WO2003016345A1

WO2003016345A1 - Regulation of human regulator of g-protein signaling

Info

Publication number: WO2003016345A1
Application number: PCT/EP2002/009173
Authority: WO
Inventors: Rainer H. KÖHLER
Original assignee: Bayer Healthcare Ag
Priority date: 2001-08-16
Filing date: 2002-08-16
Publication date: 2003-02-27

Abstract

Reagents that regulate human regulator of G-protein signaling and reagents which bind to human regulator of G-protein signaling gene products can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, asthma and diabetes.

Description

REGULATION OF HUMAN REGULATOR OF G-PROTEIN SIGNALING

TECHNICAL FIELD OF THE INVENTION

The invention relates to the regulation of human regulator of G-protein signaling.

BACKGROUND OF THE INVENTION

G protein-coupled receptors (GPCRs) play a major role in signal transduction and are targets of many therapeutic drugs. The regulator of G protein signaling (RGS) proteins form a recently identified protein family, and they strongly modulate the activity of G proteins. Their best known function is to inhibit G protein signaling by accelerating GTP hydrolysis [GTPase activating protein (GAP)] thus turning off G protein signals. RGS proteins also possess non-GAP functions, through both their

RGS domains and various non-RGS domains and motifs (e.g., GGL, DEP, DH/PH, PDZ domains and a cysteine string motif). They are a highly diverse protein family, have unique tissue distributions, are strongly regulated by signal transduction events, and will likely play diverse functional roles in living cells.

Most RGS proteins are GTPase accelerating proteins (GAPs) for Gi and Gq class G protein alpha subunits, and thereby terminate signaling. However, RGS proteins also provide scaffolding properties to help assemble or maintain signaling complexes. Thus, RGS proteins are kinetic regulators that may sharpen both signal activation and termination. The five subfamilies of mammalian RGS proteins contain a characteristic RGS domain and distinct flanking domains that convey lipid and/or protein interactions within receptor complexes. The RGS domain provides GAP activity and additional interactions with the receptor complex. SUMMARY OF THE INVENTION

It is an object of the invention to provide reagents and methods of regulating a human regulator of G-protein signaling. This and other objects of the invention are provided by one or more of the embodiments described below.

One embodiment of the invention is a regulator polypeptide of G-protein signaling (RGS) comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2;

the amino acid sequence shown in SEQ ID NO: 2;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; and

the amino acid sequence shown in SEQ ID NO: 5.

Yet another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a regulator polypeptide of G-protein signaling (RGS) comprising an amino acid sequence selected from the group consisting of:

the amino acid sequence shown in SEQ ID NO: 2;

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence shown in SEQ ID NO: 5.

Binding between the test compound and the regulator polypeptide of G-protein signaling (RGS) is detected. A test compound which binds to the regulator polypeptide of G-protein signaling (RGS) is thereby identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the activity of the regulator of G-protein signaling (RGS).

Another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a polynucleotide encoding a regulator polypeptide of G-protein signaling (RGS), wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; and

the nucleotide sequence shown in SEQ ID NO: 1;

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 4; and

the nucleotide sequence shown in SEQ ID NO: 4.

Binding of the test compound to the polynucleotide is detected. A test compound which binds to the polynucleotide is identified as a potential agent for decreasing extracellular matrix degradation. The agent can work by decreasing the amount of the regulator of G-protein signaling (RGS) through interacting with the regulator of G- protein signaling (RGS) mRNA. Another embodiment of the invention is a method of screening for agents which regulate extracellular matrix degradation. A test compound is contacted with a regulator polypeptide of G-protein signaling (RGS) comprising an amino acid sequence selected from the group consisting of:

the amino acid sequence shown in SEQ ID NO: 2;

the amino acid sequence shown in SEQ ID NO: 5.

A regulator polypeptide of G-protein signaling (RGS) activity is detected. A test compound which increases regulator polypeptide of G-protein signaling (RGS) activity relative to regulator of G-protein signaling (RGS) activity in the absence of the test compound is thereby identified as a potential agent for increasing extra- cellular matrix degradation. A test compound which decreases regulator polypeptide of G-protein signaling (RGS) activity relative to regulator of G-protein signaling (RGS) activity in the absence of the test compound is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Even another embodiment of the invention is a method of screening for agents which decrease extracellular matrix degradation. A test compound is contacted with a regulator of G-protein signaling (RGS) product of a polynucleotide which comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1; the nucleotide sequence shown in SEQ ID NO: 1;

the nucleotide sequence shown in SEQ ID NO: 4.

Binding of the test compound to the regulator of G-protein signaling (RGS) product is detected. A test compound which binds to the regulator of G-protein signaling

(RGS) product is thereby identified as a potential agent for decreasing extracellular matrix degradation.

Still another embodiment of the invention is a method of reducing extracellular matrix degradation. A cell is contacted with a reagent which specifically binds to a polynucleotide encoding a regulator polypeptide of G-protein signaling (RGS) or the product encoded by the polynucleotide, wherein the polynucleotide comprises a nucleotide sequence selected from the group consisting of:

nucleotide sequences which are at least about 50% identical to the nucleotide sequence shown in SEQ ID NO: 1;

the nucleotide sequence shown in SEQ ID NO: 1;

the nucleotide sequence shown in SEQ ID NO: 4.

Regulator of G-protein signaling (RGS) activity in the cell is thereby decreased. The

thus provides a human regulator of G-protein signaling that can be used to identify test compounds that may act, for example, as activators or inhibitors at the enzyme's active site. Human regulator of G-protein signaling and fragments thereof also are useful in raising specific antibodies that can block the enzyme and effectively reduce its activity.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the DNA-sequence encoding a regulator polypeptide of G-protein signaling (RGS) (SEQ ID NO:l).

Fig. 2 shows the amino acid sequence deduced from the DNA-sequence of Fig.1

(SEQ ID NO:2). Fig. 3 shows the amino acid sequence of the protein identified by swissnew|P41220|RGS2_HUMAN REGULATOR OF G_PROTEIN SIGNALING 2 (RGS2) (SEQ ID NO :3).

Fig. 4 shows the DNA-sequence encoding a regulator polypeptide of G-protein signaling (RGS) (SEQ ID NO:4). Fig. 5 shows the amino acid sequence deduced from the DNA-sequence of Fig. 4

(SEQ ID NO:5). Fig. 6 shows the BLASTP - alignment of SEQ ID NO:2 against swissnew|P41220|RGS2_HUMAN REGULATOR OF G-PROTEIN

SIGNALING 2 (RGS2) (G0/G1 SWITCH REGULATORY PROTEIN 8). Fig. 7 shows the HMMPFAM - alignment of SEQ ID NO:2 against pfam|hmm|RGS

Regulator of G protein signaling domain. Fig. 8 shows the BLASTP - alignment of SEQ ID NO:2 against pdb|lEZT|lEZT-A regulator of g-protein signaling 4fragment: core rgs domain; Fig. 9 shows the Genewise exon-intron structure prediction using genomic sequence

AL390957.10 with Human RGS2 DETAILED DESCRIPTION OF THE INVENTION

The invention relates to an isolated polynucleotide from the group consisting of:

a) a polynucleotide encoding a regulator polypeptide of G-protein signaling (RGS) comprising an amino acid sequence selected from the group consisting of:

amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ID NO: 2; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence shown in SEQ ID NO: 5.

b) a polynucleotide comprising the sequence of SEQ ID NO: 1 or 4;

c) a polynucleotide which hybridizes under stringent conditions to a poly- nucleotide specified in (a) and (b) and encodes a regulator of G-protein signaling (RGS) polypeptide;

d) a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a regulator polypeptide of G-protein signaling (RGS); and

e) a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a regulator polypeptide of G-protein signaling (RGS). Furthermore, it has been discovered by the present applicant that a novel regulator of G-protein signaling, particularly a human regulator of G-protein signaling, can be used in therapeutic methods to treat asthma and diabetes. Human regulator of G- protein signaling comprises the amino acid sequence shown in SEQ ID NO:2 and 5. A coding sequence for human regulator of G-protein signaling is shown in SEQ ID

NO:l. An alternative spliced sequence for human regulator of G-protein signaling is shown in SEQ ID NO:4. This sequence is located on chromosome 1.

Human regulator of G-protein signaling is 58% identical over 136 amino acids to regulator of G-protein signaling (FIG. 1). Human regulator of G-protein signaling of the invention is expected to be useful for the same purposes as previously identified regulator of G-protein signaling enzymes. Human regulator of G-protein signaling is believed to be useful in therapeutic methods to treat disorders such as asthma and diabetes. Human regulator of G-protein signaling also can be used to screen for human regulator of G-protein signaling activators and inhibitors.

Polypeptides

Human regulator of G-protein signaling polypeptides according to the invention comprise at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, or 249 contiguous amino acids selected from the amino acid sequence shown in SEQ ID NO: 2 or 5 or a biologically active variant thereof, as defined below. A regulator of G-protein signaling polypeptide of the invention therefore can be a portion of a regulator of G-protein signaling protein, a full-length regulator of G-protein signaling protein, or a fusion protein comprising all or a portion of a regulator of G-protein signaling protein.

Biologically Active Variants

Regulator of G-protein signaling polypeptide variants that are biologically active, i.e., retain the ability to bind a ligand to produce a biological effect, such as cyclic AMP formation, mobilization of intracellular calcium, or phosphoinositide metabolism, also are regulator of G-protein signaling polypeptides. Preferably, naturally or non- naturally occurring regulator of G-protein signaling polypeptide variants have amino acid sequences which are at least about 50, preferably at least about 59, 65, 70. 75. 90, 96, or 98%) identical to the amino acid sequence shown in SEQ ID NO: 2 or 5 or a fragment thereof. Percent identity between a putative regulator of G-protein signaling polypeptide variant and an amino acid sequence of SEQ ID NO: 2 or 5 is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), and Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA SP:10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff & Henikoff, 1992.

Those skilled in the art appreciate that there are many established algorithms available to align two amino acid sequences. The "FASTA" similarity search algorithm of Pearson & Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant. The FASTA algorithm is described by Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 55:2444(1988), and by Pearson, Meth. Enzymol. 183:63 (1990). Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO: 2 or 5) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score. If there are several regions with scores greater than the "cutoff value (calculated by a predetermined formula based upon the length of the sequence the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman- Wunsch- Sellers algorithm (Needleman & Wunsch, J. MoL Biol.48:444 (1970); Sellers, SIAM J. Appl. Math.26:181 (1974)), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=l, gap opening penalty^lO, gap extension penalty=l, and substitution matrix=BLOSUM62. These parameters can be introduced into a FASTA program by modifying the scoring matrix file ("SMATRIX"), as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.

Variations in percent identity can be due, for example, to amino acid substitutions, insertions, or deletions. Amino acid substitutions are defined as one for one amino acid replacements. They are conservative in nature when the substituted amino acid has similar structural and/or chemical properties. Examples of conservative replacements are substitution of a leucine with an isoleucine or valine, an aspartate with a glutamate, or a threonine with a serine.

Amino acid insertions or deletions are changes to or within an amino acid sequence. They typically fall in the range of about 1 to 5 amino acids. Guidance in determining which amino acid residues can be substituted, inserted, or deleted without abolishing biological or immuno logical activity of a regulator of G-protein signaling polypeptide can be found using computer programs well known in the art, such as DNASTAR software.

The invention additionally, encompasses regulator of G-protein signaling poly- peptides that are differentially modified during or after translation, e.g., by glycosy- lation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications can be carried out by known techniques including, but not limited, to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, N8 protease, ΝaBH₄, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunica- mycin, etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N- terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of prokaryotic host cell expression. The regulator of G-protein signaling polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

The invention also provides chemically modified derivatives of regulator of G- protein signaling polypeptides that may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U.S. Patent No. 4,179,337). The chemical moieties for derivitization can be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like. The polypeptides can be modified at random or predetermined positions within the molecule and can include one, two, three, or more attached chemical moieties.

Whether an amino acid change or a polypeptide modification results in a biologically active regulator of G-protein signaling polypeptide can readily be determined by assaying for binding to a ligand or by conducting a functional assay, as described for example, in Woodson, J. Biol. Chem, 276:20160-6 (2001). Fusion Proteins

Fusion proteins are useful for generating antibodies against regulator of G-protein signaling polypeptide amino acid sequences and for use in various assay systems.

For example, fusion proteins can be used to identify proteins that interact with portions of a regulator of G-protein signaling polypeptide. Protein affinity chromatography or library-based assays for protein-protein interactions, such as the yeast two- hybrid or phage display systems, can be used for this purpose. Such methods are well known in the art and also can be used as drug screens.

