75-80% homologous to SEQ ID NO: 8, even more preferably to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. At least about 85-90% homologous to it, and even more preferably SEQ ID NO: 2, SEQ ID NO: 5, or
It may be at least about 95%, 98% or higher and homologous to SEQ ID NO: 8.

An isolated nucleic acid molecule encoding an IRAP-BP polypeptide homologous to a protein of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 comprises one or more nucleotide substitutions , Additions or deletions are introduced into the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, or one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. It may be produced in this manner. Introduction of mutations into SEQ ID NO: 1 or SEQ ID NO: 4 can be performed by standard techniques, eg, site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-critical amino acid residues. "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), amino acids with acidic side chains (eg, aspartic acid, glutamic acid), and amino acids with polar, uncharged side chains (eg, For example, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (eg, alanine,
Valine, leucine, isoleucine, proline, phenylalanine, methionine,
Tryptophan), amino acids with beta-branched side chains (eg, threonine, valine, isoleucine), and amino acids with aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Thus, IRAP-BP
To substitute a predicted non-critical amino acid residue in a polypeptide, it is preferable to substitute another amino acid residue of the same side chain family. Depending on the selection, in another example, mutations can be introduced randomly over all or part of the IRAP-BP codon sequence, such as saturation mutagenesis, and the resulting mutant
Screening for biological activity of -BP may identify variants that retain activity. After mutagenesis is performed on SEQ ID NO: 1 or SEQ ID NO: 4, the encoded protein may be expressed recombinantly and its protein activity examined.

In one preferred embodiment, the mutant IRAP-BP polypeptide is interacted with (1) a non-IRAP-BP molecule, eg, in a cell, and an IRAP-BP target molecule (eg, IRAP and / or GLUT4). Ability to act, (2) ability to bind to non-IRAP-BP molecules, eg, intracellular, and IRAP-BP target molecules (eg, IRAP and / or GLUT4, etc.), (3) eg, non-IRA, intracellular
The ability to recognize transport motifs in P-BP molecules and IRAP-BP target molecules (such as IRAP and / or GLUT4), (4) non-IRAP-BP molecules, eg, in cells, and IRAP-BP target molecules (eg, Ability to bind to transport motifs within IRAP and / or GLUT4), (5) I
Ability to modulate transposition of RAP-BP target molecule, (6), ability to modulate GLUT4 translocation (
Eg, exocytosis), (7) ability to modulate translocation of IRAP (eg, exocytosis), (8) ability to modulate IRAP-BP target molecule classification, (9) IRAP
And / or the ability to modulate the classification of GLUT4, (10) the ability to modulate retention of IRAP-BP target molecules, (11) the ability to modulate retention of IRAP and / or GLUT4 (eg, circulating compartments of endocytosis, etc.) , In a specialized intracellular compartment),
(12) ability to modulate the entry of IRAP-BP target molecules into circulating vesicles, (13)
The ability to modulate the entry of IRAP and / or GLUT4 into circulating vesicles, (15) IRAP-
(16) ability to regulate intracellular transport and / or retention of BP target molecule, (16) ability to regulate intracellular transport and / or retention of IRAP and / or GLUT4, or (77) GLUT4 and / or
Alternatively, vesicles containing IRAP can be assayed for their ability to modulate intracellular trafficking.

[0052] In addition to the nucleic acid molecules encoding an IRAP-BP polypeptide described above, another aspect of the invention pertains to isolated nucleic acid molecules that are antisense thereto. An "antisense" nucleic acid is complementary to a "sense" nucleic acid that encodes a single protein, e.g., complementary to a codon strand of a double-stranded cDNA molecule or to an mRNA sequence. Includes nucleotide sequence. Thus, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. Antisense nucleic acid is IRAP-B
It may be complementary to the entire P codon chain or to a small portion thereof. In one embodiment, the antisense nucleic acid molecule is antisense to the "codon region" of the codon strand of the nucleotide sequence encoding IRAP-BP.
The term "codon region" refers to a region of a nucleotide sequence that includes a codon that is translated into an amino acid residue (eg, the codon region of mouse IRAP-BP-1 corresponding to SEQ ID NO: 3). In another embodiment, the antisense nucleic acid molecule is antisense to a "non-codon region" of the codon strand of the nucleotide sequence encoding IRAP-BP. The term “non-codon region” refers to the 5 ′ and 3 ′ sequences flanking the codon region that are not translated into amino acids (ie, also referred to as 5 ′ and 3 ′ untranslated regions). .

The codon chain sequences encoding the IRAP-BPs disclosed herein (eg, SEQ ID NO: 3 and SE
With Q ID NO: 6), the antisense nucleic acid of the present invention can also be designed based on the Watson-Crick base pairing rule. The antisense nucleic acid molecule may be complementary to the entire codon region of the IRAP-BP mRNA, but more preferably is antisense to a small portion of the codon or non-codon region of the IRAP-BP mRNA. It may be a certain oligonucleotide. For example, the antisense oligonucleotide may be complementary to a region around the translation start site of IRAP-BP mRNA. Antisense oligonucleotides can be, for example, about 5, 10, 15, 20, 25, 30, 3
It may be 5, 40, 45 or 50 nucleotides in length. The antisense nucleic acid of the present invention can be constructed using a chemical synthesis method or an enzyme ligation method using a method known in the art. For example, antisense nucleic acids (eg, antisense oligonucleotides) use naturally occurring nucleotides, or are designed to increase the biological stability of the molecule, or can be used in combination with antisense and sense nucleic acids. Various modified nucleotides designed to increase the physical stability of the duplex formed between them may be used, for example, phosphorothioate derivatives and acridine substituted nucleotides. Examples of modified nucleotides that can be used to make this antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine , 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosin (
Original: beta-D-galactosylqueosine), inosine, N6-isopentenyl adenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7
-Methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-
Thiouracil, beta-D-mannosylqueosine (original: beta-D-mannosylqueosine)
), 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine (original: wybutoxosine), pseudo uracil (original: pseudouracil) , Keosin (original: queosine), 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v ), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-
(Carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine. Depending on the selection, the antisense nucleic acids can also be made biologically (ie, RNA transcribed from the inserted nucleic acid) using an expression vector in which a single nucleic acid has been subcloned in the antisense orientation. Will be in the antisense orientation for the target nucleic acid of interest, which is described in detail in the following section).

An antisense nucleic acid molecule of the invention typically binds to or binds to mRNA and / or genomic DNA in a cell encoding an IRAP-BP polypeptide,
Thus, the protein may be administered to a subject or produced in situ to inhibit expression of the protein, such as by inhibiting transcription and / or translation. Hybridization can be by conventional nucleotide complementarity forming a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to a DNA duplex, specific between the major grooves of the duplex. It may be due to interaction. One example of a route of administration of the antisense nucleic acid molecules of the present invention includes direct injection into a tissue site. Optionally, the antisense nucleic acid molecule can be administered systemically after modification to target selected cells. For systemic administration, for example, linking the antisense nucleic acid molecule to a peptide or antibody that binds to a cell surface receptor or antigen,
The antisense molecule may be modified to specifically bind to a receptor or antigen expressed on the selected cell surface. In addition, the antisense nucleic acid molecules can be delivered to cells using the vectors described herein. To achieve a sufficient intracellular concentration of the antisense molecule, the antisense nucleic acid molecule must have strong pol II or po.
Vector constructs that are under the control of the lIII promoter are preferred.

In yet another embodiment, an antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule specifically forms a double-stranded hybrid with complementary RNA, where the strands are parallel to one another, in contrast to the normal beta unit (Gaultier et al. (1987) Nucleic Acids. Res. 15: 6625-664.
1). In addition, antisense nucleic acid molecules include 2'-o-methyl ribonucleotides (Inoue e
t al. (1987) Nucleic Acids Res. 15: 6131-6148) or chimeric RNA-DNA analog
(Inoue et al. (1987) FEBS Lett. 215: 327-330).

[0056] In yet another embodiment, the antisense nucleic acids of the invention are ribozymes. Riboenzymes are catalytic RNA molecules that have ribonuclease activity and can cleave single-stranded nucleic acids such as mRNAs that have regions complementary to them. Thus, using a riboenzyme, such as a hammerhead riboenzyme (discussed in Haselhoff and Gerlach (1988) Nature 334: 585-591), catalytically cleaves the IRAP-BP mRNA transcript, thereby producing -Can inhibit the translation of BP mRNA. A riboenzyme having specificity for a nucleic acid encoding IRAP-BP is IRAP-BP-1 or IAP disclosed herein.
It can be designed based on the nucleotide sequence of the RAP-BP-2 cDNA (ie, SEQ ID NO: 1 or SEQ ID NO: 4). For example, a derivative of Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active position therein is complementary to the nucleotide sequence to be cleaved in the mRNA encoding IRAP-BP. C
See U.S. Pat. No. 4,987,071 to Ech et al. and U.S. Pat. No. 5,116,742 to Cech et al. Depending on the selection, IRAP-BP mRNA can also be used to select catalytic RNA with specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel, D. and Szostak, JW (1993) Science 261: 1411-1418.

Depending on the selection, expression of the IRAP-BP gene may be regulated by the IRAP-BP regulatory region (eg, IRAP-BP).
BP promoter and / or enhancer) can be inhibited by targeting a nucleotide sequence that forms a triple helix to prevent transcription of the IRAP-BP gene in target cells. Schematically, Helene, C. (1991) Antica
ncer Drug Des. 6 (6): 569-84; Helene, C. et al. (1992) Ann. NY Acad. Sci.
660: 27-36; and Maher, LJ (1992) Bioassays 14 (12): 807-15.

II. Isolated IRAP-BP polypeptides and anti-IRAP-BP antibodies One aspect of the present invention relates to isolated IRAP-BP polypeptides, and biologically active portions thereof, and anti-IRAP-BP antibodies. Polypeptide fragments suitable for use as the immunogens to be generated. In one example, a native IRAP-BP polypeptide can be isolated from a cell or tissue source by a suitable purification scheme using standard protein purification techniques. In another example, an IRAP-BP polypeptide is made by recombinant DNA technology. As an alternative to recombinant expression, IRAP-BP polypeptides may be synthesized chemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portion thereof is defined as a cellular material or other contaminant present in the cell or tissue source from which the IRAP-BP polypeptide was obtained. If there is virtually no protein, or if chemically synthesized,
It is substantially free of chemical precursors or other chemicals. The term "substantially free of cellular material" refers to an IRAP-BP polypeptide in which the protein is isolated from the cellular components of the cells from which it was isolated or recombinantly produced. Preparations are included. In one embodiment, the term "substantially free of cellular material" refers to less than about 30% (by dry weight) of a non-IRAP-BP polypeptide (also referred to herein as a "contaminating protein"), Preferably less than about 20% non-I
RAP-BP polypeptides, even more preferably less than about 10% of non-IRAP-BP polypeptides, and most preferably preparations of IRAP-BP polypeptides with less than about 5% non-IRAP-BP polypeptides. If the IRAP-BP polypeptide or a biologically active portion thereof is produced recombinantly, it is further preferred that the medium be substantially free of medium, i.e., that the medium is about 20% of the volume of the protein preparation. %, More preferably less than about 10%, and most preferably less than about 5%.

[0060] The phrase "substantially free of chemical precursors or other chemicals" refers to the separation of the protein from the chemical precursors or other chemicals involved in the synthesis of the protein. Preparations of various IRAP-BP polypeptides. In one embodiment, the phrase "substantially free of chemical precursors or other chemicals" includes less than about 30% (by dry weight) of chemical precursors or non-IRAP-BP chemicals, more preferably Is less than about 20% chemical precursor or non-IRAP-BP chemical, even more preferably less than about 10% chemical precursor or non-IRAP-BP chemical, and most preferably less than about 5% chemical Preparations of IRAP-BP polypeptides with precursors or non-IRAP-BP chemicals are included.

[0061] Biologically active portions of the IRAP-BP polypeptide include SEQ ID NO: 2, SEQ ID NO:
5, or an amino acid sequence as set forth in SEQ ID NO: 8, comprising a smaller number of amino acids than the full-length IRAP-BP polypeptide, and exhibiting at least one activity of the IRAP-BP polypeptide, Peptides containing an amino acid sequence sufficiently homologous to the amino acid sequence of the peptide or derived from the amino acid sequence of the IRAP-BP polypeptide are included. Typically, a biologically active portion includes a domain or motif that has at least one of the activities of the IRAP-BP polypeptide. IRAP
The biologically active portion of a -BP polypeptide can be, for example, 50, 100, 150, 200, 250, 3
The polypeptide may be 00, 350, 400, 450 or more amino acids long. For example, in one embodiment, the biologically active portion of the IRAP-BP polypeptide is
It contains at least one helix-turn-helix domain. In addition, other biologically active portions, such as those from which other regions of the protein have been deleted, have been made by recombinant techniques to provide one or more of the functional activities of the native IRAP-BP polypeptide. The above may be evaluated.

[0062] In certain preferred embodiments, the IRAP-BP polypeptide comprises SEQ ID NO: 2, SEQ ID NO: 5
Or the amino acid sequence shown in SEQ ID NO: 8. In another embodiment, the IRAP-BP polypeptide is SEQ ID NO: 2, SEQ ID NO: 5, or SE, as detailed in Section I above.
SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO, which is substantially homologous to Q ID NO: 8
The amino acid sequences may differ due to natural allelic differences or mutagenesis, while retaining the functional activity of the NO: 8 protein. Thus, in another embodiment, the IRAP-BP polypeptide is SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 5.
: 8 comprising an amino acid sequence that is at least about 60% homologous to the amino acid sequence of
It is a protein that retains the functional activity of the IRAP-BP polypeptide of EQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. Preferably, the protein is
At least about 65-70% homologous to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, more preferably at least to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8 About 75-80%
Homologous, even more preferably at least about 85-90% homologous to SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8, and most preferably SEQ ID NO: 2, SEQ ID NO: 5, or
It may be at least about 95%, 98% or more homologous to SEQ ID NO: 8.

To determine the percent homology or identity of two amino acid sequences, or two nucleotide or amino acid sequences, the sequences are aligned (eg, with a second amino acid or nucleic acid) for optimal comparison. Gaps may be introduced into the first amino acid sequence or nucleic acid sequence for optimal alignment with the sequence). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, of the reference sequence. And even more preferably at least 70%, 80%, or 90% in length (e.g., 499
At least 150, preferably at least 200, more preferably at least 250, even more preferably at least 299, and even more preferably when aligned to the IRAP-BP amino acid sequence of SEQ ID NO: 2 having At least 349, 399, or 449 amino acid residues are aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When one position in the first sequence is occupied by the same amino acid residue or nucleotide as at the corresponding position in the second sequence, then the molecules are homologous at that position (ie, where The "homology" of an amino acid or nucleic acid used is equivalent to the "identity" of the amino acid or nucleic acid). The percent homology between two sequences is a function of the number of identical positions shared by the sequences (ie,% homology = number of identical positions / total number of positions x 100).

[0065] Sequence comparison between two sequences and determination of percent homology can be performed using a mathematical algorithm. One suitable, but non-limiting example of a mathematical algorithm used for comparing sequences is described in Karlin and Altschul (1993) Proc.
Karlin and Altschul (1990) improved in Natl. Acad. Sci. USA 90: 5873-77.
Proc. Natl. Acad. Sci. USA 87: 2264-68. Alt for such an algorithm
Incorporated into the NBLAST and XBLAST programs (version 2.0) of schul, et al. (1990) J. Mol. Biol. 215: 403-10. When a BLAST nucleotide search is performed using the NBLAST program with a score of 100 and a word length of 12, a nucleotide sequence homologous to the IRAP-BP nucleic acid molecule of the present invention can be obtained. BLAST protein search, XBLAST
When the program is performed with a score = 50 and a word length = 3, an amino acid sequence homologous to the IRAP-BP polypeptide molecule of the present invention can be obtained. To perform gapped alignments for comparison, use Gapped BLAST with Alt
Schul et al., (1997) Nucleic Acids Research 25 (17): 3389-3402. When utilizing BLAST and gapped BLAST programs, the default parameters of each program (eg, XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another suitable but non-limiting example of a mathematical algorithm used for sequence comparison is Myers and Mi
ller, algorithm of CABIOS (1989). Such an algorithm is described in the ALIGN program (part of the GCG sequence alignment software package)
Version 2.0). When using the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length / penalty of 12, and a gap penalty of 4 can be used.

The present invention further provides an IRAP-BP chimeric or fusion protein. As used herein, IRAP-BP "chimeric protein" or "fusion protein" includes an IRAP-BP polypeptide operatively linked to a non-IRAP-BP polypeptide. "IRAP-BP polypeptide" refers to a polypeptide having an amino acid sequence corresponding to IRAP-BP, while "non-IRAP-BP polypeptide"
For example, a polypeptide that differs from an IRAP-BP polypeptide and has an amino acid sequence corresponding to a protein that is not substantially homologous to the IRAP-BP polypeptide, such as a protein derived from the same or a different organism. In the IRAP-BP fusion protein,
The IRAP-BP polypeptide may correspond to all or a part of one IRAP-BP polypeptide. In a preferred embodiment, the IRAP-BP fusion protein comprises at least one biologically active portion of an IRAP-BP polypeptide. In another preferred embodiment, the IRAP-BP fusion protein comprises at least two biologically active portions of an IRAP-BP polypeptide. In this fusion protein, the term "operably linked" refers to the fact that an IRAP-BP polypeptide and a non-IRAP-BP polypeptide are mutually in-
Intended to refer to being fused in a frame. Non-IRAP-BP polypeptides may be fused to the N-terminus or C-terminus of the IRAP-BP polypeptide.

For example, in one embodiment, the fusion protein is a GST-IRAP-BP fusion protein in which an IRAP-BP sequence is fused to the C-terminus of a GST sequence. The presence of such a fusion protein facilitates the purification of the recombinant IRAP-BP.

In another embodiment, the fusion protein is an IRAP-BP polypeptide containing a heterologous signal sequence at its N-terminus. For example, the native IRAP-BP signal sequence may be removed and replaced with a signal sequence from another protein. In certain host cells (eg, mammalian host cells), expression and / or secretion of IRAP-BP is
It can be increased by using a heterologous signal sequence.

An IRAP-BP fusion protein of the invention may be incorporated into a pharmaceutical composition and administered to a subject in vivo. IRAP-BP fusion proteins may be used to affect the bioavailability of an IRAP-BP substrate. The use of IRAP-BP fusion proteins may be therapeutically useful for treatments for respiratory diseases (eg, asthma). Further, the IRAP-BP of the present invention
The fusion protein can be used as an immunogen to generate anti-IRAP-BP antibodies in a subject, used to purify IRAP-BP ligand, and IRAP-BP of IRAP-BP.
It can also be used in screening assays to identify molecules that inhibit interaction with the ligand.

Preferably, the IRAP-BP chimeric or fusion proteins of the present invention may be made by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences can be ligated in-frame based on the prior art.
), Such techniques include the use of blunt or cohesive ends for ligation, restriction enzyme digestion to provide suitable ends, and, where appropriate, filling of cohesive ends. Alkaline phosphatase treatment to prevent unwanted conjugation, and enzyme ligation. In another embodiment, the fusion gene is
It can be synthesized by conventional techniques, including synthesizers. Depending on the selection, PCR amplification of the gene fragment can be performed using an anchor primer, resulting in a complementary overhang between two consecutive gene fragments,
Then, the overhang portion is annealed and re-amplified to produce a chimeric gene sequence (for example, Current Protocols in Molecular Biology, eds. Aus
ubel et al. John Wiley & Sons: 1992). In addition, a number of expression vectors are commercially available that already encode a fusion component (eg, a GST polypeptide). When the nucleic acid encoding IRAP-BP is cloned into such an expression vector, the fusion component is ligated in-frame to the IRAP-BP polypeptide.

The present invention further relates to variants of the IRAP-BP polypeptide that act as either IRAP-BP agonists (mimetics) or IRAP-BP antagonists. Mutants of the IRAP-BP polypeptide can be made by mutagenesis, eg, by discrete point mutation or truncation of the IRAP-BP polypeptide. IR
An agonist of an AP-BP polypeptide may retain substantially the same or a smaller set of biological activities as a naturally occurring IRAP-BP polypeptide. An antagonist of an IRAP-BP polypeptide inhibits one or more of the activities of a naturally occurring IRAP-BP polypeptide, such as by competitively inhibiting the protease activity of the IRAP-BP polypeptide. be able to. Thus, specific biological effects can be elicited by treatment with mutants of limited function. In one example, treatment of a subject with a variant having a smaller set of biological activities than the naturally occurring protein of interest results in the treatment of the subject as compared to treatment with the naturally occurring IRAP-BP polypeptide. There are few side effects on the body.

In one example, a variant of an IRAP-BP polypeptide that acts as either an IRAP-BP agonist (mimetic) or an IRAP-BP antagonist is described in terms of IRAP-BP polypeptide agonist or antagonist activity. One may identify the IRAP-BP polypeptide by screening a combinatorial library of variants, eg, a truncation variant. In one embodiment, IRAP-BP
A variagated library of variants has been generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of IRAP-BP variants can be created, for example, by enzymatically linking a mixture of synthetic oligonucleotides into a gene sequence, resulting in a denatured set of potential IRAP -BP sequences can be expressed as individual polypeptides or, alternatively, as a larger set of fusion proteins containing the set of IRAP-BP sequences therein (eg, for phage display). it can. There are a variety of methods available for generating libraries of latent IRAP-BP variants from degenerate oligonucleotide sequences. Chemical synthesis of the degenerate gene sequence may be performed on an automatic DNA synthesizer, and the synthetic gene may then be ligated into a suitable expression vector. With a modified set of genes, all of the sequences encoding the desired set of potential IRAP-BP sequences can be provided in one mixture. Methods for synthesizing modified oligonucleotides are known in the art (eg, Narang, SA (
1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323
; Itakura et al. (1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid
Res. 11: 477.).

In addition, using a fragment library of sequences encoding the IRAP-BP polypeptide,
A variegated population of RAP-BP fragments can be generated, and after screening this population, variants of the IRAB-BP polypeptide can be selected. In one embodiment, a library of codon sequence fragments can be generated, but the method involves double-stranded PCR of the sequence encoding IRAP-BP under conditions such that nicking occurs only about once per molecule. Treating the fragment with nuclease, denaturing the double-stranded DNA, and religating the DNA to form a double-stranded strand that may contain a sense / antisense pair made of different nicking products Forming a DNA, removing the single-stranded portion from the reformed duplex by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, expression vectors encoding N-terminal, C-terminal, and internal fragments of IRAP-BP polypeptides of various sizes can be obtained.

Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutation or truncation and for screening cDNA libraries for gene products having selected properties. is there. Such techniques can be adapted to rapidly screen gene libraries generated by combinatorial mutagenesis of IRAP-BP polypeptides. The most widely used technique for screening large gene libraries, which is amenable to high throughput analysis, typically involves cloning the gene library into a replicable expression vector,
Transforming a suitable cell with the resulting library of vectors;
Detecting the desired activity, expressing the combined gene under conditions that facilitate isolation of the vector encoding the gene whose product was detected. Using a new technique, recruisive ensemble mutagenesis (REM), to increase the frequency of functional variants in the library, in conjunction with this screening assay, IRAP-BP variants can be Can also be identified (Arkin and Yourvan (1992) PNAS 89: 7811-7815; Delgrave et al.
(1993) Protein Engineering 6 (3): 327-331).

In one embodiment, a cell-based assay may be used to analyze a variegated library. For example, a library of expression vectors can be transfected into a cell line that normally synthesizes IRAP-BP. Next, the transfected cells are cultured. As a result of the culturing, it is confirmed that the specific mutant IRAP-BP is expressed, and that the mutant is expressed in the cells.
Although the effect on BP activity can be detected, the detection may be performed using any of a number of activity assays directed toward the native IRAP-BP polypeptide. The plasmid DNA responsible for the modulated IRAP-BP activity may then be removed from the cells and individual clones may be further characterized.

Using standard techniques for polyclonal and monoclonal antibody formulations,
An antibody that binds to IRAP-BP can be produced using the isolated IRAP-BP polypeptide, or a portion thereof or one fragment thereof, as an immunogen. Although a full-length IRAP-BP polypeptide may be utilized, or alternatively, the present invention provides an antigenic peptide fragment of IRAP-BP for use as an immunogen. The antigenic peptide of IRAP-BP contains at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2, and an antibody raised against the peptide is
Includes an epitope of IRAP-BP that forms a specific immune complex with IRAP-BP. Preferably, the antigenic peptide has at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues. It may contain a group.

A suitable epitope encompassed by the antigenic peptide is a region of IRAP-BP located on the surface of the protein, for example a hydrophilic region.

[0077] IRAP-BP immunogens are typically used to generate antibodies by immunizing a suitable subject (eg, rabbit, goat, mouse or other mammal) with the immunogen. Suitable immunogenic preparations may include, for example, a recombinantly expressed IRAP-BP polypeptide or a chemically synthesized IRAP-BP polypeptide. The formulation may further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent. Immunogenic IRAP-B
Immunization of a suitable subject with a P formulation elicits a polyclonal anti-IRAP-BP antibody response.

Thus, another aspect of the invention pertains to anti-IRAP-BP antibodies. As used herein, the term “antibody” refers to an immunoglobulin molecule and an immunologically active portion of the immunoglobulin molecule, ie, an antigen-binding antigen that specifically binds (immunoreacts with) an antigen, such as IRAP-BP. A molecule containing a site. Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F (ab ') 2 fragments, which can be made by treating the antibody with an enzyme such as pepsin. It is. The present invention provides polyclonal and monoclonal antibodies that bind to IRAP-BP.
As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a population of antibody molecules that includes only one antigen binding site capable of immunoreacting with a particular epitope of IRAP-BP. Thus, the composition of a monoclonal antibody is typically one that exhibits a single binding affinity for the particular IRAP-BP polypeptide with which it is immunoreactive.

[0079] Polyclonal anti-IRAP-BP antibodies can be produced by immunizing a suitable subject with IRAP-BP immunogen, as described above. The anti-IRAP-BP antibody titer of the immunized subject is monitored over a period of time by standard techniques, eg, enzyme-linked immunosorbent assay (ELISA) using immobilized IRAP-BP. You may. If necessary, apply antibody molecules directed to IRAP-BP to mammals (eg, blood).
And further purified by known techniques such as protein A chromatography to obtain an IgG fraction. After a suitable time after immunization, e.g., anti-IRAP-BP
When the antibody titer is highest, the cells producing the antibody may be collected from the subject and used to produce monoclonal antibodies by standard techniques, including, for example, , Kohler and Milstein (1975) Nature 256: 495-497) (
Brown et al. (1981) J. Immunol. 127: 539-46; Brown et al. (1980) J. Biol.
Chem .255: 4980-83; Yeh et al. (1976) PNAS 76: 2927-31; and Yeh et al. (
1982) Int. J. Cancer 29: 269-75), the more recently described human B cell hybridoma technology (Kozbor et al. (1983
) Immunol Today 4:72), EBV-hybridoma technology (Cole et al. (1985), Monocl
onal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma technology. Techniques for producing monoclonal antibody hybridomas are known (roughly RH Kenneth, in Monoclonal Antibodies: A New Dimensi).
on In Biological Analyses, Plenum Publishing Corp., New York, New York (
1980); EA Lerner (1981) Yale J. Biol. Med., 54: 387-402; ML Gefter
et al. (1977) Somatic Cell Genet. 3: 231-36). Briefly, an immortal cell line (typically myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with IRAP-BP immunogen as described above,
The resulting culture supernatant of the hybridoma cells was screened, and IRAP-BP
A hybridoma producing a monoclonal antibody that binds to is identified.