A regulator of G-protein signaling polypeptide fusion protein comprises two polypeptide segments fused together by means of a peptide bond. The first polypeptide segment comprises at least 6, 10, 15, 20, 25, 50, 75, 100, 125, 150, 175, 200, 225, or 249 contiguous amino acids of SEQ ID NO:2 or 5 or of a biologically active variant, such as those described above. The first polypeptide segment also can comprise full- length regulator of G-protein signaling protein.

The second polypeptide segment can be a full-length protein or a protein fragment. Proteins commonly used in fusion protein construction include β-galactosidase, β- glucuronidase, green fluorescent protein (GFP), autofluorescent proteins, including blue fluorescent protein (BFP), glutathione-S-transferase (GST), luciferase, horseradish peroxidase (HRP), and chlorarnphenicol acetyltransferase (CAT). Additionally, epitope tags are used in fusion protein constructions, including histidine (His) tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, NSN-

G tags, and thioredoxin (Trx) tags. Other fusion constructions can include maltose binding protein (MBP), S-tag, Lex a DΝA binding domain (DBD) fusions, GAL4 DΝA binding domain fusions, and herpes simplex virus (HSN) BP16 protein fusions. A fusion protein also can be engineered to contain a cleavage site located between the regulator of G-protein signaling polypeptide-encoding sequence and the hetero- logous protein sequence, so that the regulator of G-protein signaling polypeptide can be cleaved and purified away from the heterologous moiety.

A fusion protein can be synthesized chemically, as is known in the art. Preferably, a fusion protein is produced by covalently linking two polypeptide segments or by standard procedures in the art of molecular biology. Recombinant DNA methods can be used to prepare fusion proteins, for example, by making a DNA construct which comprises coding sequences selected from SEQ ID NO:l or 4 in proper reading frame with nucleotides encoding the second polypeptide segment and expressing the DNA construct in a host cell, as is known in the art. Many kits for constructing fusion proteins are available from companies such as Promega Corporation (Madison, WI), Stratagene (La Jolla, CA), CLONTECH (Mountain View, CA), Santa Cruz Biotechnology (Santa Cruz, CA), MBL International Corporation (MIC; Watertown, MA), and Quantum Biotechnologies (Montreal, Canada; 1-888-DNA-KITS).

Identification of Species Homologs

Species homologs of human regulator of G-protein signaling polypeptide can be obtained using regulator of G-protein signaling polypeptide polynucleotides

(described below) to make suitable probes or primers for screening cDNA expression libraries from other species, such as mice, monkeys, or yeast, identifying cDNAs which encode homologs of regulator of G-protein signaling polypeptide, and expressing the cDNAs as is known in the art.

Polynucleotides

A regulator of G-protein signaling polynucleotide can be single- or double-stranded and comprises a coding sequence or the complement of a coding sequence for a regulator of G-protein signaling polypeptide. A coding sequence for human regulator of G-protein signaling is shown in SEQ ID NO:l and 4. Degenerate nucleotide sequences encoding human regulator of G-protein signaling polypeptides, as well as homologous nucleotide sequences which are at least about 50. 55, 60, 65, 70, preferably about 75, 90, 96, 98, or 99% identical to the nucleotide sequence shown in SEQ ID NO:l or 4 or its complement also are regulator of G- protein signaling polynucleotides. Percent sequence identity between the sequences of two polynucleotides is determined using computer programs such as ALIGN which employ the FASTA algorithm, using an affine gap search with a gap open penalty of -12 and a gap extension penalty of -2. Complementary DNA (cDNA) molecules, species homologs, and variants of regulator of G-protein signaling polynucleotides that encode biologically active regulator of G-protein signaling polypeptides also are regulator of G-protein signaling polynucleotides. Polynucleotide fragments comprising at least 8, 9, 10, 11, 12, 15, 20, or 25 contiguous nucleotides of SEQ ID NO:l or 4 or its complement also are regulator of G-protein signaling polynucleotides. These fragments can be used, for example, as hybridization probes or as antisense oligonucleotides.

Identification of Polynucleotide Variants and Homologs

Variants and homologs of the regulator of G-protein signaling polynucleotides described above also are regulator of G-protein signaling polynucleotides. Typically, homologous regulator of G-protein signaling polynucleotide sequences can be identified by hybridization of candidate polynucleotides to known regulator of G- protein signaling polynucleotides under stringent conditions, as is known in the art. For example, using the following wash conditions~2X SSC (0.3 M NaCl, 0.03 M sodium citrate, pH 7.0), 0.1% SDS, room temperature twice, 30 minutes each; then 2X SSC, 0.1% SDS, 50 °C once, 30 minutes; then 2X SSC, room temperature twice, 10 minutes each— homologous sequences can be identified which contain at most about 25-30%) basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. Species homologs of the regulator of G-protein signaling polynucleotides disclosed herein also can be identified by making suitable probes or primers and screening cDNA expression libraries from other species, such as mice, monkeys, or yeast. Human variants of regulator of G-protein signaling polynucleotides can be identified, for example, by screening human cDNA expression libraries. It is well known that the T_m of a double-stranded DNA decreases by 1-1.5 °C with every 1% decrease in homology (Bonner et al., J. Mol. Biol. 81, 123 (1973). Variants of human regulator of G-protein signaling polynucleotides or regulator of G-protein signaling polynucleotides of other species can therefore be identified by hybridizing a putative homologous regulator of G-protein signaling polynucleotide with a polynucleotide having a nucleotide sequence of SEQ ID NO:l or 4 or the complement thereof to form a test hybrid. The melting temperature of the test hybrid is compared with the melting temperature of a hybrid comprising polynucleotides having perfectly complementary nucleotide sequences, and the number or percent of basepair mismatches within the test hybrid is calculated.

Nucleotide sequences which hybridize to regulator of G-protein signaling polynucleotides or their complements following stringent hybridization and/or wash conditions also are regulator of G-protein signaling polynucleotides. Stringent wash conditions are well known and understood in the art and are disclosed, for example, in Sambrook et al, MOLECULAR CLONING: A LABORATORY MANUAL, 2d ed., 1989, at pages 9.50-9.51.

Typically, for stringent hybridization conditions a combination of temperature and salt concentration should be chosen that is approximately 12-20 °C below the calculated T_m of the hybrid under study. The T_m of a hybrid between a regulator of G-protein signaling polynucleotide having a nucleotide sequence shown in SEQ ID NO:l or 4 or the complement thereof and a polynucleotide sequence which is at least about 50, preferably about 75, 90, 96, or 98%) identical to one of those nucleotide sequences can be calculated, for example, using the equation of Bolton and McCarthy, Proc. Natl. Acad. Sci. U.S.A. 48, 1390 (1962):

T_m = 81.5 °C - 16.6(logιo[Na⁺]) + 0.41(%G + C) - 0.63(%formamide) - 600//). where / = the length of the hybrid in basepairs.

Stringent wash conditions include, for example, 4X SSC at 65 °C, or 50% formamide, 4X SSC at 42 °C, or 0.5X SSC, 0.1% SDS at 65 °C. Highly stringent wash conditions include, for example, 0.2X SSC at 65 °C.

Preparation of Polynucleotides

A regulator of G-protein signaling polynucleotide can be isolated free of other cellular components such as membrane components, proteins, and lipids. Poly- nucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the poly- merase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated regulator of G-protein signaling polynucleotides. For example, restriction enzymes and probes can be used to isolate polynucleotide fragments, which comprise regulator of G-protein signaling nucleotide sequences. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.

Human regulator of G-protein signaling cDNA molecules can be made with standard molecular biology techniques, using regulator of G-protein signaling mRNA as a template. Human regulator of G-protein signaling cDNA molecules can thereafter be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of polynucleotides of the invention, using either human genomic DNA or cDNA as a template. Alternatively, synthetic chemistry techniques can be used to synthesize regulator of G-protein signaling polynucleotides. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized which will encode a regulator of G- protein signaling polypeptide having, for example, an amino acid sequence shown in

SEQ ID NO:2 or 5 or a biologically active variant thereof.

Extending Polynucleotides

Narious PCR-based methods can be used to extend the nucleic acid sequences disclosed herein to detect upstream sequences such as promoters and regulatory elements. For example, restriction-site PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, PCR Methods Applic. 2, 318-322, 1993). Genomic DΝA is first amplified in the presence of a primer to a linker sequence and a primer specific to the known region. The amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one. Products of each round of PCR are transcribed with an appropriate RΝA polymerase and sequenced using reverse transcriptase.

Inverse PCR also can be used to amplify or extend sequences using divergent primers based on a known region (Triglia et al., Nucleic Acids Res. 16, 8186, 1988). Primers can be designed using commercially available software, such as OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, Minn.), to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 °C. The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.

Another method which can be used is capture PCR, which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al, PCR Methods Applic. 1, 111-119, 1991). In this method, multiple restriction enzyme digestions and ligations also can be used to place an engineered double-stranded sequence into an unknown fragment of the DNA molecule before performing PCR.

Another method which can be used to retrieve unknown sequences is that of Parker et al., Nucleic Acids Res. 19, 3055-3060, 1991). Additionally, PCR, nested primers, and PROMOTERFINDER libraries (CLONTECH, Palo Alto, Calif.) can be used to walk genomic DNA (CLONTECH, Palo Alto, Calif). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. Randomly-primed libraries are preferable, in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA. Genomic libraries can be useful for extension of sequence into 5' non-transcribed regulatory regions.

Commercially available capillary electrophoresis systems can be used to analyze the size or confirm the nucleotide sequence of PCR or sequencing products. For example, capillary sequencing can employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) that are laser activated, and detection of the emitted wavelengths by a charge coupled device camera. Output/light intensity can be converted to electrical signal using appropriate software (e.g. GENOTYPER and Sequence NAVIGATOR, Perkin Elmer), and the entire process from loading of samples to computer analysis and electronic data display can be computer controlled. Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample. Obtaining Polypeptides

Human regulator of G-protein signaling polypeptides can be obtained, for example, by purification from human cells, by expression of regulator of G-protein signaling polynucleotides, or by direct chemical synthesis.

Protein Purification

Human regulator of G-protein signaling polypeptides can be purified from any cell that expresses the polypeptide, including host cells that have been transfected with regulator of G-protein signaling expression constructs. A purified regulator of G- protein signaling polypeptide is separated from other compounds that normally associate with the regulator of G-protein signaling polypeptide in the cell, such as certain proteins, carbohydrates, or lipids, using methods well-known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified regulator of G-protein signaling polypeptides is at least 80% pure; preferably, the preparations are 90%o,

95%>, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis.

Expression of Polynucleotides

To express a regulator of G-protein signaling polynucleotide, the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding regulator of G-protein signaling polypeptides and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1989.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding a regulator of G-protein signaling polypeptide. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculo virus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector ~ enhancers, promoters, 5' and 3' untranslated regions ~ which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORTl plasmid (Life

Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a regulator of G-protein signaling polypeptide, vectors based on SV40 or EB V can be used with an appropriate selectable marker. Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selected depending upon the use intended for the regulator of G-protein signaling polypeptide. For example, when a large quantity of a regulator of G-protein signaling polypeptide is needed for the induction of antibodies, vectors which direct high level expression of fusion proteins that are readily purified can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors such as BLUE- SCRIPT (Stratagene). In a BLUESCRIPT vector, a sequence encoding the regulator of G-protein signaling polypeptide can be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of β-galactosidase so that a hybrid protein is produced. pIN vectors (Van Heeke & Schuster, J Biol. Chem. 264, 5503-5509, 1989) or pGEX vectors (Promega, Madison, Wis.) also can be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems can be designed to include heparin, thrombin, or factor Xa protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH can be used. For reviews, see Ausubel et al. (1989) and Grant et al., Methods Enzymol. 153, 516-544, 1987.

Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequences encoding regulator of G-protein signaling polypeptides can be driven by any of a number of promoters.

For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6, 307-311, 1987). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters can be used (Coruzzi et al., EMBO J. 3, 1671-1680, 1984; Broglie et al., Science 224, 838-843, 1984; Winter et al., Results Probl. Cell Differ. 17, 85-105, 1991). These constructs can be introduced into plant cells by direct DNA transformation or by pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (e.g., Hobbs or Murray, in MCGRAW HILL YEARBOOK OF SCIENCE AND TECHNOLOGY, McGraw Hill, New York, N.Y., pp. 191-196, 1992).

An insect system also can be used to express a regulator of G-protein signaling polypeptide. For example, in one such system Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. Sequences encoding regulator of G-protein signaling polypeptides can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of regulator of G-protein signaling polypeptides will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein. The recombinant viruses can then be used to infect S. frugiperda cells or Trichoplusia larvae in which regulator of G-protein signaling polypeptides can be expressed (Engelhard et al., Proc. Nat. Acad. Sci. 91, 3224-3227, 1994).