[0080] Any of a number of known protocols used to fuse lymphocytes and immortal cell lines may be applied for the purpose of generating anti-IRAP-BP monoclonal antibodies (see, eg, supra). G. Galfre et al. (1977) Nature 266: 55052
Gefter et al. Somatic Cell Genet .; Lerner, Yale J. Biol. M cited above.
ed .; see Kenneth, Monoclonal Antibodies, cited above). further,
Those skilled in the art will appreciate that there are many variations on such methods, which will also be useful. Typically, an immortal cell line (eg, a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, mouse hybridomas can be made by fusing lymphocytes from mice immunized with an immunogenic preparation of the invention to an immortal mouse cell line. A preferred immortal cell line is a medium containing hypoxanthine, aminopterin and thymidine (
This is a mouse myeloma cell line that is sensitive to HAT medium). For example, P3-NS1 / 1-Ag4-1
Any of a number of myeloma cell lines, such as the P3-x63-Ag8.653 or Sp2 / O-Ag14 myeloma lines, may be used as fusion partners based on standard techniques. These myeloma cell lines are available from the ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Thus, hybridoma cells born from this fusion are selected using HAT medium, and unfused myeloma cells and non-proliferatively fused myeloma cells will die in HAT medium (not fused). Spleen cells die after a few days because they have not been transformed). Hybridoma cells producing the monoclonal antibody of the present invention,
Detection is achieved by screening the hybridoma culture supernatants for antibodies that bind to IRAP-BP, eg, using a standard ELISA assay.

[0081] Instead of producing a hybridoma that secretes a monoclonal antibody, a recombinant combinatorial immunoglobulin library (eg, an antibody phage display library) is prepared using IRAP-B.
By screening with P, monoclonal anti-IRAP-BP antibodies can be identified and isolated, and thus immunoglobulin libraries that bind to IRAP-BP
Members can be isolated. Kits for preparing and screening a phage display library are commercially available (for example, Recomb manufactured by Pharmacia).
inant Phage Antibody System, Catalog No. 27-9400-01; and SurfZAPTM Phage Display Kit, manufactured by Stratagene, Catalog No. 240612). In addition, examples of methods and reagents particularly adapted for use in producing and screening antibody display libraries include, for example, Radner et al., U.S. Patent No. 5,223,409, Kang et al., PCT International Publication No.WO 92/18619, Dower et al. PCT International Publication No.WO 91/17271,
Winter et al. PCT International Publication No. WO 92/20791, Markland et al. PCT International Publication No. WO 92/15679, Braitling et al. PCT International Publication No. WO 93/01
No. 288, PCT International Publication No. WO 92/01047 from McAferti et al., PCT International Publication No.WO 92/09690 from Garrard et al., PCT International Publication No.WO 90/0 from Ladner et al.
No. 2809, Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992
) Hum. Antibod. Hybridomas 3: 81-85; Huse et al. (1989) Science 246: 1275-
1281; Griffiths et al. (1993) EMBO J 12: 725-734; Hawkins et al. (1992) J
Mol. Biol. 226: 889-896; Clarkson et al. (1991) Nature 352: 624-628; Gra.
m et al. (1992) PNAS 89: 3576-3580; Garrad et al. (1991) Bio / Technology 9
: 1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res. 19: 4133-4137; Barbas
et al. (1991) PNAS 88: 7978-7982; and McCafferty et al. Nature (1990) 3
48: 552-554.

In addition, recombinant anti-IRAP-BP antibodies, including chimeric and humanized monoclonal antibodies, including both human and non-human moieties, that can be made using standard recombinant DNA techniques are also provided by the present invention. Within the range. Such chimeric and humanized monoclonal antibodies can be produced using recombinant DNA techniques known in the art, including, for example, Robinson et al. In International Application No. The method described in US86 / 02269, and Akira et al., European Patent Application No. 18
No. 4,187, Taniguchi, M. European Patent Application No. 171,496, Morrison et al. European Patent Application No. 173,494, Neuberger et al. PCT International Publication No.
WO 86/01533, Caviley et al., U.S. Patent No. 4,816,567, Caviley et al., European Patent Application No. 125,023, Better et al. (1988) Science 240: 1041-1.
043; Liu et al. (1987) PNAS 84: 3439-3443; Liu et al. (1987) J. Immunol.
139: 3521-3526; Sun et al. (1987) PNAS 84: 214-218; Nishimura et al. (1987
) Canc. Res. 47: 999-1005; Wood et al. (1985) Nature 314: 446-449; and Sha.
w et al. (1988) J. Natl. Cancer Inst. 80: 1553-1559); Morrison, SL (1
985) Science 229: 1202-1207; Oi et al. (1986) BioTechniques 4: 214; Winter
US Patent 5,225,539; Jones et al. (1986) Nature 321: 552-525; Verhoeya
n et al. (1988) Science 239: 1534; and Beidler et al. (1988) J. Immunol.
141: 4053-4060, there is a method described.

Using an anti-IRAP-BP antibody (eg, a monoclonal antibody), IRAP-BP can be isolated by standard techniques, such as affinity chromatography or immunoprecipitation. With an anti-IRAP-BP antibody, it is easy to purify native IRAP-BP from cells or to purify recombinantly produced IRAP-BP expressed in host cells. In addition, anti-IRAP-BP antibodies may be used to detect IRAP-BP polypeptides (eg, in cell lysates or cell supernatants) to assess IRAP-BP polypeptide abundance and expression patterns. it can. Using anti-IRAP-BP antibody in diagnosis,
Protein mass in tissues can also be observed as part of a laboratory procedure, for example, to determine the efficacy of a particular treatment regimen. Detection can be facilitated by binding the antibody to a detectable substance (ie, physically linked). Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, emitting materials, bioluminescent materials and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, alkaline galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; Examples of fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials Are luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125I, 131I, 35S, or 3H.

III. Recombinant Expression Vectors and Host Cells Another aspect of the invention relates to a vector, preferably an expression vector, comprising a nucleic acid encoding an IRAP-BP polypeptide (or a portion thereof). The term “vector” as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which is a circular double-stranded DNA loop into which additional DNA portions can be ligated. Another type of vector is a virus vector,
Therein additional DNA portions can be ligated into the viral genome.
Some vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a recent origin of replication and episomal mammalian vectors). Other vectors, such as non-episomal mammalian vectors, are integrated into the genome of a host cell upon introduction into the host cell, and thus are replicated along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Generally, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to encompass other forms of expression vectors that perform equivalent functions, such as, for example, viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

A recombinant expression vector of the invention comprises a nucleic acid of the invention in a form suitable for expressing the nucleic acid in a host cell, which means that the recombinant expression vector It is meant to include one or more regulatory sequences operably linked to the desired nucleic acid sequence, selected based on the host cell to be utilized for expression. “Operatively linked” in a recombinant expression vector means that the nucleotide sequence can be expressed (in an in vitro transcription / translation system, or in the host cell when the vector is introduced into the host cell). In such an embodiment, it is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence. "
The term "regulatory sequence" is intended to include a promoter, enhancer, or other expression control element, such as a polyadenylation signal. Such regulatory sequences include, for example, Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in particular host cells (eg, tissue-specific regulatory sequences). , Is included. Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the host cell to be transformed, the desired level of protein expression, and the like. By introducing the expression vector of the present invention into a host cell, as described herein, a nucleic acid-encoded protein or peptide including a fusion protein or peptide (eg, IRAP-BP polypeptide, mutant IRAP- BP polypeptides, fusion proteins, and the like).

[0086] The recombinant expression vectors of the invention can also be designed for expression of an IRAP-BP polypeptide in prokaryotic or eukaryotic cells. For example, an IRAP-BP polypeptide can be expressed in bacterial cells, such as, for example, E. coli, insect cells (using a baculovirus expression vector), yeast cells, or mammalian cells. Suitable host cells are further described in Goeddel, Gene Expression Technology: Methods in Enzymolog
y 185, Academic Press, San Diego, CA (1990). Depending on the selection, the recombinant expression vector may be transcribed and translated in vitro, for example, using a T7 promoter regulatory sequence and T7 polymerase.

The expression of prokaryotic proteins is exclusively performed in E. coli having a vector containing a constitutive or inducible promoter that directs the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, but often at the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes. That is, 1) increase the expression of the recombinant protein, 2) increase the solubility of the recombinant protein, and 3) act as a ligand in the affinity purification method to help the purification of the recombinant protein. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction between the fusion component and the recombinant protein so that the recombinant protein can be separated from the fusion component after purification of the fusion protein. Have been. Such enzymes, and the recognition sequences they recognize, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include glutathione-S-transferase (GS
T), maltose E binding protein, or pGEX, which fuses protein A to the target recombinant protein (Pharmacia Biotech Inc; Smith, DB and Johnson, KS (
1988) Gene 67: 31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5
(Pharmacia, Piscataway, NJ).

[0088] The purified fusion protein may be used in an IRAP-BP activity assay (eg, a direct assay as detailed below, or a competition assay, etc.), or for example, IRAP-B.
It may be used to generate antibodies specific for the P polypeptide. In one preferred embodiment, the IRAP-BP fusion protein expressed with the retroviral expression vector of the present invention is used to infect bone marrow cells, which can then be transplanted into irradiated recipients. Good. Thus, the pathology of the recipient is examined after a sufficient time has elapsed (eg, six (6) weeks later).

Examples of suitable inducible non-fused E. coli expression vectors include pTrc (Amann et al., (1988
) Gene 69: 301-315) and pET 11d (Studier et al., Gene Expression Technolog)
y: Methods in Enzymology 185, Academic Press, San Diego, California (199
0) 60-89). Expression of the target gene from the pTrc vector was determined using the hybrid trp-lac
It depends on the transcription of the host RNA polymerase from the fusion promoter. Expression of the target gene from the pET 11d vector was determined by co-expressed viral RNA polymerase (T7g
n1) -mediated transcription from the T7 gn10-lac fusion promoter. This viral polymerase was lacUV5 by host strain BL21 (DE3) or HMS174 (DE3).
Supplied from a resident prophage carrying the T7 gn1 gene under the transcriptional control of the promoter.

One strategy for maximizing the expression of a recombinant protein in E. coli is to express the protein in a host bacterium that has impaired its ability to proteolytically cleave the recombinant protein. (Gottesman, S., Gene Expression Techn
ology: Methods in Enzymology 185, Academic Press, San Diego, California
(1990) 119-128). Another strategy is that individual codons for each amino acid are E. coli.
This is a method in which the nucleic acid sequence of a nucleic acid inserted into an expression vector is changed so as to be used preferentially in E. coli (Wada et al., (1992) Nucleic Acids Res. 20: 2
111-2118). Such alterations of the nucleic acid sequence of the present invention can be made by standard DNA synthesis techniques.

[0091] In another embodiment, the IRAP-BP expression vector is a yeast expression vector. Examples of vectors used for expression in the yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987)
Embo J. 6: 229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30: 933-943
), PJRY88 (Schultz et al., (1987) Gene 54: 113-123), pYES2 (Invitrogen, San Diego, CA), and picZ (Invitrogen, San Diego, CA).

[0092] Optionally, the IRAP-BP polypeptide may be expressed in insect cells using a baculovirus expression vector. Baculovirus vectors that can be used to express proteins in cultured insect cells (eg, Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3: 2156-2165) and the pVL series ( Luckl
ow and Summers (1989) Virology 170: 31-39).

[0093] In yet another embodiment, the nucleic acids of the invention are expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Natur
e 329: 840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6: 187-195. When used in mammalian cells, the control function of this expression vector is often provided by viral regulatory elements. For example, commonly used promoters are polyoma,
It is derived from adenovirus 2, cytomegalovirus and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see Sambrook, J., Fritsh, EF, and Maniatis, T. Molecular Cloning: A La
boratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Ha
rbor Laboratory Press, Cold Spring Harbor, NY, 1989, Chapters 16 and 17
See chapter.

In another embodiment, the recombinant mammalian expression vector is one that can preferentially direct the expression of the nucleic acid in a particular cell type (eg, to express the nucleic acid,
Tissue specific regulatory elements are used). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include albumin promoter (liver-specific, Pinkert et al. (1987) Genes Dev. 1: 268-277), lymphoid-specific promoter (Calame and Eaton (1988) Adv. Immunol. 43: 235-275), especially T cell receptor promoters (Winoto and Baltimore (1989) EMBO J. 8: 729-733) and immunoglobulins (Banerji et al. (1983) Cell 33: 729-). 740; Queen and Baltimore
(1983) Cell 33: 741-748), a neuron-specific promoter (eg, a neurofilament promoter, Byrne and Ruddle (1989) PNAS 86: 5473-5477), a pancreas-specific promoter (Edlund et al. (1985) Science 230: 912-916), and mammary gland-specific promoters (eg, whey promoter, US Pat. No. 4,873,316 and European Patent Publication No. 264,166). For example, the mouse hox promoter (Kessel and Gruss
(1990) Science 249: 374-379) and the α-fetoprotein promoter (Campes and T
Developmentally regulated promoters, such as ilghman (1989) Genes Dev. 3: 537-546), are also included.

Further, the present invention provides a recombinant expression vector containing the DNA molecule of the present invention cloned therein in the antisense direction. That is, this DNA molecule has an Rsense that is antisense to IRAP-BP mRNA (by transcription of the DNA molecule of interest).
It is operatively linked to a regulatory sequence in such a way that expression of the NA molecule is possible. The regulatory sequence operatively linked to the nucleic acid cloned in the antisense orientation may be selected from those that direct the continuous expression of the antisense RNA molecule in various cell types, such as viral promoters and / or enhancers, Alternatively, a regulatory sequence may be selected that directs constitutive, tissue-specific or cell type-specific expression of the antisense RNA. The antisense expression vector may be in the form of a recombinant plasmid, phagemid or attenuated virus, wherein the antisense nucleic acid comprises
It will be produced under the control of a highly efficient regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a description of the regulation of gene expression using antisense genes, see Weintraub, H. et al., Antisens
e RNA as a molecular tool for genetic analysis, Reviews-Trends in Gene
tics, Vol. 1 (1) 1986.

[0096] Another embodiment of the present invention relates to a host cell into which the recombinant expression vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. Such terminology is understood to refer not only to the particular subject cell, but also to the progeny or potential progeny of such cell. In effect, such progeny may not be identical to the parent cell, since certain modifications may occur in subsequent generations either due to mutations or the effects of the environment. It is encompassed within the terminology used herein.

[0097] The host cell can be any prokaryotic or eukaryotic cell. For example, an IRAP-BP polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast cells or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known in the art.

[0098] Vector DNA may be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, "transformation" and "transformation"
The term "transfection" is used in the art for introducing foreign nucleic acids (eg, DNA) into host cells, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. It is intended to refer to the various technologies that are recognized in. Suitable methods for transforming or transfecting host cells are described in Sambr
ook, et al. (Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spr.
ing Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY, 1989) and other research manuals.

For stable transfection of mammalian cells, depending on the expression vector and transfection technique used, only a small fraction of the cells
It is known that foreign DNA is not integrated into their genomes. To identify and select for these integrants, it is common to introduce a gene encoding a selectable marker (eg, resistance to antibiotics) into the host cell along with the gene of interest. Suitable selectable markers include those that confer resistance to the drug, such as, for example, G418, hygromycin, and methotrexate. The nucleic acid molecule encoding the selectable marker can be introduced into the host cell on the same vector as that encoding the IRAP-polypeptide, or can be introduced on a separate vector.
Cells stably transfected with the introduced nucleic acid can be distinguished by drug selection (eg, cells that have incorporated the selectable marker gene will survive and others will die).

A host cell according to the present invention, such as a prokaryotic or eukaryotic host cell in culture, can also be used to produce (ie, express) an IRAP-BP polypeptide. Accordingly, the present invention further provides IRAP-B using the host cells of the present invention.
It also provides a method for producing a P polypeptide. In one embodiment, the method comprises transforming a host cell according to the present invention (pre-transfected with a recombinant expression vector encoding an IRAP-BP polypeptide) into a medium suitable for producing the IRAP-BP polypeptide. Culturing in the medium. In another embodiment, the method further comprises isolating the IRAP-BP polypeptide from the medium or the host cell.

Furthermore, the host cells of the present invention can also be used to produce transgenic non-human animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or embryonic stem cell into which a sequence encoding IRAP-BP has been previously introduced. Thus, using such a host cell, the exogenous IRAP-BP sequence is pre-introduced into its genome, a non-human transgenic animal, or a pre-endogenous IRAP-BP sequence is altered. Some homologous recombinant animals can be produced. Such animals can be used to study the function and / or activity of IRAP-BP and
It is useful in identifying and / or evaluating modulators of RAP-BP activity. As used herein, a "transgenic animal" is a non-human animal, preferably a mammal, more preferably a rat or mouse, such as a rat or mouse, wherein one or more of the cells of the animal contains the transgene. Teeth. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA that integrates into the genome of a single cell, from which transgenic animals are generated, which exogenous DNA remains in the genome of a mature animal and, therefore, It directs the expression of the encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, the term "homologous recombinant animal" refers to an endogenous gene of the animal, such as an embryonic cell of the animal, and an exogenous DNA molecule introduced into the cell of the animal before the animal develops. Endogenous I
Non-human animals, preferably mammals, more preferably mice, wherein the RAP-BP gene has been altered.

The transgenic animal of the present invention can be prepared by introducing a nucleic acid encoding IRAP-BP into the male pronucleus of a fertilized oocyte, for example, by microinjection, retroviral infection, etc., and transferring the oocyte to pseudopregnancy. By developing in female Foster animals,
Can be made. IRAP-BP-1 such as those of SEQ ID NO: 4 or SEQ ID NO: 6
The cDNA sequence can be introduced as a transgene into the genome of a non-human animal. Alternatively, for example, a mouse IRAP-BP gene (eg, SEQ ID NO: 1 or SEQ ID NO:
Non-human homologs of the human IRAP-BP gene, such as 3), may be used as the transgene. Depending on the selection, for example, a homologue of the IRAP-BP gene such as the IRAP-BP-2 gene,
It may be isolated based on the hybridization of SEQ ID NO: 1 or SEQ ID NO: 4 to the IRAP-BP cDNA and used as a transgene (further described in subsection I above). Furthermore, by including an intron sequence and a polyadenylation signal in the transgene, the expression efficiency of the transgene can be increased. A tissue-specific regulatory sequence may be operatively linked to the IRAP-BP transgene to direct the expression of an IRAP-BP polypeptide to a particular cell. Methods for the manipulation of embryos and the production of transgenic animals by microinjection, particularly animals such as mice, are well known in the art, for example, both U.S. Pat.Nos. 4,736,866 and 4,870,009 to Leder et al. U.S. Patent No. 4,873,191 and Hogan, B., M.
anipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, NY, 1986). Similar methods have been used to generate other transgenic animals. The transgenic founder animal is responsible for the presence of the IRAP-BP transgene in its genome and / or
Alternatively, it can be identified based on the expression of IRAP-BP mRNA in the tissues or cells of the animal. Thus, the transgenic founder animal can be used to breed additional animals with the transgene. Further, the transgenic animal having the transgene encoding the IRAP-BP polypeptide may be another transgenic animal having another transgene.

In order to produce a homologous recombinant animal, deletion, addition or substitution is introduced in advance, and the IRAP
A vector is created that contains at least a portion of the IRAP-BP gene that has been altered, eg, by functionally disrupting the BP gene. The IRAP-BP gene may be a human gene (eg, cDNA of SEQ ID NO: 4 or SEQ ID NO: 6), but more preferably human IRAP
It may be a non-human homolog of the BP gene. For example, to construct a homologous recombination vector suitable for altering the endogenous IRAP-BP gene in the mouse genome,
The mouse IRAP-BP gene (SEQ ID NO: 1) can be used. In one preferred embodiment, the vector is such that the endogenous IRAP-BP gene is functionally disrupted (ie, no longer encodes a functional protein) when homologous recombination occurs. (This is also called a "knockout" vector.) In some selections, the endogenous IRAP-BP gene is mutated or altered when homologous recombination occurs, but still encodes a functional protein (eg, upstream Regulatory region has been altered and therefore this endogenous IR
Vectors can be designed such that the expression of the AP-BP polypeptide has been altered). In this homologous recombination vector, the changed part of the IRAP-BP gene has the additional nucleic acid sequence of the IRAP-BP gene on both sides of its 5 'end and 3' end, so that the vector has Exogenous IRAP-BP gene and endogenous IRA in embryonic stem cells
Homologous recombination with the P-BP gene is to take place. This additional, flanking, or flanking, IRAP-BP nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5 'and 3' ends) are included in the vector (for homologous recombination vectors, see, eg, Thomas, KR and Capecchi, MR (1987) Cell 51:
See 503). This vector is introduced (eg, by electroporation) into an embryonic stem cell line, and cells in which the introduced IRAP-BP gene has been homologously recombined with the endogenous IRAP-BP gene are selected (eg, Li , E. et al. (1992) Cell 69: 915). Next, the selected cells are injected into blastocysts of an animal (eg, a mouse) to form an aggregation chimera (eg, Bradley, A. in Teratocarcinomas and
Embryonic Stem Cells: A Practical Approach, EJ Robertson, ed. (IRL, Ox
ford, 1987) pp. 113-152). The chimeric embryo is then implanted into a suitable pseudopregnant female Foster animal and the embryo brought to term. If progeny that have homologously recombined DNA in their germ cells are used, the transgene is transmitted in the germ line, so that all of the animal's cells contain homologously recombined DNA. Animals can be bred. Methods for constructing homologous recombination vectors and animals are further described in Bradley, A. (1991) Current Opinion in Biot.
echnology 2: 823-829 and PCT International Publication No. WO 90 by Le Murek et al.
No. 11354, WO 91/01140 by Smithies et al., WO 92/0 by Jillstra et al.
968 and WO 93/04169 by Burns et al.

In another example, transgenic non-human animals can be made that contain selected systems that allow for regulated expression of the transgene. One example of such a system is the cre / loxP recombinase system of bacteriophage P1. For a description of the cre / loxP recombinase system, see, eg, Lakso et al. (1992) PNAS 89: 6232-623.
Please refer to 6. Another example of a recombinase system is the Saccharomyces cerevisiae FLP recombinase system (O'Gorman et al. (1991) Science 251: 1351-1355).
). When regulating the expression of a transgene using the cre / loxP recombinase system, use Cr
There is a need for an animal that contains a transgene that encodes both the e-recombinase and the selected protein. Such animals may be bred by crossing two transgenic animals, for example, one containing the transgene encoding the selected protein and the other containing the transgene encoding the recombinase. It can be provided through the construction of "heavy" transgenic animals.

The non-human transgenic animal clones described herein are Wilmut clones.
, I. et al. (1997) Nature 385: 810-813. Briefly, one cell is isolated from a transgenic animal, such as a somatic cell, and induced to exit the growth cycle and enter the G0 phase. The quiescent cells are then fused to enucleated oocytes from an animal of the same species from which the quiescent cells were isolated, for example, through the use of electric pulses. The reconstructed oocytes are cultured until they have developed into morula or undifferentiated germ cells, and then transferred to pseudopregnant female Foster animals. A child born of this female Foster animal will be a clone of the original animal from which the cells were isolated, for example, somatic cells.

IV. Drug composition IRAP-BP nucleic acid molecule, IRAP-BP polypeptide, anti-IRAP-BP antibody, IRAP based on the present invention
-BP ligand, and an IRAP-BP modulator (also referred to herein as an "active compound") can be incorporated into a pharmaceutical composition suitable for administration.
Typically, it will include the nucleic acid molecule, protein, antibody, or modulatory compound of interest, and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" refers to any solvent, dispersion medium, coating, antibacterial and antifungal agent, isotonic agent, and absorption delay compatible with pharmaceutical administration. It is intended to include agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in compositions is contemplated. In addition, auxiliary active compounds may be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, etc., and oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration. Parenteral, intradermal,
Alternatively, the solution or suspension used for subcutaneous administration may contain the following components. That is, for example, water for injection, physiological saline, fixed oil, sterile diluents such as polyethylene glycol, glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl paraben, ascorbic acid or sodium bisulfite, etc. Antioxidants, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetate, citrate or phosphate, and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. . For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremopho
r ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and mold. The carrier can be a solvent or dispersion medium, including, for example, water, ethanol, polyols (eg, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin, maintaining the required particle size in the case of dispersion, and utilizing a surfactant. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, etc., and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

A sterile injectable solution is obtained by bringing the required amount of the active compound (eg, an IRAP-BP polypeptide or an anti-IRAP-BP antibody) to one or more of the components listed above, as appropriate. Together with the above, it can be prepared by incorporating it into a suitable solvent followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, so that
A powder is produced which comprises the active ingredient and the further desired ingredients from the solution which have been previously sterile filtered.

[0110] Oral compositions generally include an inert diluent or an edible carrier. They may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. For use as a mouthwash, an oral composition can be prepared by using a fluid carrier. In this case, the compound in the fluid carrier is used orally and swished (original: swish). Or expectorated or swallowed. A pharmaceutically compatible binding agent, and / or
Adjuvant materials can also be included as part of the composition. Tablets, pills, capsules, troches, and the like, may contain any of the following ingredients, or compounds of a similar nature: That is, microcrystalline cellulose, binders such as tragacanth gum or gelatin, excipients such as starch or lactose, alginic acid, Prim
ogel, or disintegrants such as corn starch, magnesium stearate or Ster
lubricants such as otes, glidants such as colloidal silicon dioxide
), Sweeteners such as sucrose or saccharin, or flavors such as peppermint, methyl salicylate, or orange flavors.

For administration by inhalation, the compounds are delivered in the form of a pressurized container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or an aerosol spray from a nebulizer.

Further, systemic administration may be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be through nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, gels, or creams as generally known in the art.

The compounds may also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or fixed enemas for rectal delivery.

In one example, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. . For example, biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester, and polylactic acid can also be used. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals. Liposomal suspensions (including liposomes targeting monoclonal antibodies to viral antigens to infected cells) may be used as pharmaceutically acceptable carriers. These can be prepared according to methods known in the art, for example, as described in US Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral resuscitation in unit dosage form for ease of administration and uniformity of dosage. Unit dose form as used herein refers to physically discrete units prepared as unit doses for the subject to be treated, each unit being associated with a required pharmaceutical carrier. It contains a predetermined amount of the active compound which is calculated to produce the desired therapeutic effect. Specific details of the unit dosage forms of the present invention are due to the particular characteristics of the compounds, the particular therapeutic effect sought to be achieved and the formulation of such active compounds for treatment of an individual. It is determined by and directly dependent on the limitations inherent in the industry.

The toxic and therapeutic effects of such compounds may be determined by cell culture or cell culture, eg, by determining the LD50 (lethal dose for 50% of the population) and ED50 (therapeutically effective dose for 50% of the population). It can be determined by standard pharmaceutical techniques in laboratory animals. The dose ratio between toxic and therapeutic effects is the therapeutic index and the ratio LD50 / E
It can be represented by D50. Compounds that exhibit large therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used, but in order to minimize potential damage to uninfected cells and thus reduce side effects, delivery systems targeting such compounds to diseased tissue sites are needed. Care must be taken to design.

[0117] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. Dosages will vary within this range depending on the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose may be estimated initially from cell culture assays. To obtain a circulating plasma concentration range that includes the IC50 as determined in cell culture (ie, the concentration of the test compound that inhibited half of the symptoms—maximal), doses may be made in animal models. Such information can be used to more accurately determine useful doses in humans. The amount in plasma may be measured, for example, by high performance liquid chromatography.

A nucleic acid molecule of the present invention may be inserted into a vector and used as a gene therapy vector. Gene therapy vectors can be, for example, intravenous, topical (see US Pat. No. 5,328,470) or stereotactic (eg, Chen et al. (1994) PNAS 91: 3054-3057).
) Can be delivered to a subject. Pharmaceutical formulations of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a delayed release matrix with a genetic carrier embedded therein and having a delivery vehicle embedded therein. Depending on the selection, the pharmaceutical formulation may contain one or more of the gene delivery systems that produce the gene delivery system, provided that a complete gene delivery vector, such as a retroviral vector, can be produced intact from the recombinant cells. Cells may be included.

The pharmaceutical composition may be included in a container, pack, or dispenser together with instructions for administration.