Mammalian Expression Systems

A number of viral-based expression systems can be used to express regulator of G- protein signaling polypeptides in mammalian host cells. For example, if an adenovirus is used as an expression vector, sequences encoding regulator of G- protein signaling polypeptides can be ligated into an adenovirus transcription/translation complex comprising the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome can be used to obtain a viable virus that is capable of expressing a regulator of G-protein signaling polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. 81, 3655-3659, 1984). If desired, transcription enhancers, such as the Rous sarcoma virus (RSN) enhancer, can be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) also can be used to deliver larger fragments of DΝA than can be contained and expressed in a plasmid. HACs of 6M to 10M are constructed and delivered to cells via conventional delivery methods (e.g., liposomes, polycationic amino polymers, or vesicles).

Specific initiation signals also can be used to achieve more efficient translation of sequences encoding regulator of G-protein signaling polypeptides. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding a regulator of G-protein signaling polypeptide, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals (including the ATG initiation codon) should be provided. The initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used (see Scharf et al., Results Probl. Cell Differ. 20, 125-162, 1994).

Host Cells

A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed regulator of G-protein signaling polypeptide in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells that have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, NA 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines which stably express regulator of G-protein signaling polypeptides can be transformed using expression vectors which can contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before they are switched to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced regulator of G- protein signaling sequences. Resistant clones of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type. See, for example, ANIMAL CELL CULTURE, R.I. Freshney, ed., 1986.

Any number of selection systems can be used to recover transformed cell lines.

These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11, 223-32, 1977) and adenine phosphoribosyltransferase (Lowy et al., Cell 22, 817-23, 1980) genes which can be employed in tic or aprf cells, respectively. Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection. For example, dhfr confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. 77, 3567-70, 1980), npt confers resistance to the aminoglycosides, neomycin and G-418 (Colbere-Garapin et ah, J. Mol. Biol. 150, 1-14, 1981), and als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murray, 1992, supra). Additional selectable genes have been described. For example, trpB allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. 85, 8047-51, 1988). Visible markers such as anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, can be used to identify transformants and to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al, Methods Mol. Biol. 55, 121-131, 1995).

Detecting Expression

Although the presence of marker gene expression suggests that the regulator of G- protein signaling polynucleotide is also present, its presence and expression may need to be confirmed. For example, if a sequence encoding a regulator of G-protein signaling polypeptide is inserted within a marker gene sequence, transformed cells containing sequences that encode a regulator of G-protein signaling polypeptide can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding a regulator of G-protein signaling polypeptide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the regulator of G- protein signaling polynucleotide.

Alternatively, host cells which contain a regulator of G-protein signaling polynucleotide and which express a regulator of G-protein signaling polypeptide can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques that include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a polynucleotide sequence encoding a regulator of G-protein signaling polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding a regulator of G-protein signaling polypeptide. Nucleic acid amplification-based assays involve the use of oligonucleotides selected from sequences encoding a regulator of G-protein signaling polypeptide to detect transformants that contain a regulator of G-protein signaling polynucleotide.

A variety of protocols for detecting and measuring the expression of a regulator of G- protein signaling polypeptide, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a regulator of G-protein signaling polypeptide can be used, or a competitive binding assay can be employed. These and other assays are described in Hampton et al., SEROLOGICAL METHODS: A LABORATORY MANUAL, APS Press, St. Paul, Minn., 1990) and Maddox et al, J. Exp. Med. 755, 1211-1216, 1983).

A wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding regulator of G-protein signaling polypeptides include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, sequences encoding a regulator of G-protein signaling polypeptide can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like. Expression and Purification of Polypeptides

Host cells transformed with nucleotide sequences encoding a regulator of G-protein signaling polypeptide can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode regulator of G-protein signaling polypeptides can be designed to contain signal sequences which direct secretion of soluble regulator of G-protein signaling polypeptides through a prokaryotic or eukaryotic cell membrane or which direct the membrane insertion of membrane-bound regulator of G-protein signaling polypeptide.

As discussed above, other constructions can be used to join a sequence encoding a regulator of G-protein signaling polypeptide to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system

(Immunex Corp., Seattle, Wash.). Inclusion of cleavable linker sequences such as those specific for Factor Xa or enterokmase (Invitrogen, San Diego, CA) between the purification domain and the regulator of G-protein signaling polypeptide also can be used to facilitate purification. One such expression vector provides for expression of a fusion protein containing a regulator of G-protein signaling polypeptide and 6 histidine residues preceding a thioredoxin or an enterokmase cleavage site. The histidine residues facilitate purification by IMAC (immobilized metal ion affinity chromatography, as described in Porath et al., Prot. Exp. Purif. 3, 263-281, 1992), while the enterokinase cleavage site provides a means for purifying the regulator of G-protein signaling polypeptide from the fusion protein. Vectors that contain fusion proteins are disclosed in Kroll et al., DNA Cell Biol. 12, 441-453, 1993. Chemical Synthesis

Sequences encoding a regulator of G-protein signaling polypeptide can be synthesized, in whole or in part, using chemical methods well known in the art (see

Caruthers et al., Nucl. Acids Res. Symp. Ser. 215-223, 1980; Horn et al. Nucl. Acids Res. Symp. Ser. 225-232, 1980). Alternatively, a regulator of G-protein signaling polypeptide itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149-2154, 1963; Roberge et al, Science 269,

202-204, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of regulator of G-protein signaling polypeptides can be separately synthesized and combined using chemical methods to produce a full-length molecule.

The newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic regulator of G-protein signaling polypeptide can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; see Creighton, supra). Additionally, any portion of the amino acid sequence of the regulator of G-protein signaling polypeptide can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.

Production of Altered Polypeptides

As will be understood by those of skill in the art, it may be advantageous to produce regulator of G-protein signaling polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce an RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence.

The nucleotide sequences disclosed herein can be engineered using methods generally known in the art to alter regulator of G-protein signaling polypeptide- encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the polypeptide or mRNA product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to engineer the nucleotide sequences. For example, site-directed mutagenesis can be used to insert new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, introduce mutations, and so forth.

Antibodies

Any type of antibody known in the art can be generated to bind specifically to an epitope of a regulator of G-protein signaling polypeptide. "Antibody" as used herein includes intact immunoglobulin molecules, as well as fragments thereof, such as Fab,

F(ab')₂, and Fv, which are capable of binding an epitope of a regulator of G-protein signaling polypeptide. Typically, at least 6, 8, 10, or 12 contiguous amino acids are required to form an epitope. However, epitopes which involve non-contiguous amino acids may require more, e.g., at least 15, 25, or 50 amino acids.

An antibody which specifically binds to an epitope of a regulator of G-protein signaling polypeptide can be used therapeutically, as well as in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Various immunoassays can be used to identify antibodies having the desired specificity. Numerous protocols for competitive binding or immunoradiometric assays are well known in the art. Such immunoassays typically involve the measurement of complex formation between an immunogen and an antibody that specifically binds to the immunogen.

Typically, an antibody which specifically binds to a regulator of G-protein signaling polypeptide provides a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies which specifically bind to regulator of G-protein signaling polypeptides do not detect other proteins in immunochemical assays and can immunoprecipitate a regulator of G-protein signaling polypeptide from solution.

Human regulator of G-protein signaling polypeptides can be used to immunize a mammal, such as a mouse, rat, rabbit, guinea pig, monkey, or human, to produce polyclonal antibodies. If desired, a regulator of G-protein signaling polypeptide can be conjugated to a carrier protein, such as bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin. Depending on the host species, various adjuvants can be used to increase the immunological response. Such adjuvants include, but are not limited to, Freund's adjuvant, mineral gels (e.g., aluminum hydroxide), and surface active substances (e.g. lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol). Among adjuvants used in humans, BCG (bacilli Calmette-Gueri ) and Corynebacterium parvum are especially useful.

Monoclonal antibodies that specifically bind to a regulator of G-protein signaling polypeptide can be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These techniques include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler et al, Nature 256, 495-497, 1985; Kozbor et al, J. Immunol. Methods 81, 31-42, 1985; Cote et al, Proc. Natl. Acad. Sci. 80, 2026-2030, 1983; Cole et al, Mol. Cell Biol. 62, 109-120, 1984). In addition, techniques developed for the production of "chimeric antibodies," the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity, can be used (Morrison et al, Proc. Natl. Acad. Sci. 81. 6851-6855, 1984; Neuberger et al, Nature 312, 604-608, 1984; Takeda et al, Nature 314, 452-454, 1985). Monoclonal and other antibodies also can be "humanized^" to prevent a patient from mounting an immune response against the antibody when it is used therapeutically. Such antibodies may be sufficiently similar in sequence to human antibodies to be used directly in therapy or may require alteration of a few key residues. Sequence differences between rodent antibodies and human sequences can be minimized by replacing residues which differ from those in the human sequences by site directed mutagenesis of individual residues or by grating of entire complementarity determining regions. Alternatively, humanized antibodies can be produced using recombinant methods, as described in GB2188638B. Antibodies that specifically bind to a regulator of G-protein signaling polypeptide can contain antigen binding sites which are either partially or fully humanized, as disclosed in U.S. 5,565,332.

Alternatively, techniques described for the production of single chain antibodies can be adapted using methods known in the art to produce single chain antibodies that specifically bind to regulator of G-protein signaling polypeptides. Antibodies with related specificity, but of distinct idiotypic composition, can be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci. 88, 11120-23, 1991).

Single-chain antibodies also can be constructed using a DNA amplification method, such as PCR, using hybridoma cDNA as a template (Thirion et al, 1996, Eur. J. Cancer Prev. 5, 507-11). Single-chain antibodies can be mono- or bispecific, and can be bivalent or tetravalent. Construction of tetravalent, bispecific single-chain antibodies is taught, for example, in Coloma & Morrison, 1997, Nat. Biotechnol. 15, 159-63. Construction of bivalent, bispecific single-chain antibodies is taught in

Mallender & Voss, 1994, J Biol. Chem. 269, 199-206. A nucleotide sequence encoding a single-chain antibody can be constructed using manual or automated nucleotide synthesis, cloned into an expression construct using standard recombinant DNA methods, and introduced into a cell to express the coding sequence, as described below. Alternatively, single-chain antibodies can be produced directly using, for example, filamentous phage technology (Verhaar et al, 1995, Int. J. Cancer 61, 497-501; Nicholls et al, 1993, J Immunol. Meth. 165, 81-91).

Antibodies which specifically bind to regulator of G-protein signaling polypeptides also can be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi et al, Proc. Natl. Acad. Sci. 86, 3833-3837, 1989; Winter et al, Nature 349, 293-299, 1991).

Other types of antibodies can be constructed and used therapeutically in methods of the invention. For example, chimeric antibodies can be constructed as disclosed in WO 93/03151. Binding proteins which are derived from immunoglobulins and which are multivalent and multispecific, such as the "diabodies" described in WO 94/13804, also can be prepared.

Antibodies according to the invention can be purified by methods well known in the art. For example, antibodies can be affinity purified by passage over a column to which a regulator of G-protein signaling polypeptide is bound. The bound antibodies can then be eluted from the column using a buffer with a high salt concentration.

Antisense Oligonucleotides

Antisense oligonucleotides are nucleotide sequences that are complementary to a specific DNA or RNA sequence. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form complexes and block either transcription or translation. Preferably, an antisense oligonucleotide is at least 11 nucleotides in length, but can be at least 12, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides long. Longer sequences also can be used. Antisense oligonucleotide molecules can be provided in a DNA construct and introduced into a cell as described above to decrease the level of regulator of G-protein signaling gene products in the cell.

Antisense oligonucleotides can be deoxyribonucleotides, ribonucleotides, or a combination of both. Oligonucleotides can be synthesized manually or by an automated synthesizer, by covalently linking the 5' end of one nucleotide with the 3' end of another nucleotide with non-phosphodiester internucleotide linkages such alkyl- phosphonates, phosphorothioates, phosphorodithioates, alkylphosphonothioates, alkylphosphonates, phosphoramidates, phosphate esters, carbamates, acetamidate, carboxymethyl esters, carbonates, and phosphate triesters. See Brown, Meth. Mol. Biol. 20, 1-8, 1994; Sonveaux, Meth. Mol. Biol. 26, 1-72, 1994; Uhlmann et al, Chem. Rev. 90, 543-583, 1990.