V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologs, and antibodies described herein can be used in at least one of the following methods. A) screening assays, b) diagnostic and / or prognostic assays, or c) methods of treatment (
For example, treatment and prevention). As described herein, an IRAP-BP polypeptide of the invention has one or more of the following activities. (I) an activity that interacts with, for example, a non-IRAP-BP molecule in a cell, and an IRAP-BP target molecule (eg, such as IRAP and / or GLUT4); IRAP-
Activity binding to a BP target molecule (such as IRAP and / or GLUT4), (iii) non-IRAP-BP molecules in cells and IRAP-BP target molecule (such as IRAP and / or GLUT4)
And (iv) the activity of recognizing a transport motif within, and (iv) non-IRAP-BP
Molecules, and have the activity of binding to a transport motif within an IRAP-BP target molecule (eg, IRAP and / or GLUT4), such as (1) modulation of translocation of IRAP-BP target molecule, (2), GLUT4 (Eg, exocytosis), (3) modulation of IRAP transposition (eg, exocytosis), (4) modulation of the classification of IRAP-BP target molecule, (5) IRAP and / or GLUT4 Modulation of classification, (6) modulation of retention of IRAP-BP target molecules, (7) modulation of retention of IRAP and / or GLUT4 (eg, in specialized intracellular compartments, such as the circulating compartment of endocytosis). (8) Modulation of IRAP-BP target molecule entry into circulating vesicles, (9) Modulation of IRAP and / or GLUT4 entry into circulating vesicles, (10) Intracellular IRAP-BP target molecule Regulation of transport and / or retention, (11) intracellular transport and / or IRAP and / or GLUT4 Regulation of lifting, and (12) may be used to modulate, intracellular transport of vesicles containing GLUT4 and / or IRAP. An isolated nucleic acid molecule according to the present invention can, for example, express an IRAP-BP polypeptide (eg, via a recombinant expression vector in a host cell in gene therapy applications), as described further below. Or (in biological samples)
It can be used to detect genetic changes in IRAP-BP mRNA or IRAP-BP gene and to modulate IRAP-BP activity. The IRAP-BP polypeptide is an IRAP-BP
It can be used to treat disorders characterized by insufficient or excessive production of polypeptides and / or IRAP-BP target molecules. In addition, the present IRAP-BP polypeptides can be used to screen for agents or compounds that modulate IRAP-BP activity,
Also used to treat disorders characterized by insufficient or excessive production of a BP polypeptide or production of an IRAP-BP polypeptide type having reduced or abnormal activity compared to an IRAP-BP wild-type protein. Can be. Furthermore, the anti-IRAP-BP antibody of the present invention
It can also be used to detect and isolate RAP-BP polypeptides, modulate the bioavailability of IRAP-BP polypeptides, and modulate IRAP-BP activity.

A. Screening Assays The present invention provides for binding to an IRAP-BP polypeptide, or for example, IRAP-BP expression or IRAP
A method for identifying modulators having a stimulatory or inhibitory effect on -BP activity, ie, candidate or test compounds or agonists (eg, peptides, peptidomimetics, small molecules or other agents) (herein "screening assays") Also referred to). Such modulators include, for example, (1)
Modulation of insulin sensitivity (eg, increased insulin sensitivity), (2) IRAP-B
It is useful for modulating the translocation of a P target molecule, and (3) controlling diabetes (eg, type II diabetes).

In one embodiment, the present invention provides an assay for screening candidate or test compounds that bind to or modulate an IRAP-BP polypeptide or polypeptide or a biologically active portion thereof. I will provide a. The test compounds of the present invention can be used in biological libraries, spatially assignable parallel solid or solution phase libraries, synthetic library methods requiring deconvolution, "one bead one compound" (original: one-
It can be obtained using any of a number of combinatorial library methods known in the art, including "bead one-compound" library methods, and synthetic library methods using affinity chromatography sorting. Biological library approaches are limited to peptide libraries, while the other four approaches are applicable to libraries of compound peptides, non-peptide oligomers or small molecules (Lam, KS (1997) Anticancer Drug Des. 12: 145). ).

Examples of methods for synthesizing molecular libraries are described, for example, in DeWitt et al. (1993) Proc. Natl.
Acad. Sci. USA 90: 6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA
91: 11422; Zuckermann et al. (1994) .J. Med.Chem. 37: 2678; Cho et al.
(1993) Science 261: 1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Eng
l. 33: 2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33: 2061; an
d in Gallop et al. (1994) J. Med. Chem. 37: 1233.

The library of compounds is in solution (eg, Houghten (1992) Biotechniques 13: 412).
-421) or on a bead (Lam (1991) Nature 354: 82-84), chip (Fodor (1993) N
ature 364: 555-556), on bacteria (Ladner USP 5,223,409), on spores (Ladner USP '409)
) On the plasmid (Cull et al. (1992) Proc Natl Acad Sci USA 89: 1865-1869)
Or on phage (Scott and Smith (1990) Science 249: 386-390); (Devlin (19
90) Science 249: 404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.
87: 6378-6382); (Felici (1991) J. Mol. Biol. 222: 301-310); (Ladner above)
May be presented.

In one example, one assay involves contacting a cell expressing an IRAP-BP polypeptide or a biologically active portion thereof with a test compound, wherein the test compound comprises:
A cell-based assay for determining the ability of an IRAP-BP polypeptide or a biologically active portion thereof to modulate the activity. The cell may be, for example, from a mammal or a yeast cell. For example, an IRAP-BP polypeptide may be expressed heterologously to a cell or expressed natively to a cell.
The ability of the test compound to modulate the activity of an IRAP-BP polypeptide or a biologically active portion thereof can be assayed for any of the activities of the IRAP-BP polypeptide described herein. You can find out. In addition, IRAP
The ability of the test compound to modulate the activity of a -BP polypeptide or a biologically active portion thereof can be determined by assaying for the activity of an IRAP-BP target molecule.
You can find out. In one example, the ability of the test compound to modulate the activity of an IRAP-BP polypeptide or a biologically active portion thereof is assayed for the activity of IRAP (eg, assaying for aminopeptidase activity). Determination) by performing the assay. In one preferred embodiment, the cells expressing the IRAP-BP polypeptide or a biologically active portion thereof further comprise IRAP-B
The P target molecule or a biologically active part thereof was expressed. In another preferred embodiment, the cells express IRAP and / or GLUT4, or a biologically active portion thereof. In yet another preferred example, the cells are contacted with a compound that stimulates an activity associated with IRAP-BP, such as insulin, and a test compound is tested for the ability of IRAP-BP to modulate the associated activity. .

In another example, one assay involves translating cells expressing an IRAP-BP polypeptide or a biologically active portion thereof with a bioactive peptide derived from an IRAP-BP target molecule. Contacting a test compound to determine the ability of the test compound to modulate the activity of an IRAP-BP polypeptide or a biologically active portion thereof.
This is a cell-based assay. In one embodiment, the bioactive peptide is
It is obtained from the amino acid sequence of GLUT4. In another embodiment, the bioactive peptide is derived from the amino acid sequence of IRAP. In another embodiment, the bioactive peptide corresponds to the cytoplasmic domain of either GLUT4 or IRAP. In yet another embodiment, the bioactive peptide is GLUT4
Or it corresponds to the transport motif of IRAP.

According to a cell-based assay according to the present invention, the ability of a test compound to modulate the activity of an IRAP-BP polypeptide, or a biologically active portion thereof, is determined by measuring the activity of the IRAP-BP polypeptide described herein. It can be determined by assaying for any of the natural activities of the BP polypeptide. For example, transpose GLUT4, transpose IRAP, IRAP
And / or GLUT4 classification, IRAP and / or GLUT4 retention, or IRAP and / or GL
Fig. 3 is an assay for intracellular transport of vesicles containing UT4. In addition, IRAP-
To determine the activity of a BP polypeptide or a biologically active portion thereof, use IRAP-BP
Assays may be performed for indirect activity consistent with the activity of the polypeptide.
For example, the effect of a test compound on the ability of an IRAP-BP-expressing cell to take up glucose in an insulin-dependent manner can be assayed in the presence and in the assay of the test compound. In addition, the ability of a test compound to modulate the activity of an IRAP-BP polypeptide or a biologically active portion thereof
Although unnatural activity for polypeptides, cells may be engineered for activity and assayed for certain activities. For example, non-IRAP-B
Cells may be engineered to express an IRAP-BP target molecule, which is a recombinant protein containing a biologically active portion of the IRAP-BP target molecule operatively linked to a P target molecule polypeptide. In an exemplary embodiment, the cytoplasmic domain of the IRAP-BP target molecule GLUT4 or IRAP is operatively linked to a transmembrane domain and an extracellular domain, for example, a transferrin receptor, to determine the chimeric protein's ability to transport intracellularly. The effect of the test compound may be determined. (Jonhson et al. (1998)
J. Biol. Chem. 273: 17968-17977 provides an example of making such a chimera. Furthermore, in a preferred embodiment, it is contemplated that the cell-based assay according to the present invention comprises a final step of identifying the compound as a modulator of IRAP-BP activity.

In yet another embodiment, the assay of the invention comprises contacting an IRAP-BP polypeptide or a biologically active portion thereof with a test compound, and contacting the IRAP-BP polypeptide or a biologically active portion thereof. Cell-free assays, such as testing the ability of the test compound to bind to a chemically active moiety. The binding of the test compound to the IRAP-BP polypeptide, for example, by binding the test compound or IRAP-BP polypeptide to a radioisotope or enzyme label, the binding of the test compound to the IRAP-BP polypeptide, The determination can be performed by detecting a labeled compound or polypeptide in the complex, or the like. For example, the test compound or polypeptide may be directly or indirectly labeled with 125I, 35S, 14C, or 3H, and the radioisotope may be detected by directly counting radiation or performing scintillation counting. Good. Alternatively, a test compound or polypeptide can be enzymatically labeled, such as with horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzyme label detected by determining the conversion of a suitable substrate to the product. Good.

Further, the binding of the test compound to the IRAP-BP polypeptide can also be performed using a technique such as real-time biomolecular interaction analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991
Anal. Chem. 63: 2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. B
iol. 5: 699-705. As used herein, "BIA" is a technique for studying a physiologically specific interaction in real time without labeling any of the interacting substances (for example, BIAcoreTM). Changes in optical phenomena caused by surface plasmon resonance (SPR) can be used as indicators of real-time reactions between biological molecules.

In a preferred embodiment, the assay comprises contacting an IRAP-BP polypeptide, or a biologically active portion thereof, with an IRAP-BP target molecule that binds to IRAP-BP to form an assay mixture. Forming, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the IRAP-BP polypeptide, wherein the test compound , IRAP-B
Determining the ability to interact with a P polypeptide comprises determining the ability of the test compound to preferentially bind to IRAP-BP or a biologically active portion thereof, as compared to IRAP-BP. Including the step of examining. In one embodiment, the IRAP-BP target molecule is G
LUT4 protein or a biologically active portion thereof (eg, N-terminal cytoplasmic domain or transport motif). In another embodiment, the IRAP-BP target molecule is an IRAP protein or a biologically active portion thereof (eg, an N-terminal cytoplasmic domain or a transport motif).

In another example, the assay comprises contacting an IRAP-BP polypeptide or a biologically active portion thereof with a test compound, and contacting the IRAP-BP polypeptide or a biologically active portion thereof. Cell-free assays, such as testing the ability of the test compound to modulate (eg, stimulate or inhibit) the activity of a particular moiety. The ability of a test compound to modulate the activity of an IRAP-BP polypeptide can be measured, for example, by one of the methods described above for cell-based assays, by measuring the activity of a downstream IRAP-BP binding partner or target molecule. This can be determined by examining the ability of the IRAP-BP polypeptide to modulate. For example, the catalytic / enzymatic activity of the target molecule on a suitable substrate may be determined as described above (eg, the aminopeptidase activity of IRAP).

In yet another example, the cell-free assay involves contacting the IRAP-BP polypeptide or a biologically active portion thereof with an IRAP-BP target molecule that binds the IRAP-BP polypeptide. Forming an assay mixture, contacting the assay mixture with a test compound, and preferentially modulating the activity of an IRAP-BP binding partner or target molecule when compared to IRAP-BP. Determining the performance of the test compound.

In two or more embodiments of the assay method according to the invention described above, the IRAP
Immobilization of -BP or any of its binding partners / target molecules facilitates the separation of one or both proteins from their uncomplexed form and facilitates assay automation. It may be preferable to adapt. Test compound
Binding to IRAP-BP polypeptide, or IRAP- in the presence or absence of a candidate compound
Interaction of the BP polypeptide with the target molecule may be effected in any vessel suitable for containing the reactants. Examples of such containers include microtiter and
There are plates, test tubes, and microcentrifuge tubes. In one embodiment, one domain may be added to provide a fusion protein that binds one or both of these proteins to a matrix. For example, glutathione-S-transferase / IRAP-BP fusion protein or glutathione-S-transferase / target fusion protein is adsorbed to glutathione sepharose beads (Sigma Chemical Co., St. Louis, Mo.) or glutathione-derivatized microtiter plates. And then mix them with the test compound, or with the test compound and any non-adsorbed target protein or IRAP-BP polypeptide, and mix this mixture
Incubation may be under conditions such that complexes are formed (eg, physiological conditions for salt and pH). After incubation, the beads or the wells of the microtiter plate are washed to remove unbound components, in the case of beads, the matrix is fixed and the complexes are examined directly or indirectly, for example as described above. Alternatively, the complex can be dissociated from the matrix and analyzed using standard techniques.
The level of AP-BP binding or activity may be determined.

Other techniques for immobilizing proteins on matrices may also be used in the screening assays of the present invention. For example, either an IRAP-BP polypeptide or an IRAP-BP target molecule can be immobilized utilizing conjugation of biotin and streptavidin. The biotinylated IRAP-BP polypeptide or target molecule can be obtained from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (eg, Biotinylation Kit, manufactured by Pierce Chemicals, Rockford, Ill.). And fixed in wells of a 96-well plate (Pierce Chemical) coated with streptavidin. In some selections, antibodies that are reactive to the IRAP-BP polypeptide or target molecule but do not interfere with the binding of the IRAP-BP polypeptide to its target molecule are induced in the wells of the plate. Thus, unconjugated target or
IRAP-BP polypeptide may be captured in this well. In addition to those described above for the GST-immobilized complex, methods for detecting such a complex include methods for immunodetecting the complex using antibodies reactive against IRAP-BP polypeptides or target molecules,
There are enzyme binding assays that rely on detecting enzyme activity associated with the IRAP-BP polypeptide or target molecule.

In another example, modulators of IRAP-BP expression are identified, such as by contacting a cell with a candidate compound and determining the expression of IRAP-BP mRNA or protein in the cell. The expression level of IRAP-BP mRNA or protein in the presence of the candidate compound,
Compare the expression level of IRAP-BP mRNA or protein in the absence of the candidate compound.
Based on this comparison, the candidate compound can thus be identified as a modulator of IRAP-BP expression. For example, if the expression of IRAP-BP mRNA or protein is higher (statistically significantly higher) in the presence than in the absence of the candidate compound, the candidate compound will express IRAP-BP mRNA or protein. Stimulant.
Alternatively, if the expression of IRAP-BP mRNA or protein is less (statistically significantly less) in the presence than in the absence of the candidate compound, the candidate compound
Identified as an inhibitor of AP-BP mRNA or protein expression. IRAP-BP in cells
mRNA or protein expression levels can be determined by the methods described herein for detecting IRAP-BP mRNA or protein.

In yet another embodiment of the present invention, the IRAP-BP polypeptide is used as a “bait protein” in a two- or three-hybrid assay (see, eg, US Patent No. No. 5,283,317, Zervos et al. (19
93) Cell 72: 223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054
; Bartel et al. (1993) Biotechniques 14: 920-924; Iwabuchi et al. (1993)
Oncogene 8: 1693-1696; and Brent WO94 / 10300).
Other proteins that bind to or interact with BP and that are involved in IRAP-BP activity ("IRAP-BP binding proteins" or "IRAP-BP target molecules") can be identified. Such IRAP-BP target molecules may also be involved in regulating cellular activities modulated by the IRAP-BP polypeptide.

The two-hybrid system is based on the modular nature of most transcription factors, consisting of a separable DNA binding domain and an activation domain. Briefly, this assay utilizes two + different DNA constructs. In one construct, the gene encoding the IRAP-BP polypeptide is fused to a gene encoding the DNA binding domain of a known transcription factor (eg, GAL-4). In the other construct, a DNA sequence from a library of DNA sequences encoding an unknown protein ("prey" or "sample") is transformed into a gene encoding the activation domain of a known transcription factor. Fuse. If the "feed" and "prey" proteins can interact in vivo to form an IRAP-BP-dependent complex, bring the DNA-binding and activation domains of this transcription factor into close proximity . This proximity allows the transcription of a reporter gene (eg, LacZ) operatively linked to a transcriptional regulatory site responsive to the transcription factor, detects reporter gene expression, and contains this functional transcription factor. The isolated cell colony can be used to obtain a cloned gene encoding a protein that interacts with the IRAP-BP polypeptide.

The invention further relates to novel agents identified by the screening assays described above. Therefore, it is within the scope of the present invention to utilize an agent identified as described herein in a suitable animal model. For example, an agent identified as described herein (eg, an IRAP-BP modulating agent, an antisense IRAP-BP nucleic acid molecule, an IRAP-BP specific antibody, or an IRAP-BP target molecule) can be used in an animal model. The efficacy, toxicity, or side effects of treatment with such agents can be determined. Alternatively, the mechanism of action of such an agent can be examined using an agent identified as described herein in an animal model. Furthermore, the present invention relates to the use of the novel agents identified by the above-described screening assays for treatment as described herein.

B. Diagnostic Assays The portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) and antibodies against the polypeptides identified herein can be used in various ways as diagnostic and / or detection reagents. Can be used. In one embodiment, a polynucleotide reagent (eg, a portion or fragment of an IRAP-BP nucleotide sequence described herein) is used in a method for detecting an IRAP-BP polynucleotide in a biological sample.
In another embodiment, an IRAP-BP antibody is used in a method for detecting an IRAP-BP polypeptide in a biological sample. One example of a method of detecting the presence or absence of an IRAP-BP polypeptide or nucleic acid in a biological sample comprises obtaining a biological sample from a test subject,
An IRAP-BP polypeptide or a nucleic acid encoding an IRAP-BP polypeptide (eg, mRNA, genomic DNA) such that the presence of the -BP polypeptide or nucleic acid is detected in the biological sample
Contacting the biological sample with a compound or agent capable of detecting Agents suitable for detecting IRAP-BP mRNA or genomic DNA are IRAP-BP
It is a labeled nucleic acid probe that can hybridize to BP mRNA or genomic DNA. The nucleic acid probe can be a full-length IRAP-BP nucleic acid, such as, for example, the nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 4, or at least 15, 30, 50, 100, 250 or 500 nucleotides in length. Or a fragment or portion of an IRAP-BP nucleic acid, such as an oligonucleotide sufficient to specifically hybridize under stringent conditions to IRAP-BP mRNA or genomic DNA. Other probes suitable for use in the diagnostic assays of the invention are described herein.

A suitable agent for detecting an IRAP-BP polypeptide is an antibody capable of binding to the IRAP-BP polypeptide, preferably with a detectable label. Antibodies may be polyclonal, but are more preferably monoclonal. An intact antibody, or one fragment thereof (eg, Fab or F (ab ') 2) may be used.
The term "labeling" with respect to a probe or antibody refers to directly labeling or directly labeling the probe or antibody by attaching (ie, physically linking) a detectable compartment to the probe or antibody. It is intended to include indirect labeling of the probe or antibody by reactivity with another reagent. Examples of indirect labeling include primary antibody detection using a fluorescently labeled secondary antibody or biotin-based DNase detection, such as by fluorescently labeled streptavidin.
And end-labeling the A probe. The term "biological sample" is intended to include tissues, cells and biological fluids separated from a subject, as well as tissues, cells and fluids within a subject. That is, using the detection method of the present invention, in v
It is possible to detect IRAP-BP mRNA, protein or genomic DNA in a biological sample in vitro or in vivo. For example, techniques for detecting IRAP-BP mRNA in vitro include Northern hybridizations and in situ hybridizations. Techniques for detecting IRAP-BP polypeptides in vitro include enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitations and immunofluorescence. Techniques for detecting IRAP-BP genomic DNA in vitro include Southern hybridizations. In addition, techniques for detecting IRAP-BP polypeptides in vivo include:
There is a method of introducing a labeled anti-IRAP-BP antibody into a subject. For example, antibodies include
It can be labeled with a radioactive marker such that its presence and location within the subject can be detected with standard imaging techniques.

[0141] In one embodiment, the biological sample comprises protein molecules taken from a test subject. Alternatively, the biological sample can include mRNA molecules from a subject or genomic DNA from a test subject. A suitable biological sample is a tissue sample (eg, fat muscle) separated from the subject by conventional means (eg, a muscle biopsy, etc.).

In another example, the method further comprises obtaining a control biological sample from the control subject, and detecting the presence of the IRAP-BP polypeptide, mRNA, or genomic DNA in the biological sample. Contacting a control sample with a compound or agent capable of detecting an IRAP-BP polypeptide, mRNA, or genomic DNA;
Comparing the presence of the BP polypeptide, mRNA or genomic DNA with the presence of the IRAP-BP polypeptide, mRNA or genomic DNA in the test sample.

The invention further includes a kit for detecting the presence of IRAP-BP in a biological sample. For example, the kit includes a labeled compound or an agent capable of detecting an IRAP-BP polypeptide or mRNA in a biological sample, and an IRAP in the sample.
Means for determining the amount of -BP and means for comparing the amount of IRAP-BP in a sample with a standard can be included. The compound or agent may be packaged in a suitable container. Further, the kit may include instructions for using the kit to detect an IRAP-BP polypeptide or nucleic acid.

B.1. Prognostic Assays The diagnostic methods described herein may further be used to determine subjects having or at risk of developing a disease or disorder associated with abnormal IRAP-BP expression or activity. Can be used. For example, using the assays described herein, such as the diagnostic assays described above or the assays below, for example, IRAP-BP, such as insulin resistance or diabetes.
Subjects having or at risk of developing a disorder associated with the expression or activity of a polypeptide or nucleic acid can be determined. Thus, the present invention relates to abnormal IRAP-B
Provide a method for determining a disease or disorder related to P expression or activity,
In this method, a test sample is taken from a subject and the IRAP-BP polypeptide or nucleic acid (
(E.g., mRNA, genomic DNA), wherein the presence of the IRAP-BP polypeptide or nucleic acid is such that the subject has, or is at risk of developing, a disease or disorder associated with abnormal IRAP-BP expression or activity. It is for diagnosing. As used here,
“Test sample” refers to a biological sample obtained from a subject of interest. For example, the test sample may be a biological fluid, a cell sample, or a tissue (eg, muscle or fat).

In addition, using the prognostic assays described herein, a subject can be treated with an agent (eg, an agonist, antagonist, peptidomimetic) to treat a disease or disorder associated with abnormal IRAP-BP expression or activity. Ticks, proteins, peptides, nucleic acids, small molecules, or other drug candidates). For example, such a method can be used to determine whether a subject can be effectively treated with an agent for a disorder, such as, for example, insulin resistance or diabetes. Thus, the present invention provides a method for treating a subject with an abnormal IRAP-BP
The present invention provides a method for determining whether a disorder related to expression or activity can be effectively treated with a certain agent. In this method, a test sample is obtained and IRAP-B is obtained.
Detect expression or activity of a P polypeptide or nucleic acid (eg, abundant expression or activity of an IRAP-BP polypeptide or nucleic acid can be used to treat disorders associated with abnormal IRAP-BP expression or activity). And a subject to whom the agent can be administered.)

Furthermore, the method of the present invention is used to detect a genetic alteration in the IRAP-BP gene, and thus, a subject having the altered gene is at risk of developing a disorder such as insulin resistance or diabetes. You can find out if there is. In a preferred embodiment, the method comprises detecting in a cell or tissue sample from a subject at least one change that affects the integrity of the gene encoding the IRAP-BP protein. Or the presence or absence of a change in the IRAP-BP gene or the presence or absence of a misexpression of the IRAP-BP gene. For example, such a genetic change is 1
1) deletion of one or more nucleotides from the IRAP-BP gene; 2) addition of one or more nucleotides to the IRAP-BP gene; 3) one or more nucleotides of the IRAP-BP gene. 4) Rearrangement of the chromosome of the IRAP-BP gene, 5) Changes in the amount of messenger RNA transcripts of the IRAP-BP gene, 6) Abnormality of the IRAP-BP gene such as abnormal methylation pattern of genomic DNA. Modification, 7) IRAP-B
Presence of non-wild-type splicing pattern of P gene messenger RNA transcript; 8) non-wild-type amount of IRAP-BP protein; 9) allele loss of IRAP-BP gene; and 10) IRAP-BP protein. By detecting the presence of at least one of the following: an inappropriate post-translational modification. As described herein, many assay techniques are known in the art that can be used to detect changes in the IRAP-BP gene. Suitable biological samples are tissue or cell samples that have been separated from a subject by conventional means.

In some embodiments, detection of the alteration includes polymerase chain reaction (PCR) such as, for example, PCR or RACE PCR (see, eg, US Pat. Nos. 4,683,195 and 4,683,202) or ligation. Chain reaction (LCR) (eg, Landegran et al. (198
8) Science 241: 1077-1080; and Nakazawa et al. (1994) PNAS 91: 360-364), which involves a point mutation in the IRAP-BP gene. May be particularly advantageous in detecting
vaya et al. (1995) Nucleic Acids Res. 23: 675-682). The method includes the steps of collecting a sample of cells from a patient and extracting nucleic acids (
Isolating, for example, genomic nucleic acids, mRNA, or both), and under conditions such that hybridization and amplification of the IRAP-BP gene (if present) occurs.
Contacting the nucleic acid sample with one or more primers that specifically hybridize to the RAP-BP gene, and detecting the presence or absence of the amplification product, or detecting the size of the amplification product. Comparing its length with a control sample. It is expected that PCR and / or LCR will preferably be used as a preliminary amplification step in combination with any of the techniques used to detect mutations described herein.

Alternative amplification methods include self-sustaining sequence replication (Guatelli, JC et al., 199).
Natl. Acad. Sci. USA 87: 1874-1878), transcription amplification system (Kwoh, DY et al.
Natl. Acad. Sci. USA 86: 1173-1177), Q-beta replicase (Lizardi, PM et all, 1988, Bio / Technology 6: 1197), or any other nucleic acid amplification method. But then, using techniques known in the art,
The amplified molecule will be detected. These detection methods are particularly useful when detecting nucleic acid molecules, where the number of such molecules present is very small.

In an alternative embodiment, mutations in the IRAP-BP gene taken from a sample cell can be determined by a change in the restriction enzyme cleavage pattern. For example, sample and control DNA can be isolated, amplified (if selected), digested with one or more restriction endonucleases, fragment size determined by gel electrophoresis, and compared. I do. Any difference in fragment length between the sample and the control DNA is an indication that there is a mutation in the sample DNA. In addition, the use of sequence-specific riboenzymes (see, for example, US Pat. No. 5,498,531) may be used to score for the presence of specific mutations by generating or eliminating riboenzyme cleavage sites.

In another example, by hybridizing a sample and a control nucleic acid, such as DNA or RNA, to a high-density array containing hundreds or thousands of oligonucleotide probes, IRAP-BP gene mutation may be determined (Cronin
, MT et al. (1996) Human Mutation 7: 244-255; Kozal, MJ et al. (1996
) Nature Medicine 2: 753-759). For example, as discussed by Cronin, MT et al. Above, a two-dimensional array containing lightly generated (original: light-generated) DNA probes can be used to determine gene mutations in IRAP-BP. Briefly, a first hybridization array of probes is used to detect DNA in samples and controls.
By scanning over a long strand of DNA to create a linear array of probes with overlapping sequences, one can discriminate base changes between sequences. This step makes it possible to determine the point mutation. This step is followed by a second hybridization that allows the characterization of a particular mutation by using a smaller and specialized probe array that is complementary to all the mutants or mutations detected. Perform an array. Each mutation array consists of a set of parallel probes, one complementary to the wild-type gene and the other complementary to the mutated gene.