Modifications of regulator of G-protein signaling gene expression can be obtained by designing antisense oligonucleotides that will form duplexes to the control, 5', or regulatory regions of the regulator of G-protein signaling gene. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or chaperons. Therapeutic advances using triplex DNA have been described in the literature (e.g., Gee et al, in Huber & Carr,

MOLECULAR AND IMMUNOLOGIC APPROACHES, Futura Publishing Co., Mt. Kisco, N.Y., 1994). An antisense oligonucleotide also can be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Precise complementarity is not required for successful complex formation between an antisense oligonucleotide and the complementary sequence of a regulator of G- protein signaling polynucleotide. Antisense oligonucleotides which comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides which are precisely complementary to a regulator of G-protein signaling polynucleotide, each separated by a stretch of contiguous nucleotides which are not complementary to adjacent regulator of G-protein signaling nucleotides, can provide sufficient targeting specificity for regulator of G-protein signaling mRNA. Preferably, each stretch of complementary contiguous nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences are preferably 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an antisense-sense pair to determine the degree of mismatching which will be tolerated between a particular antisense oligonucleotide and a particular regulator of G-protein signaling polynucleotide sequence.

Antisense oligonucleotides can be modified without affecting their ability to hybridize to a regulator of G-protein signaling polynucleotide. These modifications can be internal or at one or both ends of the antisense molecule. For example, inter- nucleoside phosphate linkages can be modified by adding cholesteryl or diamine moieties with varying numbers of carbon residues between the amino groups and terminal ribose. Modified bases and/or sugars, such as arabinose instead of ribose, or a 3', 5 '-substituted oligonucleotide in which the 3' hydroxyl group or the 5' phosphate group are substituted, also can be employed in a modified antisense oligonucleotide. These modified oligonucleotides can be prepared by methods well known in the art. See, e.g., Agrawal et al, Trends Biotechnol. 10, 152-158, 1992;

Uhlmann et al, Chem. Rev. 90, 543-584, 1990; Uhlmann et al, Tetrahedron. Lett. 215, 3539-3542, 1987.

Ribozymes

Ribozymes are RNA molecules with catalytic activity. See, e.g., Cech, Science 236, 1532-1539; 1987; Cech, Ann. Rev. Biochem. 59, 543-568; 1990, Cech, Curr. Opin.

Struct. Biol. 2, 605-609; 1992, Couture & Stinchcomb, Trends Genet. 12, 510-515, 1996. Ribozymes can be used to inhibit gene function by cleaving an RNA sequence, as is known in the art (e.g., Haseloff et al, U.S. Patent 5,641,673). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of specific nucleotide sequences.

The coding sequence of a regulator of G-protein signaling polynucleotide can be used to generate ribozymes that will specifically bind to mRNA transcribed from the regulator of G-protein signaling polynucleotide. Methods of designing and constructing ribozymes which can cleave other RNA molecules in trans in a highly sequence specific manner have been developed and described in the art (see Haseloff et al. Nature 334, 585-591, 1988). For example, the cleavage activity of ribozymes can be targeted to specific RNAs by engineering a discrete "hybridization" region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA and thus specifically hybridizes with the target (see, for example, Gerlach et al, EP 321,201).

Specific ribozyme cleavage sites within a regulator of G-protein signaling RNA target can be identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target RNA containing the cleavage site can be evaluated for secondary structural features which may render the target inoperable. Suitability of candidate regulator of G-protein signaling RNA targets also can be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays. Longer complementary sequences can be used to increase the affinity of the hybridization sequence for the target. The hybridizing and cleavage regions of the ribozyme can be integrally related such that upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target.

Ribozymes can be introduced into cells as part of a DNA construct. Mechanical methods, such as microinjection, lipo some-mediated transfection, electroporation, or calcium phosphate precipitation, can be used to introduce a ribozyme-containing DNA construct into cells in which it is desired to decrease regulator of G-protein signaling expression. Alternatively, if it is desired that the cells stably retain the DNA construct, the construct can be supplied on a plasmid and maintained as a separate element or integrated into the genome of the cells, as is known in the art. A ribozyme-encoding DNA construct can include transcriptional regulatory elements, such as a promoter element, an enhancer or UAS element, and a transcriptional terminator signal, for controlling transcription of ribozymes in the cells.

As taught in Haseloff et al, U.S. Patent 5,641,673, ribozymes can be engineered so that ribozyme expression will occur in response to factors that induce expression of a target gene. Ribozymes also can be engineered to provide an additional level of regulation, so that destruction of mRNA occurs only when both a ribozyme and a target gene are induced in the cells.

Differentially Expressed Genes

Described herein are methods for the identification of genes whose products interact with human regulator of G-protein signaling. Such genes may represent genes that are differentially expressed in disorders including, but not limited to, asthma and diabetes. Further, such genes may represent genes that are differentially regulated in response to manipulations relevant to the progression or treatment of such diseases. Additionally, such genes may have a temporally modulated expression, increased or decreased at different stages of tissue or organism development. A differentially expressed gene may also have its expression modulated under control versus experimental conditions. In addition, the human regulator of G-protein signaling gene or gene product may itself be tested for differential expression.

The degree to which expression differs in a normal versus a diseased state need only be large enough to be visualized via standard characterization techniques such as differential display techniques. Other such standard characterization techniques by which expression differences may be visualized include but are not limited to, quantitative RT (reverse transcriptase), PCR, and Northern analysis.

Identification of Differentially Expressed Genes

To identify differentially expressed genes total RNA or, preferably, mRNA is isolated from tissues of interest. For example, RNA samples are obtained from tissues of experimental subjects and from corresponding tissues of control subjects. Any RNA isolation technique that does not select against the isolation of mRNA may be utilized for the purification of such RNA samples. See, for example, Ausubel et al, ed., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, Inc. New York, 1987-1993. Large numbers of tissue samples may readily be processed using techniques well known to those of skill in the art, such as, for example, the single-step RNA isolation process of Chomczynski, U.S. Patent 4,843,155.

Transcripts within the collected RNA samples that represent RNA produced by differentially expressed genes are identified by methods well known to those of skill in the art. They include, for example, differential screening (Tedder et al, Proc. Natl. Acad. Sci. U.S.A. 85, 208-12, 1988), subtractive hybridization (Hedrick et al,

Nature 308, 149-53; Lee et al, Proc. Natl. Acad. Sci. U.S.A. 88, 2825, 1984), and, preferably, differential display (Liang & Pardee, Science 257, 967-71, 1992; U.S. Patent 5,262,311).

The differential expression information may itself suggest relevant methods for the treatment of disorders involving the human regulator of G-protein signaling. For example, treatment ma)- include a modulation of expression of the differentially expressed genes and/or the gene encoding the human regulator of G-protein signaling. The differential expression information may indicate whether the expression or activity of the differentially expressed gene or gene product or the human regulator of G-protein signaling gene or gene product are up-regulated or down-regulated.

Screening Methods

The invention provides assays for screening test compounds that bind to or modulate the activity of a regulator of G-protein signaling polypeptide or a regulator of G- protein signaling polynucleotide. A test compound preferably binds to a regulator of G-protein signaling polypeptide or polynucleotide. More preferably, a test compound decreases or increases GAP activity by at least about 10, preferably about 50, more preferably about 75, 90, or 100%) relative to the absence of the test compound.

Test Compounds

Test compounds can be pharmacologic agents already known in the art or can be compounds previously unknown to have any pharmacological activity. The compounds can be naturally occurring or designed in the laboratory. They can be isolated from microorganisms, animals, or plants, and can be produced recom- binantly, or synthesized by chemical methods known in the art. If desired, test compounds can be obtained using any of the numerous combinatorial library methods known in the art, including but not limited to, biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the "one-bead one-compound" library method, and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide libraries, while the other four approaches are applicable to polypeptide, non-peptide oligomer, or small molecule libraries of compounds. See Lam, Anticancer Drug Des. 12, 145, 1997. Methods for the synthesis of molecular libraries are well known in the art (see, for example, DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al, J. Med. Chem. 37, 2678, 1994; Cho et al, Science 261, 1303, 1993; Carell et al, Angew. Chem. Int. Ed. Engl.

33, 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al, J. Med. Chem. 37, 1233, 1994). Libraries of compounds can be presented in solution (see, e.g., Houghten, BioTechniques 13, 412-421, 1992), or on beads (Lam, Nature 354, 82-84, 1991), chips (Fodor, Nature 364, 555-556, 1993), bacteria or spores (Ladner, U.S. Patent 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. U.S.A.

89, 1865-1869, 1992), or phage (Scott & Smith, Science 249, 386-390, 1990; Devlin, Science 249, 404-406, 1990); Cwirla et al, Proc. Natl. Acad. Sci. 97, 6378-6382, 1990; Felici, J Mol. Biol. 222, 301-310, 1991; and Ladner, U.S. Patent 5,223,409).

High Throughput Screening

Test compounds can be screened for the ability to bind to regulator of G-protein signaling polypeptides or polynucleotides or to affect regulator of G-protein signaling activity or regulator of G-protein signaling gene expression using high throughput screening. Using high throughput screening, many discrete compounds can be tested in parallel so that large numbers of test compounds can be quickly screened. The most widely established techniques utilize 96-well microtiter plates. The wells of the microtiter plates typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers, and plate readers are commercially available to fit the 96-well format.

Alternatively, "free format assays," or assays that have no physical barrier between samples, can be used. For example, an assay using pigment cells (melanocytes) in a simple homogeneous assay for combinatorial peptide libraries is described by Jayawickreme et al, Proc. Natl. Acad. Sci. U.S.A. 19, 1614-18 (1994). The cells are placed under agarose in petri dishes, then beads that carry combinatorial compounds are placed on the surface of the agarose. The combinatorial compounds are partially released the compounds from the beads. Active compounds can be visualized as dark pigment areas because, as the compounds diffuse locally into the gel matrix, the active compounds cause the cells to change colors.

Another example of a free format assay is described by Chelsky, "Strategies for Screening Combinatorial Libraries: Novel and Traditional Approaches," reported at the First Annual Conference of The Society for Biomolecular Screening in Philadelphia, Pa. (Nov. 7-10, 1995). Chelsky placed a simple homogenous enzyme assay for carbonic anhydrase inside an agarose gel such that the enzyme in the gel would cause a color change throughout the gel. Thereafter, beads carrying combinatorial compounds via a photolinker were placed inside the gel and the compounds were partially released by UV-light. Compounds that inhibited the enzyme were observed as local zones of inhibition having less color change.

Yet another example is described by Salmon et al, Molecular Diversity 2, 57-63 (1996). In this example, combinatorial libraries were screened for compounds that had cytotoxic effects on cancer cells growing in agar.

Another high throughput screening method is described in Beutel et al, U.S. Patent

5,976,813. In this method, test samples are placed in a porous matrix. One or more assay components are then placed within, on top of, or at the bottom of a matrix such as a gel, a plastic sheet, a filter, or other form of easily manipulated solid support. When samples are introduced to the porous matrix they diffuse sufficiently slowly, such that the assays can be performed without the test samples running together.

Binding Assays

For binding assays, the test compound is preferably a small molecule that binds to and occupies, for example, the active site of the regulator of G-protein signaling polypeptide, such that normal biological activity is prevented. Examples of such small molecules include, but are not limited to, small peptides or peptide-like molecules.

In binding assays, either the test compound or the regulator of G-protein signaling polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase. Detection of a test compound that is bound to the regulator of G-protein signaling polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product.

Alternatively, binding of a test compound to a regulator of G-protein signaling polypeptide can be determined without labeling either of the interactants. For example, a microphysiometer can be used to detect binding of a test compound with a regulator of G-protein signaling polypeptide. A microphysiometer (e.g., Cytosensor™) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and a regulator of G-protein signaling polypeptide (McConnell et al, Science 257, 1906-1912, 1992).

Determining the ability of a test compound to bind to a regulator of G-protein signaling polypeptide also can be accomplished using a technology such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338-2345, 1991, and Szabo et al, Curr. Opin. Struct. Biol. 5, 699-705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore™). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules. In yet another aspect of the invention, a regulator of G-protein signaling polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent 5,283,317; Zervos et al, Cell 72, 223-232, 1993; Madura et al, J. Biol. Chem. 268, 12046-12054, 1993; Bartel et al, BioTechniques 14, 920-924, 1993; Iwabuchi et al, Oncogene 8, 1693-1696, 1993; and Brent W094/10300), to identify other proteins which bind to or interact with the regulator of G-protein signaling polypeptide and modulate its activity.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. For example, in one construct, polynucleotide encoding a regulator of G-protein signaling polypeptide can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct a DNA sequence that encodes an unidentified protein ("prey" or "sample") can be fused to a polynucleotide that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo to form an protein-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein that interacts with the regulator of G-protein signaling polypeptide.