In yet another example, various sequencing reactions known in the art may be used to generate IRAP
Mutations can be detected by directly sequencing the sequence of the -BP gene and comparing the sequence of this sample IRAP-BP to the corresponding wild-type (control) sequence. Examples of sequencing reactions include Maxim and Gilbert ((1977) PNAS 74: 560) or Sanger ((1977
) Includes those based on technology developed by PNAS 74: 5463). Furthermore, mass spectrometry (for example, PCT International Publication No. WO 94/16101, Cohen et al. (1996) Adv. Chromatog
r. 36: 127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38: 1
Diagnostic assays are also performed, including sequencing methods (see 47-159) ((1995
) Biotechniques 19: 448) In some cases, any of a variety of automated sequencing methods could be used.

Other methods for detecting mutations in the IRAP-BP gene include RNA / RNA or
Methods that use protection from cleavage agents to detect mismatched bases in RNA / DNA heteroduplexes (Myers et al. (1985) Science 230: 1242) are included. Schematically, "
The technique of "mismatch cleavage" is formed by first hybridizing (labeled) RNA or DNA containing the wild-type IRAP-BP sequence to potential mutant RNA or DNA taken from a tissue sample. Begins with providing a heteroduplex. An agent that cleaves a single-stranded region of a duplex, such as one present due to base pair mismatches between control and sample strands, resulting in a double-stranded Process heavy chains. For example, an RNA / DNA duplex can be treated with RNase, and a DNA / DNA hybrid can be digested with an S1 nuclease to degrade the mismatched region with an enzyme. In another example, a DNA / DNA or RNA / DNA duplex can be treated with hydroxylamine or osmium tetroxide together with piperidine to degrade the mismatched region. After degradation of the mismatched regions, the resulting material is separated by size on a denaturing polyacrylamide gel to determine the mutation site. For example, T Cotton et al. (1988) Proc. Nat
l Acad Sci USA 85: 4397; Saleeba et al. (1992) Methods Enzymol. 217: 286-2
See 95. In one preferred embodiment, control DNA or RNA may be labeled for detection.

In yet another embodiment, the mismatch cleavage reaction is performed in a defined system with a double-stranded DN.
Detect and map point mutations in IRAP-BP cDNA from cell samples using one or more proteins that recognize mismatched base pairs of A (so-called "DNA mismatch repair" enzymes) Things. For example, E. coli mutY
The enzyme cleaves A in the G / A mismatch, and thymidine DNA glycosylase from Hela cells cleaves T in the G / T mismatch (Hsu et al. (1994) Carcinogene
sis 15: 1657-1662). In one exemplary embodiment, a probe based on an IRAP-BP sequence, such as, for example, a wild-type IRAP-BP sequence, is hybridized to a test cell cDNA or other DNA product. This duplex is treated with a DNA mismatch repair enzyme, and the cleavage product, if any, can be detected by methods such as electrophoresis protocols. See, for example, U.S. Patent No. 5,459,039.

In another example, alterations in electrophoretic mobility will be used to determine mutations in the IRAP-BP gene. For example, single-strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild-type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86: 2766, see also Cotto
n (1993) Mutat Res 285: 125-144; and Hayashi (1992) Genet Anal Tech Appl
9: 73-79). Single stranded DNA fragments of the sample and control IRAP-BP nucleic acids will be denatured and will be able to be reconstituted. Since the secondary structure of a single-stranded nucleic acid varies depending on the sequence, the resulting change in electrophoretic mobility can detect even a single base change. The DNA fragment may be labeled or detected with a labeled probe. The sensitivity of the assay will be improved with RNA (rather than DNA), whose secondary structure is more sensitive to sequence changes. In one preferred embodiment, the method utilizes heteroduplex analysis to separate double-stranded heteroduplex molecules based on differences in electrophoretic mobility (Keen et al. (1991
) Trends Genet 7: 5).

In yet another example, the movement of mutated and wild-type fragments in polyacrylamide gels containing gradient denaturants was determined by denaturing gradient gel electrophoresis (DGGE) (Mye
Assay using rs et al. (1985) Nature 313: 495). When DGGE is used as an analytical method, for example, a highly soluble GC-rich DNA of about 40 base pairs by PCR
The DNA will be modified to prevent complete denaturation, such as by adding a GC clamp portion consisting of In a further example, a temperature gradient is used in place of a denaturing gradient to examine differences in control and sample DNA mobility (Rosenbaum and Reissner (1987)
Biophys Chem 265: 12753).

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, an oligonucleotide primer with a known mutation in the center is made and then hybridized to the target DNA under conditions such that hybridization will occur only if a perfect match is found. (Saiki et al. (1986) Nature 324: 163); Saiki et al. (1989) Proc.
Natl Acad. Sci USA 86: 6230). Such an allele-specific oligonucleotide can be a PCR-amplified target DNA or a number of different variants if the oligonucleotide is attached to a hybridizing membrane and hybridizes to a labeled target DNA. Hybridize to

Alternatively, allele-specific amplification techniques that rely on selective PCR amplification could be used in connection with the present invention. Oligonucleotides used as primers for specific amplification may have the desired mutation at the central position of the molecule (
As a result, amplification becomes dependent on differential hybridization) (Gib
bs et al. (1989) Nucleic Acids Res. 17: 2437-2448), or, under appropriate conditions, where mismatches prevent or reduce extension by the polymerase. The target mutation may be present at the extreme 3 'end position (Prossner (1993) Tibtech 11: 238). In addition, it may be preferable to introduce a new restriction site in the mutated region in order to perform cleavage-based detection. (Gasparini et al. (1992) Mol. Cell Probes 6: 1). In some examples, it is anticipated that amplification could be performed using Taq ligase for amplification (Ba
rany (1991) Proc. Natl. Acad. Sci USA 88: 189). In such a case, ligation will occur only when there is a perfect match at the 3 'end of the 5' sequence, so that by examining the presence or absence of amplification, a known mutation is present at a particular site Can be detected.

The method described herein may be performed, for example, by using a prepackaged diagnostic kit that includes at least one probe nucleic acid or antibody reagent described herein, and the kit may be prepared using IRAP- It may be useful for use in diagnosing patients presenting symptoms of a disease or disorder involving the BP gene or diagnosing a family history thereof.

Additionally, any cell type or tissue that expresses IRAP-BP may be utilized in the prognostic assays described herein.

B. 2 Observation of effects during clinical trials Observation of the effect of an agonist (eg, drug or compound) on the expression or activity of IRAP-BP polypeptide (eg, modulation of insulin resistance) can be performed not only in basic drug screening but also in clinical studies. It can also be used for clinical trials. For example, in a screening assay as described herein, increasing the expression of IRAP-BP gene, protein mass, or up-regulating IRAP-BP activity may have the effect of an agonist, IRAP-BP gene expression,
It can be observed in clinical trials of subjects exhibiting reduced protein mass or down-regulated IRAP-BP activity. Alternatively, the effect of an agent determined to reduce IRAP-BP gene expression, protein mass, or down-regulate IRAP-BP activity in a screening assay may be assessed by increasing IRAP-BP gene expression, protein mass. Alternatively, it may be observed in clinical trials of subjects exhibiting up-regulated IRAP-BP activity. In such clinical trials, the expression or activity of the IRAP-BP gene and, preferably, of other genes that are implicated in, for example, insulin resistance,
Expression or activity may be used as a "readout" or marker of the phenotype of a particular cell.

Agents (eg, compounds, such as, but not limited to, IRAP-BP) that modulate IRAP-BP activity (identified in screening assays as described herein), including but not limited to (Drugs or small molecules) to identify genes that are modulated in cells. In this way, to study the effects of agonists on insulin resistance in clinical trials, etc., cells were isolated, RNA was prepared, and IRAP-
The expression levels of BP and other genes implicated in insulin resistance may be analyzed, respectively. The gene expression level (ie, gene expression pattern)
Quantification by Northern blot analysis or RT-PCR as described herein,
Alternatively, one of the methods described herein may be used to measure and quantify the amount of protein produced, or to quantify by measuring the activity level of IRAP-BP or other genes. Good. In this way, the expression pattern of the gene can be used as a marker as an indicator of the physiological response of the cell to the agent. Accordingly, this response state may be determined before treating the individual with the agent and at various times during the treatment.

In one preferred embodiment, the present invention provides a method for treating an agent (eg, an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule identified in the screening assays described herein). Molecule, or other drug candidate), to provide a method for observing the effects of treating a subject with (i) administering the agent prior to administering the agent. Obtaining a sample; (ii) detecting the expression level of IRAP-BP polypeptide, mRNA or genomic DNA in the pre-dose sample; and (iii) one or more post-dose samples from the subject. (Iv) detecting the expression level or activity level of the IRAP-BP polypeptide, mRNA or genomic DNA in the sample after the administration, and (v) performing the administration IRAP-BP polypeptide in a sample, mRNA, or the level of expression or activity level of genomic DNA, the one or more IRAP-BP after sample administration
Comparing the polypeptide, mRNA, or genomic DNA; and (vi)
Altering the administration of the agent to the subject. For example, increased administration of an agent may be preferred if a higher level of IRAP-BP expression or activity is detected than is detected, ie, if one wishes to enhance the effect of the agent. Alternatively, reduced administration of the agent may be preferred if lower levels of IRAP-BP expression or activity than detected are desired, ie, if the effect of the agent is to be reduced. Based on such examples, IRAP-BP expression or activity can be used as an indicator of the effect of an agonist, even when there is no observable phenotypic response.

C. Methods of Treatment: The present invention includes both prophylactic and therapeutic methods of treating subjects at risk of (or susceptible to) or having a disorder associated with abnormal IRAP-BP expression or activity. Is provided.

C. 1. Prophylaxis In one aspect, the invention relates to abnormal IRAP-BP expression or activity by administering to a subject an IRAP-BP or agonist that modulates IRAP-BP expression or at least one IRAP-BP activity. The present invention provides a method for preventing a disease or condition in a subject. A subject at risk for a disease caused by or to which aberrant IRAP-BP expression or activity can be determined, for example, by any or a combination of diagnostic or prognostic assays as described herein. it can. Administration of a prophylactic agent should prevent IRAP-B from preventing or slowing the progress of the disease or disorder.
It may be performed before the onset of symptoms characteristic of P abnormalities. For example, depending on the type of IRAP-BP abnormality, an agonist that is an IRAP-BP, IRAP-BP agonist or IRAP-BP antagonist can be used to treat a subject. Suitable agents can be determined based on the screening assays described herein. The prophylaxis according to the present invention will be further described in the following sections.

C. 2. Another aspect of the invention relates to a method of modulating IRAP-BP expression or activity for therapeutic purposes. Thus, in one embodiment, the modulation method of the invention comprises the step of contacting a cell with an IRAP-BP molecule of the invention such that the activity of IRAP-BP is modulated. Instead, the modulation method of the invention comprises contacting the cell with an agent that modulates one or more of the IRAP-BP polypeptide activities associated with the cell.
Agents that modulate IRAP-BP polypeptide activity include, for example, nucleic acids or proteins, IRA
P-BP polypeptide naturally occurring target molecules (eg, carbohydrates), IRAP-BP antibodies, I
It may be an agent as described herein, such as a RAP-BP agonist or antagonist, a peptide mimetic of an IRAP-BP agonist or antagonist, or other small molecule. In one embodiment, the agent comprises one or more
It stimulates IRAP-BP activity. Examples of such stimulants include active IRAP-
Includes BP polypeptides and nucleic acid molecules encoding IRAP-BP introduced into cells. In another embodiment, the agent is one that inhibits one or more IRAP-BP activities. Examples of such inhibitory agents include antisense IRAP-BP nucleic acid molecules and anti-IRAP-BP antibodies. These modulation methods may be performed in vitro (eg, by culturing cells with the agent) or in vivo (eg, by administering the agent to a subject). Therefore, the present invention relates to IRAP-BP
It provides a method of treating an individual suffering from a disease or disorder characterized by abnormal expression or activity of a polypeptide or nucleic acid molecule. In one embodiment, the method includes an agent that modulates (eg, up-regulates or down-regulates) IRAP-BP expression or activity (eg, an agent identified in a screening assay described herein);
Or, administering a combination of agents. In another embodiment, the method comprises:
Therapies that correct for reduced or abnormal IRAP-BP expression or activity include administering an IRAP-BP polypeptide or nucleic acid molecule.

Stimulation of IRAP-BP activity may be triggered when IRAP-BP is abnormally down-regulated and / or when enhanced IRAP-BP activity appears to have a beneficial effect (eg, insulin resistance or diabetes And so on). Similarly, inhibition of IRAP-BP activity
Is preferred if is abnormally upregulated and / or if a reduction in IRAP-BP activity appears to have a beneficial effect.

The invention will be further described in the following examples, which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application are hereby incorporated by reference. References herein to websites maintained as part of the World Wide Web are referred to herein with an http: // prefix. The information contained on such websites is widely available and can be accessed electronically by connecting to the quoted address.

EXAMPLES Example 1: Identification and Characterization of Mouse IRAP-BP-1 cDNA This example describes the identification and characterization of the gene encoding mouse IRAP-BP-1.

Isolation of Mouse IRAP-BP-1 cDNA To identify a novel protein that binds to insulin-reactive aminopeptidase, ie, IRAP, a 3T3- The sequences of several cDNA libraries derived from L1 adipocytes were analyzed. Briefly, this assay “feeds” a cDNA encoding amino acid residues 55-82 of the IRAP-BP polypeptide fused to a gene encoding the DNA binding domain of GAL-4. Used as a construct. "Prey" (original: prey
) In the construct, a cDNA sequence from a differentiated 3T3-L1 library encoding an unknown protein was fused to a gene encoding the activation domain of GAL-4. In vivo, the interaction of these "feed" and "prey" proteins results in IRA
A P-dependent complex is formed, with the DNA binding domain and the activation domain of GAL4 in close proximity. Because of this proximity, a reporter gene (eg, LacZ) operatively linked to a transcriptional regulatory site responsive to GAL4 can be transcribed.