It may be desirable to immobilize either the regulator of G-protein signaling polypeptide (or polynucleotide) or the test compound to facilitate separation of bound from unbound forms of one or both of the interactants, as well as to accommodate automation of the assay. Thus, either the regulator of G-protein signaling poly- peptide (or polynucleotide) or the test compound can be bound to a solid support.

Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the enzyme polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support. Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to a regulator of G- protein signaling polypeptide (or polynucleotide) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

In one embodiment, the regulator of G-protein signaling polypeptide is a fusion protein comprising a domain that allows the regulator of G-protein signaling polypeptide to be bound to a solid support. For example, glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed regulator of G- protein signaling polypeptide; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).

Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.

Other techniques for immobilizing proteins or polynucleotides on a solid support also can be used in the screening assays of the invention. For example, either a regulator of G-protein signaling polypeptide (or polynucleotide) or a test compound can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated regulator of G-protein signaling polypeptides (or polynucleotides) or test compounds can be prepared from biotin-NHS(N-hydroxysuccinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, 111.) and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies which specifically bind to a regulator of G-protein signaling polypeptide, polynucleotide, or a test compound, but which do not interfere with a desired binding site, such as the active site of the regulator of G-protein signaling polypeptide, can be derivatized to the wells of the plate. Unbound target or protein can be trapped in the wells by antibody conjugation.

Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies which specifically bind to the regulator of G-protein signaling polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of the regulator of G-protein signaling polypeptide, and SDS gel electrophoresis under non- reducing conditions.

Screening for test compounds which bind to a regulator of G-protein signaling polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a regulator of G-protein signaling polypeptide or polynucleotide can be used in a cell-based assay system. A regulator of G-protein signaling poly- nucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to a regulator of G-protein signaling polypeptide or polynucleotide is determined as described above.

Enzyme Assays

Test compounds can be tested for the ability to increase or decrease the GAP activity of a human regulator of G-protein signaling polypeptide. GAP activity can be measured, for example, as described in Woodson, J. Biol. Chem, 276:20160-6 (2001). Enzyme assays can be carried out after contacting either a purified regulator of G- protein signaling polypeptide, a cell membrane preparation, or an intact cell with a test compound. A test compound that decreases a GAP activity of a regulator of G- protein signaling polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for decreasing regulator of G-protein signaling activity. A test compound which increases a GAP activity of a human regulator of G-protein signaling polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100% is identified as a potential therapeutic agent for increasing human regulator of G-protein signaling activity.

Gene Expression

In another embodiment, test compounds that increase or decrease regulator of G- protein signaling gene expression are identified. A regulator of G-protein signaling polynucleotide is contacted with a test compound, and the expression of an RNA or polypeptide product of the regulator of G-protein signaling polynucleotide is determined. The level of expression of appropriate mRNA or polypeptide in the presence of the test compound is compared to the level of expression of mRNA or polypeptide in the absence of the test compound. The test compound can then be identified as a modulator of expression based on this comparison. For example, when expression of mRNA or polypeptide is greater in the presence of the test compound than in its absence, the test compound is identified as a stimulator or enhancer of the mRNA or polypeptide expression. Alternatively, when expression of the mRNA or polypeptide is less in the presence of the test compound than in its absence, the test compound is identified as an inhibitor of the mRNA or polypeptide expression.

The level of regulator of G-protein signaling mRNA or polypeptide expression in the cells can be determined by methods well known in the art for detecting mRNA or polypeptide. Either qualitative or quantitative methods can be used. The presence of polypeptide products of a regulator of G-protein signaling polynucleotide can be determined, for example, using a variety of techniques known in the art, including immunochemical methods such as radioimmunoassay, Western blotting, and immunohistochemistry. Alternatively, polypeptide synthesis can be determined in vivo, in a cell culture, or in an in vitro translation system by detecting incorporation of labeled amino acids into a regulator of G-protein signaling polypeptide.

Such screening can be carried out either in a cell-free assay system or in an intact cell. Any cell that expresses a regulator of G-protein signaling polynucleotide can be used in a cell-based assay system. The regulator of G-protein signaling polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Either a primary culture or an established cell line, such as CHO or human embryonic kidney 293 cells, can be used.

Pharmaceutical Compositions

The invention also provides pharmaceutical compositions that can be administered to a patient to achieve a therapeutic effect. Pharmaceutical compositions of the invention can comprise, for example, a regulator of G-protein signaling polypeptide, regu- lator of G-protein signaling polynucleotide, ribozymes or antisense oligonucleotides, antibodies which specifically bind to a regulator of G-protein signaling polypeptide, or mimetics, activators, or inhibitors of a regulator of G-protein signaling polypeptide activity. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means. Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained through combination of

active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxy- propylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents can be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.

Dragee cores can be used in conjunction with suitable coatings, such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments can be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol. Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optional ly. stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Non-lipid polycationic amino polymers also can be used for delivery. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used, in the formulation. Such penetrants are generally known in the art.

The pharmaceutical compositions of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. The pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms. In other cases, the preferred preparation can be a lyophilized powder which can contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. Further details on techniques for formulation and administration can be found in the latest edition of REMINGTON'S PHARMACEUTICAL SCIENCES (Maack Publishing Co., Easton, Pa.). After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

Therapeutic Indications and Methods

Human regulator of G-protein signaling can be regulated to treat asthma and diabetes.

Asthma. Allergy is a complex process in which environmental antigens induce clinically adverse reactions. The inducing antigens, called allergens, typically elicit a specific IgE response and, although in most cases the allergens themselves have little or no intrinsic toxicity, they induce pathology when the IgE response in turn elicits an IgE-dependent or T cell-dependent hypersensitivity reaction. Hypersensitivity reactions can be local or systemic and typically occur within minutes of allergen exposure in individuals who have previously been sensitized to an allergen. The hypersensitivity reaction of allergy develops when the allergen is recognized by IgE antibodies bound to specific receptors on the surface of effector cells, such as mast cells, basophils, or eosinophils, which causes the activation of the effector cells and the release of mediators that produce the acute signs and symptoms of the reactions. Allergic diseases include asthma, allergic rhinitis (hay fever), atopic dermatitis, and anaphylaxis.

Asthma is though to arise as a result of interactions between multiple genetic and environmental factors and is characterized by three major features: 1) intermittent and reversible airway obstruction caused by bronchoconstriction, increased mucus production, and thickening of the walls of the airways that leads to a narrowing of the airways, 2) airway hyperresponsiveness caused by a decreased control of airway caliber, and 3) airway inflammation. Certain cells are critical to the inflammatory reaction of asthma and they include T cells and antigen presenting cells, B cells that produce IgE, and mast cells, basophils, eosinophils, and other cells that bind IgE. These effector cells accumulate at the site of allergic reaction in the airways and release toxic products that contribute to the acute pathology and eventually to the tissue destruction related to the disorder. Other resident cells, such as smooth muscle cells, lung epithelial cells, mucus-producing cells, and nerve cells may also be abnormal in individuals with asthma and may contribute to the pathology. While the airway obstruction of asthma, presenting clinically as an intermittent wheeze and shortness of breath, is generally the most pressing symptom of the disease requiring immediate treatment, the inflammation and tissue destruction associated with the disease can lead to irreversible changes that eventually make asthma a chronic disabling disorder requiring long-term management.

Despite recent important advances in our understanding of the pathophysiology of asthma, the disease appears to be increasing in prevalence and severity (Gergen and Weiss, Am. Rev. Respir. Dis. 146, 823-24, 1992). It is estimated that 30-40% of the population suffer with atopic allergy, and 15%ι of children and 5% of adults in the population suffer from asthma (Gergen and Weiss, 1992). Thus, an enormous burden is placed on our health care resources. However, both diagnosis and treatment of asthma are difficult. The severity of lung tissue inflammation is not easy to measure and the symptoms of the disease are often indistinguishable from those of respiratory infections, chronic respiratory inflammatory disorders, allergic rhinitis, or other respiratory disorders. Often, the inciting allergen cannot be determined, making removal of the causative environmental agent difficult. Current pharmacological treatments suffer their own set of disadvantages. Commonly used therapeutic agents, such as beta agonists, can act as symptom relievers to transiently improve pulmonary function, but do not affect the underlying inflammation. Agents that can reduce the underlying inflammation, such as anti-inflammatory steroids, can have major drawbacks that range from immunosuppression to bone loss (Goodman and Gilman's

THE PHARMACOLOGIC BASIS OF THERAPEUTICS, Seventh Edition, MacMillan Publishing Company, NY, USA, 1985). In addition, many of the present therapies, such as inhaled corticosteroids, are short-lasting, inconvenient to use, and must be used often on a regular basis, in some cases for life, making failure of patients to comply with the treatment a major problem and thereby reducing their effectiveness as a treatment.

Because of the problems associated with conventional therapies, alternative treatment strategies have been evaluated. Glycophorin A (Chu and Sharom, Cell. Immunol. 145, 223-39, 1992), cyclosporin (Alexander et al, Lancet 339, 324-28, 1992), and a nonapeptide fragment of IL-2 (Zav'yalov et al, Immunol. Lett. 31, 285-88, 1992) all inhibit interleukin-2 dependent T lymphocyte proliferation; however, they are known to have many other effects. For example, cyclosporin is used as a irnmuno- suppressant after organ transplantation. While these agents may represent alternatives to steroids in the treatment of asthmatics, they inhibit interleukin-2 dependent T lymphocyte proliferation and potentially critical immune functions associated with homeostasis. Other treatments that block the release or activity of mediators of bronchochonstriction, such as cromones or anti-leukotrienes, have recently been introduced for the treatment of mild asthma, but they are expensive and not effective in all patients and it is unclear whether they have any effect on the chronic changes associated with asthmatic inflammation. What is needed in the art is the identification of a treatment that can act in pathways critical to the development of asthma that both blocks the episodic attacks of the disorder and preferentially dampens the hyperactive allergic immune response without immunocompromising the patient.

Diabetes: Diabetes mellitus is a common metabolic disorder characterized by an abnormal elevation in blood glucose, alterations in lipids and abnormalities (complications) in the cardiovascular system, eye, kidney and nervous system. Diabetes is divided into two separate diseases: type 1 diabetes (juvenile onset) that results from a loss o f cells which make and secrete insulin, and type 2 diabetes (adult onset) which is caused by a defect in insulin secretion and a defect in insulin action. Type I diabetes is initiated by an autoimmune reaction that attacks the insulin secreting cells (beta cells) in the pancreatic islets. Agents that prevent this reaction from occurring or that stop the reaction before destruction of the beta cells has been accomplished are potential therapies for this disease. Other agents that induce beta cell proliferation and regeneration are also potential therapies.

Type II diabetes is the most common of the two diabetic conditions (6% of the population). The defect in insulin secretion is an important cause of the diabetic condition and results from an inability of the beta cells to properly detect and respond to rises in blood glucose levels with insulin release. Therapies that increase the response by the beta cells to glucose would offer an important new treatment for this disease.

The defect in insulin action in Type II diabetic subjects is another target fro therapeutic intervention. Agents that increase the activity of the insulin receptor in muscle, liver, and fat will cause a decrease in blood glucose and a normalization of plasma lipids. The receptor activity can be increased by agents that directly stimulate the receptor or that increase the intracellular signals from the receptor. Other therapies can directly activate the cellular end process, i.e., glucose transport or various enzyme systems, to generate an insulin-like effect and therefore produce a beneficial outcome. Because overweight subjects have a greater susceptibility to Type II diabetes, any agent that reduces body weight is a possible therapy.

Both Type I and Type II diabetes can be treated with agents that mimic insulin action or that treat diabetic complications by reducing blood glucose levels. Likewise, agents that reduce new blood vessel growth can be used to treat the eye complications that develop in both diseases.

This invention further pertains to the use of novel agents identified by the screening assays described above. Accordingly, it is within the scope of this invention to use a test compound identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a modulating agent, an antisense nucleic acid molecule, a specific antibody, ribozyme, or a regulator of G- protein signaling polypeptide binding molecule) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

A reagent which affects regulator of G-protein signaling activity can be administered to a human cell, either in vitro or in vivo, to reduce regulator of G-protein signaling activity. The reagent preferably binds to an expression product of a human regulator of G-protein signaling gene. If the expression product is a protein, the reagent is preferably an antibody. For treatment of human cells ex vivo, an antibody can be added to a preparation of stem cells that have been removed from the body. The cells can then be replaced in the same or another human body, with or without clonal propagation, as is known in the art.

In one embodiment, the reagent is delivered using a liposome. Preferably, the liposome is stable in the animal into which it has been administered for at least about

30 minutes, more preferably for at least about 1 hour, and even more preferably for at least about 24 hours. A liposome comprises a lipid composition that is capable of targeting a reagent, particularly a polynucleotide, to a particular site in an animal, such as a human. Preferably, the lipid composition of the liposome is capable of targeting to a specific organ of an animal, such as the lung, liver, spleen, heart brain, lymph nodes, and skin.

A liposome useful in the present invention comprises a lipid composition that is capable of fusing with the plasma membrane of the targeted cell to deliver its contents to the cell. Preferably, the transfection efficiency of a liposome is about

0.5 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, more preferably about 1.0 μg of DNA per 16 nmole of liposome delivered to about 10⁶ cells, and even more preferably about 2.0 μg of DNA per 16 nmol of liposome delivered to about 10⁶ cells. Preferably, a liposome is between about 100 and 500 nm, more preferably between about 150 and 450 nm, and even more preferably between about 200 and 400 nm in diameter.

Suitable liposomes for use in the present invention include those liposomes standardly used in, for example, gene delivery methods known to those of skill in the art. More preferred liposomes include liposomes having a polycationic lipid com- position and/or liposomes having a cholesterol backbone conjugated to polyethylene glycol. Optionally, a liposome comprises a compound capable of targeting the liposome to a particular cell type, such as a cell-specific ligand exposed on the outer surface of the liposome.

Complexing a liposome with a reagent such as an antisense oligonucleotide or ribozyme can be achieved using methods that are standard in the art (see, for example, U.S. Patent 5,705,151). Preferably, from about 0.1 μg to about 10 μg of polynucleotide is combined with about 8 nmol of liposomes, more preferably from about 0.5 μg to about 5 μg of polynucleotides are combined with about 8 nmol liposomes, and even more preferably about 1.0 μg of polynucleotides is combined with about 8 nmol liposomes.

In another embodiment, antibodies can be delivered to specific tissues in vivo using receptor-mediated targeted delivery. Receptor-mediated DNA delivery techniques are taught in, for example, Findeis et al. Trends in Biotechnol. 11, 202-05 (1993);

Chiou et al, GENE THERAPEUTICS: METHODS AND APPLICATIONS OF DIRECT GENE TRANSFER (J.A. Wolff, ed.) (1994); Wu & Wu, J. Biol. Chem. 263, 621-24 (1988); Wu et al, J. Biol. Chem. 269, 542-46 (1994); Zenke et al, Proc. Natl. Acad. Sci. U.S.A. 87, 3655-59 (1990); Wxι et al., J Biol. Chem. 266, 338-42 (1991). Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient which increases or decreases regulator of G-protein signaling activity relative to the regulator of G-protein signaling activity which occurs in the absence of the therapeutically effective dose.

For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

Therapeutic efficacy and toxicity, e.g., ED₅₀ (the dose therapeutically effective in 50% of the population) and LD₅₀ (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD₅o/ED₅₀.

Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

Normal dosage amounts can vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

If the reagent is a single-chain antibody, polynucleotides encoding the antibody can be constructed and introduced into a cell either ex vivo or in vivo using well- established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome- mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 μg to about 50 μg/kg, about 50 μg to about 5 mg/kg, about 100 μg to about 500 μg/kg of patient body weight, and about 200 to about 250 μg/kg of patient body weight. For administration of polynucleotides encoding single-chain antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA. If the expression product is mRNA, the reagent is preferably an antisense oligonucleotide or a ribozyme. Polynucleotides that express antisense oligonucleotides or ribozymes can be introduced into cells by a variety of methods, as described above.

Preferably, a reagent reduces expression of a regulator of G-protein signaling gene or the activity of a regulator of G-protein signaling polypeptide by at least about 10, preferably about 50, more preferably about 75, 90, or 100%) relative to the absence of the reagent. The effectiveness of the mechanism chosen to decrease the level of expression of a regulator of G-protein signaling gene or the activity of a regulator of

G-protein signaling polypeptide can be assessed using methods well known in the art, such as hybridization of nucleotide probes to regulator of G-protein signaling- specific mRNA, quantitative RT-PCR, immunologic detection of a regulator of G- protein signaling polypeptide, or measurement of regulator of G-protein signaling activity.

In any of the embodiments described above, any of the pharmaceutical compositions of the invention can be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy can be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents can act synergistically to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

Any of the therapeutic methods described above can be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans. Diagnosiic Methods

Human regulator of G-protein signaling also can be used in diagnostic assays for detecting diseases and abnormalities or susceptibility to diseases and abnormalities related to the presence of mutations in the nucleic acid sequences that encode the enzyme. For example, differences can be determined between the cDNA or genomic sequence encoding regulator of G-protein signaling in individuals afflicted with a disease and in normal individuals. If a mutation is observed in some or all of the afflicted individuals but not in normal individuals, then the mutation is likely to be the causative agent of the disease.

Sequence differences between a reference gene and a gene having mutations can be revealed by the direct DNA sequencing method. In addition, cloned DNA segments can be employed as probes to detect specific DNA segments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer can be used with a double-stranded PCR product or a single-stranded template molecule generated by a modified PCR. The sequence determination is performed by conventional procedures using radiolabeled nucleotides or by automatic sequencing procedures using fluorescent tags.

Genetic testing based on DNA sequence differences can be carried out by detection of alteration in electrophoretic mobility of DNA fragments in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized, for example, by high resolution gel electrophoresis. DNA fragments of different sequences can be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al, Science 230, 1242, 1985). Sequence changes at specific locations can also be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85,

4397-4401, 1985). Thus, the detection of a specific DNA sequence can be performed by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes and Southern blotting of genomic DNA. In addition to direct methods such as gel-electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.

Altered levels of regulator of G-protein signaling also can be detected in various tissues. Assays used to detect levels of the receptor polypeptides in a body sample, such as blood or a tissue biopsy, derived from a host are well known to those of skill in the art and include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

All patents and patent applications cited in this disclosure are expressly incorporated herein by reference. The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.

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EXAMPLE 1

Detection of human regulator polypeptide of G-Protein Signaling activity

The polynucleotide of SEQ ID NO: 1 is inserted into the expression vector pCEV4 and the expression vector pCEV4 human regulator polypeptide of G-Protein Signaling obtained is transfected into human embryonic kidney 293 cells. From these cells extracts are obtained and tested in the following assay: Briefly, 250-500 nM G- alpha-il in the assay buffer (lOmM HEPES, pH 8.0, 5 mM EDTA, 2mM DTT) is loaded with [gamma-32^p]GTP at 30°C for 20 min. The solution is placed on ice for

5 min, and all subsequent reactions are carried out on ice in a cold room. A drop of the cell extract (5-10 μl) is placed onto a wall of reaction tube next to a 5-μl drop of solution, 500 mM MgCl₂, 5 mM cold GTP, and the GAP reaction is started by addition of 175 μl of G-alpha-il-GTP solution. The final concentration of free Mg²⁺ ions in the reaction mix is 3-4 mM. To study the influence of Ca2+/calmodulin on

RGS4 GAP activity with G-alpha-il-GTP, RGS4 is preincubated with calmodulin in 10 mM HEPES, pH 8.0, 1 mM CaCl₂, 2 mM DTT on ice for 20 min. Then 175 μl of G-alpha-il loaded with [gamma-32^p]GTP is added with 5 μl of 500 mM GTP, 475 mM MgCl₂, 25 mM CaCl₂. The human regulator polypeptide of G-Protein Signaling activity of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 is demonstrated.

EXAMPLE 2

Expression of recombinant human regulator of G-protein signaling

The Pichia pastoris expression vector pPICZB (Invitrogen, San Diego, CA) is used to produce large quantities of recombinant human regulator of G-protein signaling polypeptides in yeast. The regulator of G-protein signaling-encoding DNA sequence is derived from SEQ ID NO:l. Before insertion into vector pPICZB, the DNA sequence is modified by well known methods in such a way that it contains at its - el ¬

s' -end an initiation codon and at its 3 '-end an enterokinase cleavage site, a His6 reporter tag and a termination codon. Moreover, at both termini recognition sequences for restriction endonucleases are added and after digestion of the multiple cloning site of pPICZ B with the corresponding restriction enzymes the modified DNA sequence is ligated into pPICZB. This expression vector is designed for inducible expression in Pichia pastoήs, driven by a yeast promoter. The resulting pPICZ/md-His6 vector is used to transform the yeast.

The yeast is cultivated under usual conditions in 5 liter shake flasks and the recombinantly produced protein isolated from the culture by affinity chromatography

(Ni-NTA-Resin) in the presence of 8 M urea. The bound polypeptide is eluted with buffer, pH 3.5, and neutralized. Separation of the polypeptide from the His6 reporter tag is accomplished by site-specific proteolysis using enterokinase (Invitrogen, San Diego, CA) according to manufacturer's instructions. Purified human regulator of G- protein signaling polypeptide is obtained.

EXAMPLE 3

Identification of test compounds that bind to regulator of G-protein signaling polypeptides

Purified regulator of G-protein signaling polypeptides comprising a glutathione-S- transferase protein and absorbed onto glutathione-derivatized wells of 96-well microtiter plates are contacted with test compounds from a small molecule library at pH 7.0 in a physiological buffer solution. Human regulator of G-protein signaling polypeptides comprise the amino acid sequence shown in SEQ ID NO:2. The test compounds comprise a fluorescent tag. The samples are incubated for 5 minutes to one hour. Control samples are incubated in the absence of a test compound.

The buffer solution containing the test compounds is washed from the wells.

Binding of a test compound to a regulator of G-protein signaling polypeptide is detected by fluorescence measurements of the contents of the wells. A test compound that increases the fluorescence in a well by at least 15% relative to fluorescence of a well in which a test compound is not incubated is identified as a compound which binds to a regulator of G-protein signaling polypeptide.

EXAMPLE 4

Identification of a test compound which decreases regulator of G-protein signaling gene expression

A test compound is administered to a culture of human cells transfected with a regulator of G-protein signaling expression construct and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control.

RNA is isolated from the two cultures as described in Chirgwin et al, Biochem. 18, 5294-99, 1979). Northern blots are prepared using 20 to 30 μg total RNA and hybridized with a ³²P -labeled regulator of G-protein signaling-specific probe at 65 °C in Express-hyb (CLONTECH). The probe comprises at least 11 contiguous nucleotides selected from the complement of SEQ ID NO:l. A test compound that decreases the regulator of G-protein signaling-specific signal relative to the signal obtained in the absence of the test compound is identified as an inhibitor of regulator of G-protein signaling gene expression.

EXAMPLE 5

Identification of a test compound which decreases regulator of G-protein signaling activity

A test compound is administered to a culture of human cells transfected with a regulator of G-protein signaling expression construct and incubated at 37°C for 10 to 45 minutes. A culture of the same type of cells that have not been transfected is incubated for the same time without the test compound to provide a negative control, regulator of G-protein signaling activity is measured using the method of Woodson, J. Biol. Chem, 276:20160-6 (2001).

A test compound which decreases the regulator of G-protein signaling activity of the regulator of G-protein signaling relative to the regulator of G-protein signaling activity in the absence of the test compound is identified as an inhibitor of regulator of G-protein signaling activity.

EXAMPLE 6

Tissue-specific expression of regulator of G-protein signaling

The qualitative expression pattern of regulator of G-protein signaling in various tissues is determined by Reverse Transcription-Polymerase Chain Reaction (RT- PCR).

To demonstrate that human regulator of G-protein signaling is involved in the disease process of asthma, the following whole body panel is screened to show predominant or relatively high expression in lung or immune tissues: brain, heart, kidney, liver, lung, trachea, bone marrow, colon, small intestine, spleen, stomach, thymus, mammary gland, skeletal muscle, prostate, testis, uterus, cerebellum, fetal brain, fetal liver, spinal cord, placenta, adrenal gland, pancreas, salivary gland, thyroid, peripheral blood leukocytes, lymph node, and tonsil. Once this is established, the following lung and immune system cells are screened to localize expression to particular cell subsets: lung micro vascular endothelial cells, bronchial/tracheal epithelial cells, bronchial/tracheal smooth muscle cells, lung fibroblasts, T cells (T helper 1 subset, T helper 2 subset, NKT cell subset, and cytotoxic T lymphocytes), B cells, mononuclear cells (monocytes and macrophages), mast cells, eosinophils, neutrophils, and dendritic cells. As a final step, the expression of human regulator of G-protein signaling in cells derived from normal individuals with the expression of cells derived from asthmatic individuals is compared.