In the first round of the experiment, Y190 cells were made competent by treatment with LiOAc and then co-transformed with the bait and prey constructs. Transformants about 10 ⁶ Trp-, Leu-, painted on His- plates. About 10 ² of His + colonies
Trp-, Leu-plates were re-coated and assayed for β-galactosidase activity. Plasmid DNA was isolated from nine strong galactosidase positive clones and
IRAP (55-82) cells were transformed. One positive clone, A89, was sequenced and found to be nucleotides 1-2947 of SEQ ID NO: 1.

[0171] In a second experiment, Y190 / IRAP (55-82) cells were treated with LiOAc to become competent cells and then transformed with the prey construct. Of about 10 ⁷ transformants
Trp-, Leu- and His-plates were applied. Approximately 103 His + colonies were replated on Trp-, Leu-plates and assayed for galactosidase activity. Plasmid D
NA was isolated from six strong galactosidase positive clones and transformed into Y190 / IRAP (55-82) cells. When one positive clone, E89, was sequenced, SEQ ID NO: 1
At nucleotides 451-2947.

The nucleotide sequence encoding the mouse IRAP-BP-1 protein is shown in FIGS. 1 to 6 and is represented by SEQ ID NO: 1. The protein encoded by this nucleic acid consists of about 499 amino acids and has the amino acid sequence shown in FIGS. 1 to 6 and represented by SEQ ID NO: 2. SEQ
The codon portion of ID NO: 1 (open reading frame) is shown as SEQ ID NO: 3. On the other hand, SEQ ID
The nucleotide sequence of NO: 1 encodes a protein consisting of at least 728 amino acid residues and has the amino acid sequence of SEQ ID NO: 7. The corresponding codon portion (open reading frame) is shown in SEQ ID NO: 8.

Tissue Distribution of IRAP-BP-1 mRNA In this example, IRAP-B-1 was examined by Northern blot hybridization.
The tissue distribution of P mRNA is explained.

[0174] Northern blot hybridizations were performed using various RNA samples under standard conditions and washed under stringent conditions. A 3.5 Kb mRNA transcript was detected in heart, brain, lung, liver, skeletal muscle, kidney, and testis, with highest expression in testis. In addition, Northern blot hybridization was performed on mRNA isolated from 3T3L1 preadipocytes, adipocytes differentiated for 3 days, and adipocytes differentiated for 6 days. IRAP-BP mRNA levels increased significantly with differentiation.

Example 2 Identification and Characterization of Human IRAP-BP-1 cDNA This example describes the identification and characterization of the gene encoding human IRAP-BP-1.

[0176] Two PCR primers were designed based on the nucleotide sequence of mouse IRAP-BP. The forward primer was designed based on nucleotides 508-527 of mouse IRAP-BP (SEQ ID NO: 1).

Forward Primer 5′-GGAGCCCGAAGGACAAGTTC-3 ′ 508-527 (SEQ ID NO: 9)

The reverse primer was designed based on nucleotides 231-2293 of mouse IRAP-BP (SEQ ID NO: 1).

Inverse Primer 5′-AGCGACAAAAACCTGTCCCC-3 ′ 2312-2293 (SEQ ID NO: 10)

RT-PCR was performed using human skeletal muscle total RNA as a template. A fragment of about 1805 base pairs was purified and subcloned into PCR vector 2.1 (Invitrogen). Sequencing of the insert revealed nucleotides containing the codon region of human IRAP-BP (SEQ ID NO: 4). Expression sequence tag (EST) (Original language: Expressed Seque
As a result of TBLASTN search of the dBEST database of nce Tags), an EST having significant homology to human IRAP-BP and a part of the 3′-end untranslated region of accession number W84417 was found. When the entire 912 base pair insert of this clone was sequenced, human IRAP-
This corresponds to the 3'-terminal untranslated region of BP (SEQ ID NO: 4).

The nucleotide sequence encoding the human IRAP-BP-1 protein is shown in FIGS. 7-10 and is set forth in SEQ ID NO: 4. The protein encoded by this nucleic acid is composed of about 500 amino acids and has the amino acid sequence shown in FIGS. The codon portion of SEQ ID NO: 4 (open reading frame) is listed in SEQ ID NO: 6. On the other hand, the nucleotide sequence of SEQ ID NO: 1 encodes a protein consisting of at least 561 amino acid residues and has the amino acid sequence of SEQ ID NO: 12. The corresponding codon part (open reading frame) is listed in SEQ ID NO: 11.

Alignment of mouse and human IRAP-BP shows that these proteins are very well conserved at the amino acid level. FIG.
Weight residue table, with gap length penalty = 12 and gap penalty = 4, showing such an alignment made using the ALIGN algorithm, wherein these proteins have greater than 97% identity. (97.4% identity).

Alignments of the human and mouse nucleic acid sequences are shown in FIGS. This alignment was made using the BESTFIT algorithm from the GCG software analysis package and the following parameters.

[0184]

[Table 1]

An alignment of the hypothetical amino acid sequences of human and mouse is shown in FIG. This alignment was made using the BESTFIT algorithm from the GCG software analysis package and the following parameters.

[0186]

[Table 2]

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 of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[Sequence list]

[Brief description of the drawings]

1 to 6 show the cDNA sequence and predicted amino acid sequence of mouse IRAP-BP-1. 1 to 4 show the nucleotide sequence of mouse IRAP-BP corresponding to nucleic acids 1 to 2947 of SEQ ID NO: 1. The mouse IRAP-BP amino acid sequence has residue 23
It is predicted to start at 0, end at residue 728, and correspond to amino acids 1 to 499 of SEQ ID NO: 2.

FIG. 2 is a mouse IRA corresponding to nucleic acids 721 to 1440 of SEQ ID NO: 1, following FIG.
1 shows the nucleotide sequence of P-BP.

FIG. 3 is a mouse IR corresponding to nucleic acids 1441 to 2220 of SEQ ID NO: 1, following FIG.
1 shows the nucleotide sequence of AP-BP.

FIG. 4 is a mouse IR corresponding to nucleic acids 2221 to 2947 of SEQ ID NO: 1, following FIG.
1 shows the nucleotide sequence of AP-BP.

FIG. 5 and FIG. 6 show hypothetical translation products of the mouse IRAP-BP cDNA sequence.

FIG. 6 shows the hypothetical translation product of the mouse IRAP-BP cDNA sequence, following FIG.

7 to 10 show the cDNA sequence and predicted amino acid sequence of human IRAP-BP-1. 7 to 9 show the nucleotide sequence of human IRAP-BP corresponding to nucleic acids 1 to 2064 of SEQ ID NO: 4. The human IRAP-BP amino acid sequence is predicted to begin at residue 62, end at residue 561, and correspond to amino acids 1 to 500 of SEQ ID NO: 5.

FIG. 8 shows the human IRAP-BP corresponding to nucleic acids 721 to 1440 of SEQ ID NO: 4, following FIG.
1 shows the nucleotide sequence of

FIG. 9 is a human IRAP-B corresponding to nucleic acids 1441 to 2064 of SEQ ID NO: 4, following FIG.
1 shows the nucleotide sequence of P.

FIG. 10 shows hypothetical translation products of the IRAP-BP cDNA sequence.

FIG. 11 to FIG. 14 show the alignment of nucleic acid sequences of mouse IRAP-BP (SEQ ID NO: 1, upper row) and human IRAP-BP (SEQ ID NO: 4, lower row), and mouse
The alignment of the hypothetical amino acid sequence of IRAP-BP (upper row) with the hypothetical amino acid sequence of human IRAP-BP (lower row) is shown. 11 to 13 show the BESTFIT program which is a part of the GCG software package and the gap weight of 5
The alignment of nucleic acids performed using 0 and length weight of 3 is shown.

FIG. 12 is a diagram showing BEST, which is a part of the GCG software package, similar to FIG. 11;
FIG. 4 shows nucleic acid alignments performed using the FIT program with a gap weight of 50 and a length weight of 3. FIG.

FIG. 13 is a diagram showing BEST, which is a part of the GCG software package, similar to FIG. 11;
FIG. 4 shows nucleic acid alignments performed using the FIT program with a gap weight of 50 and a length weight of 3. FIG.

FIG. 14 shows amino acid alignments performed using the same software package with a gap weight of 12 and a length weight of 4.

FIG. 15 shows mouse IRAP-BP (SEQ ID NO: 2, upper panel) and human IRAP-BP (SEQ ID NO: 2).
NO: 5, bottom). This amino acid alignment was prepared using the ALIGN algorithm with a gap penalty of -12 / -2.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/18 C12N 1/15 19/00 1/19 C12N 1/15 1/21 1/19 C12Q 1 / 68 A 1/21 G01N 33/53 D 5/10 C12N 15/00 ZNAA C12Q 1/68 5/00 A G01N 33/53 A61K 37/02 (72) Inventor Lynn Baozen United States 08648 Lawrenceville Town, New Jersey Court North 1212 F-term (reference) 4B024 AA01 AA11 BA80 CA04 CA09 CA11 CA12 DA12 EA04 GA11 GA19 HA14 HA15 4B063 QA01 QA18 QA19 QQ42 QQ52 QR32 QR56 QR62 QS03 QS34 QX07 4B065 AA72X AA91A4A01 A01A04A01 A01A04A01 BA23 CA53 CA56 DB34 NA14 ZC35 4H045 AA10 AA11 AA30 BA10 DA75 DA76 EA27 EA50 FA74

Claims (27)

[Claims] 1. An insulin-reactive aminopeptidase-binding protein (IRAP-B)
P) a polypeptide, comprising a nucleotide sequence encoding a polypeptide, which binds to an insulin-reactive aminopeptidase (IRAP) polypeptide;
An isolated nucleic acid molecule. 2. The isolated nucleic acid molecule of claim 1, comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, or a nucleotide sequence that is at least about 60% identical to the complement thereof. 3. The isolated nucleic acid molecule of claim 1, comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4. 4. The isolated nucleic acid molecule of claim 1, wherein the nucleic acid molecule specifically detects an IRAP-BP nucleic acid molecule relative to a nucleic acid molecule encoding a non-IRAP-BP polypeptide. 5. An insulin-reactive aminopeptidase-binding protein (IRAP-B).
P) An isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide, which binds to an insulin-reactive aminopeptidase (IRAP) polypeptide. 6. The isolated amino acid sequence of claim 5, comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence at least about 60% identical to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5. Nucleic acid molecule. 7. The isolated nucleic acid molecule of claim 6, which encodes a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. 8. An isolated nucleic acid molecule comprising a nucleotide sequence that hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4, wherein the isolated nucleic acid molecule comprises insulin-reactive. An isolated nucleic acid molecule encoding an aminopeptidase binding protein (IRAP-BP) polypeptide, or a biologically active portion thereof. 9. At least 550, which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 4.
An isolated nucleic acid molecule of nucleotide length. 10. An isolated nucleic acid molecule encoding a fusion protein comprising an insulin-reactive aminopeptidase binding protein (IRAP-BP) polypeptide operatively linked to a non-IRAP-BP polypeptide. 11. An isolated nucleic acid molecule that is antisense to the nucleic acid molecule of claim 1. 12. A vector comprising the nucleic acid molecule of claim 1. 13. A host cell containing the vector of claim 12. 14. An isolated insulin-reactive aminopeptidase-binding protein (IRAP-BP) polypeptide, which binds to an insulin-reactive aminopeptidase (IRAP) polypeptide. 15. The isolated polyprotein of claim 14, comprising an amino acid sequence that is at least 60% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. peptide. 16. The isolated protein of claim 14, wherein the protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence that is 60% homologous to the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. protein. 17. The isolated polypeptide of claim 14, comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 5, or SEQ ID NO: 8. 18. A fusion protein comprising an insulin-reactive aminopeptidase binding protein (IRAP-BP) polypeptide operatively linked to a non-IRAP-BP polypeptide. 19. An insulin-reactive aminopeptidase-binding protein (IRAP).
-BP) An antibody that specifically binds to a polypeptide. 20. A pharmaceutical composition comprising the protein of claim 14 and a pharmaceutically acceptable carrier. 21. A method for modulating a cell-related activity, comprising the steps of: (a) modifying a cell with an agent that modulates the activity or expression of an insulin-responsive aminopeptidase-binding protein (IRAP-BP); Contacting such that said cell-related activity is altered relative to said cell-related activity of said cell in its presence. 22. The method of claim 21, wherein said cells are in a subject and said agent is administered to said subject. 23. An insulin-reactive aminopeptidase-binding protein (IRAP)
-BP) for treating a subject having a disorder characterized by abnormal expression of activity, comprising administering to the subject an IRAP-BP modulator such that treatment of the subject is performed. Method. 24. A method for detecting the activity or expression of an insulin-reactive aminopeptidase-binding protein (IRAP-BP) in a biological sample, wherein the activity or expression of IRAP-BP is detected in the biological sample. Contacting the biological sample with an agent capable of detecting the activity or expression of IRAP-BP. 25. A kit for detecting the expression or activity of an insulin-reactive aminopeptidase binding protein (IRAP-BP) in a biological sample, the kit comprising:
A kit comprising an agent capable of detecting the activity or expression of P-BP. 26. A diagnostic assay for determining gene damage in a cell sample, wherein the presence or absence of the gene damage comprises: Characterized by at least one of abnormal modification or mutation of the encoding gene, and (ii) misregulation of said gene, or (iii) abnormal post-translational modification of the IRAP-BP polypeptide.
Diagnostic assay. 27. An insulin-reactive aminopeptidase-binding protein (IRAP
-BP) A method for identifying a compound that modulates the activity of a polypeptide, comprising: a. Providing an indicator composition comprising an IRAP-BP polypeptide, or a biologically active portion thereof; b. Contacting said indicator composition with a test compound; c. Examining the effect of the test compound on IRAP-BP activity in the indicator composition to identify compounds that modulate the activity of IRAP-BP polypeptide.