Quantitative expression profiling. Quantitative expression profiling is performed by the form of quantitative PCR analysis called "kinetic analysis" firstly described in

Higuchi et al, BioTechnology 10, 413-17, 1992, and Higuchi et al, BioTechnology 11, 1026-30, 1993. The principle is that at any given cycle within the exponential phase of PCR, the amount of product is proportional to the initial number of template copies.

If the amplification is performed in the presence of an internally quenched fluorescent oligonucleotide (TaqMan probe) complementary to the target sequence, the probe is cleaved by the 5 '-3' endonuclease activity of Taq DNA polymerase and a fluorescent dye released in the medium (Holland et al, Proc. Natl. Acad. Sci. U.S.A. 88, 7276-80, 1991). Because the fluorescence emission will increase in direct proportion to the amount of the specific amplified product, the exponential growth phase of PCR product can be detected and used to determine the initial template concentration (Heid et al, Genome Res. 6, 986-94, 1996, and Gibson et al, Genome Res. 5, 995-1001, 1996).

The amplification of an endogenous control can be performed to standardize the amount of sample RNA added to a reaction. In this kind of experiment, the control of choice is the 18S ribosomal RNA. Because reporter dyes with differing emission spectra are available, the target and the endogenous control can be independently quantified in the same tube if probes labeled with different dyes are used.

All "real time PCR" measurements of fluorescence are made in the ABI Prism 7700.

RNA extraction and cDNA preparation. Total RNA from the tissues listed above are used for expression quantification. RNAs labeled "from autopsy" were extracted from autoptic tissues with the TRIzol reagent (Life Technologies, MD) according to the manufacturer's protocol.

Fifty μg of each RNA were treated with DNase I for 1 hour at 37°C in the following reaction mix: 0.2 U/μl RNase-free DNase I (Roche Diagnostics, Germany); 0.4 U/μl RNase inhibitor (PE Applied Biosystems, CA); 10 mM Tris-HCl pH 7.9; lOmM

MgCl₂; 50 mMNaCl; and 1 mM DTT.

After incubation, RNA is extracted once with 1 volume of phenol:chloroform:iso- amyl alcohol (24:24:1) and once with chloroform, and precipitated with 1/10 volume of 3 M NaAcetate, pH5.2, and 2 volumes of ethanol.

Fifty μg of each RNA from the autoptic tissues are DNase treated with the DNA-free kit purchased from Ambion (Ambion, TX). After resuspension and spectro- photometric quantification, each sample is reverse transcribed with the TaqMan Reverse Transcription Reagents (PE Applied Biosystems, CA) according to the manufacturer's protocol. The final concentration of RNA in the reaction mix is 200ng/μL. Reverse transcription is carried out with 2.5μM of random hexamer primers.

TaqMan quantitative analysis. Specific primers and probe are designed according to the recommendations of PE Applied Biosystems; the probe can be labeled at the 5' end FAM (6-carboxy-fluorescein) and at the 3' end with TAMRA (6-carboxy-tetra- methyl-rhodamine). Quantification experiments are performed on 10 ng of reverse transcribed RNA from each sample. Each determination is done in triplicate.

Total cDNA content is normalized with the simultaneous quantification (multiplex PCR) of the 18S ribosomal RNA using the Pre-Developed TaqMan Assay Reagents (PDAR) Control Kit (PE Applied Biosystems, CA).

The assay reaction mix is as follows: IX final TaqMan Universal PCR Master Mix

(from 2X stock) (PE Applied Biosystems, CA); IX PDAR control - 18S RNA (from 20X stock); 300 nM forward primer; 900 nM reverse primer; 200 nM probe; 10 ng cDNA; and water to 25 μl.

Each of the following steps are carried out once: pre PCR, 2 minutes at 50°C, and 10 minutes at 95°C. The following steps are carried out 40 times: denaturation, 15 seconds at 95 °C, annealing/extension, 1 minute at 60°C.

The experiment is performed on an ABI Prism 7700 Sequence Detector (PE Applied Biosystems, CA). At the end of the run, fluorescence data acquired during PCR are processed as described in the ABI Prism 7700 user's manual in order to achieve better background subtraction as well as signal linearity with the starting target quantity.

REFERENCES:

1. De Vries et al. (2000) The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol 40:235-71.

2. Hepler (1999) Emerging roles for RGS proteins in cell signaling. Trends Pharmacol Sci 20(9):376-82.

3. Burchett (2000) Regulators of G protein signaling: a bestiary of modular protein binding domains. JNeurochem 75(4):1335-51.

4. Benzing et al. (2000) 14-3-3 interacts with regulator of

G protein signaling proteins and modulates their activity. J Biol Chem 275(36):28167-72. EXAMPLE 7

In vivo testing of compounds/target validation for diabetes treatment

1. Glucose Production:

Over-production of glucose by the liver, due to an enhanced rate of gluconeogenesis, is the major cause of fasting hyperglycemia in diabetes. Overnight fasted normal rats or mice have elevated rates of gluconeogenesis as do streptozotocin-induced diabetic rats or mice fed ad libitum. Rats are made diabetic with a single intravenous injection of 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kg i.p. for 5 consecutive days. Blood glucose is measured from tail-tip blood and then compounds are administered via different routes (p.o., i.p., i.v., s.c). Blood is collected at various times thereafter and glucose measured. Alternatively, com- pounds are administered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will decrease plasma glucose levels compared to the vehicle-treated control group.

2. Insulin Sensitivity:

Both ob/ob and db/db mice as well as diabetic Zucker rats are hyperglycemic, hyperinsulinemic and insulin resistant. The animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group. Compounds are administered daily either q.d. or b.i.d. by different routes (p.o., i.p., s.c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined. Compounds that improve insulin sensitivity in these models will decrease both plasma glucose and insulin levels when compared to the vehicle-treated control group. 3. Insulin Secretion:

Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the administration of a glucose load. When measuring insulin levels, compounds are administered by different routes (p.o., i.p., s.c. or i.v.) to overnight fasted normal rats or mice. At the appropriate time an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined. Compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose. When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60 and 90 minutes and plasma glucose levels determined. Compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose.

Compounds that enhance insulin secretion from the pancreas will increase plasma insulin levels and improve the disappearance of plasma glucose following the administration of a glucose load. When measuring insulin levels, test compounds which regulate triacylglycerol lipase are administered by different routes (p.o., i.p., s.c, or i.v.) to overnight fasted normal rats or mice. At the appropriate time an intravenous glucose load (0.4g/kg) is given, blood is collected one minute later. Plasma insulin levels are determined. Test compounds that enhance insulin secretion will increase plasma insulin levels compared to animals given only glucose. When measuring glucose disappearance, animals are bled at the appropriate time after compound administration, then given either an oral or intraperitoneal glucose load (lg/kg), bled again after 15, 30, 60, and 90 minutes and plasma glucose levels determined. Test compounds that increase insulin levels will decrease glucose levels and the area-under-the glucose curve when compared to the vehicle-treated group given only glucose. 4. Glucose Production:

Over-production of glucose by the liver, due to an enhanced rate of gluconeogenesis, is the major cause of fasting hyperglycemia in diabetes. Overnight fasted normal rats or mice have elevated rates of gluconeogenesis as do streptozotocin-induced diabetic rats or mice fed ad libitum. Rats are made diabetic with a single intravenous injection of 40 mg/kg of streptozotocin while C57BL/KsJ mice are given 40-60 mg/kg i.p. for 5 consecutive days. Blood glucose is measured from tail-tip blood and then compounds are administered via different routes (p.o., i.p., i.v., s.c). Blood is collected at various times thereafter and glucose measured. Alternatively, compounds are administered for several days, then the animals are fasted overnight, blood is collected and plasma glucose measured. Compounds that inhibit glucose production will decrease plasma glucose levels compared to the vehicle-treated control group.

5. Insulin Sensitivity:

Both ob/ob and db/db mice as well as diabetic Zucker rats are hyperglycemic, hyperinsulinemic and insulin resistant. The animals are pre-bled, their glucose levels measured, and then they are grouped so that the mean glucose level is the same for each group. Compounds are administered daily either q.d. or b.i.d. by different routes (p.o., i.p., s.c.) for 7-28 days. Blood is collected at various times and plasma glucose and insulin levels determined. Compounds that improve insulin sensitivity in these models will decrease both plasma glucose and insulin levels when compared to the vehicle-treated control group.

6. Insulin Secretion:

EXAMPLE 8

In vivo testing of compounds/target validation for asthma treatment

1. Activity tests in T cells for modulators of costimulatory molecules, cytokines, cytokine receptors, signalling molecules and molecules involved in T cell activation

Mouse anti-CD3 induced cytokine production model

BALB/c mice are injected with a single intravenous injection of 10 μg of 145-2C11 (purified hamster anti-mouse CD3ε monoclonal antibodies, PHARMINGEN). Compound is administered intraperitoneally 60 min prior to the anti-CD3 mAb injection. Blood is collected 90 min after the antibody injection. Serum is obtained by centrifugation at 3000 r.p.m. for 10 min. IL-2 and IL-4 levels in the serum is determined by an ELISA. 2. Activity test in B cells for modulators of the B cell receptor, signalling molecules, and molecule involved in B cell activation Ig class switching

Mouse anti-IgD induced IgE production model

BALB/c mice are injected intravenously with 0.8 mg of purified goat anti-mouse IgD antibody or PBS (defined as day 0). Compound is administered intraperitoneally from day 0 to day 6. On day 7 blood is collected and serum is obtained by centrifugation at 3000 r.p.m. for 10 min. Serum total levels of IgE are determined by YAMASA's ELISA kit and their Ig subtypes are done by an Ig ELISA KIT (Rougier

Biotech's, Montreal, Canada).

3. Activity tests in monocytes/macrophages for modulators of signalling molecules and transcription factos

Mouse LPS-induced TNF-α production model

BALB/c mice are injected intraperitoneally with LPS (200 μg/mouse). Compound is administered intraperitoneally 1 hr before the LPS injection. Blood is collected at 90 min post-LPS injection and plasma is obtained. TNF-α concentration in the sample is determined using an ELISA kit.

4. Activity tests in eosinophils for modulators of Eotaxin, eotaxin receptor (GPCR), signalling molecules, cytoskeletal molecules and adhesion molecules

Mouse eotaxin-induced eosinophilia model

BALB/c mice are injected intradermally with a 2.5 ml of air on days -6 and -3 to prepare airpouch. On day 0 compound is administered intraperitoneally 60 min before eotaxin injection (3 μg/mouse, i.d.). IL-5 (300 ng/mouse) is injected intravenously 30 min before the eotaxin injection. After 4 hr of the eotaxin injection leukocytes in exudate is collected and the number of total cells is counted. The differential cell counts in the exudate are performed by staining with May-Grunwald Gimsa solution.

5. Activity tests in Th2 cells for molecules involved in antigen presentation, costimulatory molecules, signaling molecules and transcription factors

Mouse D10 cell transfer model

D10.G4.1 cells (1 x 10⁷ cells/mouse) containing 2 mg of conalbumin in saline is administered i.v. to AKR mice. After 6 hr blood is collected and serum is obtained by centrifugation at 3000 r.p.m. for lOmin. IL-4 and IL-5 level in serum are determined by ELISA kits. Compound is administered intraperitonally at —4 and +1 hr after these cells injection.

6. Passive cutaneous anaphylaxis (PCA) test in rats

6 Weeks old male Wistar rats are sensitized intradermally (i.d.) on their shaved backs with 50 μl of 0.1 μg/ml mouse anti-DNP IgE monoclonal antibody (SPE-7) under a light anesthesia. After 24 hours, the rats are challenged intravenously with 1 ml of saline containing 0.6 mg DNP-BSA (30) (LSL CO., LTD) and 0.005 g of Evans blue.

Compounds are injected intraperitonally (i.p.) 0.5 hr prior to antigen injection. Rats without the sensitization, challenge, and compound treatment are used for a blank (control) and rats with sensitization, challenge and vehicle treatment are used to determine a value without inhibition. Thirty min after the challenge, the rats are killed, and the skin of the back is removed. Evans blue dye in the skin is extracted in formamide overnight at 63 °C. Then an absorbance at 620 nm is measured to obtain the optical density of the leaked dye. Percent inhibition of PC A with a compound is calculated as follows:

%> inhibition = {(mean vehicle value — sample value)/(mean vehicle value - mean control value)} x 100

7. Anaphylactic bronchoconstriction in rats

6 Weeks old male Wistar rats are sensitized intravenously (i.v.) with 10 μg mouse anti-DNP IgE, SPE-7, and 1 days later, the rats are challenged intravenously with 0.3 ml of saline containing 1.5 mg DNP-BSA (30) under anesthesia with urethan (1000 mg/kg, i.p.) and gallamine (50 mg/kg, i.v.). The trachea is cannulated for artificial respiration (2 ml / stroke, 70 strokes / min). Pulmonary inflation pressure (PIP) is recorded through a side-arm of cannula connected to pressure transducer. Change in PIP reflects change of both resistance and compliance of the lungs. To evaluate the drugs, each drug is given i.v. 5 min before challenge.

Claims

1. An isolated polynucleotide being selected from the group consisting of:

a. a polynucleotide encoding a regulator polypeptide of G-protein signaling (RGS) comprising an amino acid sequence selected form the group consisting of:

i. amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 2; the amino acid sequence shown in SEQ ID NO: 2; amino acid sequences which are at least about 50% identical to the amino acid sequence shown in SEQ ID NO: 5; and ii. the amino acid sequence shown in SEQ ID NO: 5.

a polynucleotide comprising the sequence of SEQ ID NO: 1 or 4;

c a polynucleotide which hybridizes under stringent conditions to a polynucleotide specified in (a) and (b) and encodes a regulator poly- peptide of G-protein signaling (RGS);

d. a polynucleotide the sequence of which deviates from the polynucleotide sequences specified in (a) to (c) due to the degeneration of the genetic code and encodes a regulator polypeptide of G-protein signaling (RGS); and

e. a polynucleotide which represents a fragment, derivative or allelic variation of a polynucleotide sequence specified in (a) to (d) and encodes a regulator polypeptide of G-protein signaling (RGS).

An expression vector containing any polynucleotide of claim 1.

3. A host cell containing the expression vector of claim 2.

4. A substantially purified regulator polypeptide of G-protein signaling (RGS) encoded by a polynucleotide of claim 1.

5. A method for producing a regulator polypeptide of G-protein signaling (RGS), wherein the method comprises the following steps:

a. culturing the host cell of claim 3 under conditions suitable for the expression of the regulator polypeptide of G-protein signaling (RGS); and

b. recovering the regulator polypeptide of G-protein signaling (RGS) from the host cell culture.

6. A method for detection of a polynucleotide encoding a regulator polypeptide of G-protein signaling (RGS) in a biological sample comprising the following steps:

a. hybridizing any polynucleotide of claim 1 to a nucleic acid material of a biological sample, thereby forming a hybridization complex; and

b. detecting said hybridization complex.

7. The method of claim 6, wherein before hybridization, the nucleic acid material of the biological sample is amplified.

8. A method for the detection of a polynucleotide of claim 1 or a regulator polypeptide of G-protein signaling (RGS) of claim 4 comprising the steps of: a. contacting a biological sample with a reagent which specifically interacts with the polynucleotide or the regulator polypeptide of G- protein signaling (RGS) and

b. detecting the interaction.

9. A diagnostic kit for conducting the method of any one of claims 6 to 8.

10. A method of screening for agents which decrease the activity of a regulator polypeptide of G-protein signaling (RGS), comprising the steps of:

a. contacting a test compound with any regulator polypeptide of G- protein signaling (RGS) encoded by any polynucleotide of claiml;

b. detecting binding of the test compound to the regulator polypeptide of

G-protein signaling (RGS), wherein a test compound which binds to the polypeptide is identified as a potential therapeutic agent for decreasing the activity of a regulator polypeptide of G-protein signaling (RGS).

11. A method of screening for agents which regulate the activity of a regulator polypeptide of G-protein signaling (RGS), comprising the steps of:

a. contacting a test compound with a regulator polypeptide of G-protein signaling (RGS) encoded by any polynucleotide of claim 1 ; and

b. detecting a regulator polypeptide of G-protein signaling (RGS) activity of the, wherein a test compound which increases the regulator polypeptide of G-protein signaling (RGS) activity is identified as a potential therapeutic agent for increasing the activity of the regulator polypeptide of G-protein signaling (RGS), and wherein a test com- pound which decreases the regulator polypeptide of G-protein signaling (RGS) activity of the polypeptide is identified as a potential therapeutic agent for decreasing the activity of the regulator polypeptide of G-protein signaling (RGS).

12. A method of screening for agents which decrease the activity of a regulator polypeptide of G-protein signaling (RGS), comprising the steps of:

a. contacting a test compound with any polynucleotide of claim 1 ; and

b. detecting binding of the test compound to the polynucleotide, wherein a test compound which binds to the polynucleotide is identified as a potential therapeutic agent for decreasing the activity of regulator polypeptide of G-protein signaling (RGS).

13. A method of reducing the activity of regulator polypeptide of G-protein signaling (RGS), comprising the step of contacting a cell with a reagent which specifically binds to any polynucleotide of claim 1 or any regulator polypeptide of G-protein signaling (RGS) of claim 4, whereby the activity of regulator polypeptide of G-protein signaling (RGS) is reduced.

14. A reagent that modulates the activity of a regulator polypeptide of G-protein signaling (RGS) or a polynucleotide wherein said reagent is identified by the method of any of the claim 10 to 12.

15. A pharmaceutical composition, comprising the expression vector of claim 2 or the reagent of claim 14 and a pharmaceutically acceptable carrier.

16. Use of the expression vector of claim 2 or the reagent of claim 14 in the preparation of a medicament for modulating the activity of a regulator polypeptide of G-protein signaling (RGS) in a disease.

7. Use of claim 16 wherein the disease is asthma or diabetes.

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Fig. 1

atgaatcctgttgcattatggagtatggcctttagaaaggaaagagaagttgc caggtcaccaactgcggaaacaatgacatggtctgaaaatatggacacgcttt tagccaaccaaggaaaggagcagctgcttggagcacatgcaaagggggcctcc ctggcaggtggaggagaaaaccaccaacgttctggaaggcaaatttctctgag tggaaaaagtgctggcatgttttactcctcagagatatacattctcaaaaata gagacagccagcgaaaggctggtctagatgcttttcgaatatttctaaaatca gagtttagtgaagaaaatgttgagttctggcttgcctgtgaagactttaagaa aacgaaaaatgcagacaaaattgcttccaaagccaagatgatt attctgaat tcattgaagctgatgcacctaaagagattaacattgacttcggtaccagagac ctcatctcaaagaatattgctgaaccaacactcaaatgctttgatgaggctca gaaattaatctattgtctcatggccaaggattctttccctcgatttctgaagt cagagatttataaaaaactgaaagctgcccaaaagattggtgtctgtcttgcc caactcaatgggaaactcattt ctttagaagcccagggagcgctgacaacaa aagagagcctagtggctcatgggagaactacagaacttgtaatactgtggaca gaagctga

Fig. 2

MNPVALWSMAFRKEREVARSPTAETMTWSENMDTLLANQGKEQLLGAHAKGAS LAGGGENHQRSGRQISLSGKSΆGM YSSEIYILKNRDSQRKΆGLDAFRIFLKS EFSEENVEFWLACEDFKKTKNADKIASKAK IYSEFIEADAPKEINIDFGTRD LISKNIAEPTLKCFDEAQKLIYCLMAKDSFPRFLKSEIYKKLKAΆQKIGVCLA QLNGKLIYFRSPGSADNKREPSGS ENYRTCNTVDRS

Fig. 3

MQSAMFLAVQHDCRPMDKSAGSGHKSEEKREK KRTLLKDWKTRLSYFLQNSS TPGKPKTGKKSKQQAFIKPSPEEAQLWSEAFDELLASKYGLAAFRAFLKSEFC EENIEFWLACEDFKKTKSPQKLSSKARKIYTDFIEKEAPKEINIDFQTKTLIA QNIQEATSGCFTTAQKRVYSLMENNSYPRFLESEFYQDLCKKPQITTEPHAT

Fig. 4

TTCTACAGGTCACCAACTGCGGAAACAATGACATGGTCTGAAAATATGGACAC GCTTTTAGCCAACCAAGCTGGTCTAGATGCTTTTCGAATATTTCTAAAATCAG AGTTTAGTGAAGAAAATGTTGAGTTCTGGCTTGCCTGTGAAGACTTTAAGAAA ACGAAAAATGCAGACAAAATTGCTTCCAAAGCCAAGATGATTTATTCTGAATT CATTGAAGCTGATGCACCTAAAGAGATTAACATTGACTTCGGTACCAGAGACC TCATCTCAAAG7AATATTGCTGAACCAACACTCAAATGCTTTGATGAGGCTCAG AAATTAATCTATTGTCTCATGGCCAAGGATTCTTTCCCTCGATTTCTGAAGTC AGAGATTTATAAAAAACTGGTAAATAGCCAACAGGTT -2/7-

Fig. 5

FYRSPTAETMTWSENMDTLLANQAGLDAFRIFLKSEFSEENVEFWLACEDFKK TKNADKIASKAKMIYSEFIEADAPKEINIDFGTRDLISKNIAEPTLKCFDEAQ KLIYCLMAKDSFPRFLKSEIYKKLVNSQQV

FIG. 6

BLASTP - alignment of SEQ ID NO: 2 against swissnew| P41220 |RGS2 HUMAN

REGULATOR OF G-PROTEIN SIGNALING 2 (RGS2) (G0/G1 SWITCH REGULATORY PROTEIN 8) .

//:swiss|P41220|RGS2_HUMAN REGULATOR OF G-PROTEIN SIGNALING 2

This hit is scoring at : 2e-40 (expectation value)

Alignment length (overlap) : 136

Identities : 58 %

Scoring matrix : BLOSUM62 (used to infer consensus pattern)

Database searched : nrdb_l_;

Q: 1 FYRSPTAETMTWSENMDTLLANQAGLDAFRIFL KSEFSEENVEFWLACEDFKKTKNADKI

F.: E...WSE .D.LLA:: GL AFR.FL KSEF.EEN:EFWLACEDFKKTK: ..K:

H: 69 FIKPSPEEAQLWSEAFDELLASKYGLAAFRAFL KSEFCEENIEFWLACEDFKKTKSPQKL

RGS PROTEIN fingerprint shown in bold

ASKAKMIYSEFIEADAPKEINIDFGTRDLISKNIAEPTLKCFDEAQKLIYCLMAKDSFPR

:SKA: .IY: :FIE. :APKEINIDF T: .LI: :NI.E.T CF..AQK :Y.LM..:S:PR SSKARKIYTDFIEKEAPKEINIDFQTKTLIAQNIQEATSGCFTTAQKRVYSLMENNϋYPR

FLKSEIYKKLVNSQQV 136

F :SE.Y:.L....Q: FLESEFYQDLCKKPQI 204

FIG. 7 HMMPFAM - alignment of SEQ ID NO: 2 against pfam|hmm|RGS

Regulator of G protein signaling domain.

This hit is scoring at : 178.4

Scoring matrix : BLOSUM62 (used to infer consensus pattern)

Q: 15 NMDTLL-ANQAGLDAFRIFLKSEFSE—ENVEFWLACEDFKKTKNA-DKIASKAKM

:.:.LL G .FR FL::EFSE ENrEFWLA.E: : :KT: : . DK : .KA:

H: 1 sfe lld qpiGrllFreFLetefsepkenleFwlaveeye tespadkrsdkare esL IYSEFIEADAPK-EINIDFGTRDLISKNIAE PTLKCFDEAQKL

IY.EFI.. :APK E:N:D .R: ...N:.E PT ..F:EAQ: aaAYGIydeFispeapkfevnldselrestqdnlleavededllksLqptkdlFeeaqre

IYCLMAKDSFPRFLKSEIYKKLV 131 IY. LM. DSFPRFL: S : .Y : . : iytlMrgDSfprFLeSdyylrfL 143

FIG. 8

BLASTP - alignment of SEQ ID NO: 2 against pdb | 1EZT | 1EZT-A regulator of g-protein signaling 4fragment: core rgs domain;

(rgs4) // :pdb I 1EZY I 1EZY-A regulator of g-protein signaling 4fragment: core domain of rgs; (rgs4)

This hit is scoring at : 3e-35 (expectation value) Alignment length (overlap) : 125 Identities : 52 %

Scoring matrix : BLOSUM62 (used to infer consensus pattern) Database searched : nrdb_l_;

Q : 8 ETMTWSENMDTLLANQAGLDAFRIFLKSEFSEENVEFWLACEDFKKTKNADKIASKAKMI

E...W:E: : : .L: : : . GL AF: .FLKSE:SEEN: :FW: : CE : :KK.K: ..K: : .KAK.I

H: 5 EVKKWAESLENLINHECGLAAFKAFLKSEYSEENIDFWISCEEYKKIKSPSKLSPKAKKI

YSEFIEADAPKEINIDFGTRDLISKNIAEPTLKCFDEAQKLIYCLMAKDSFPRFLKSEIY Y:EFI...A.KE:N:D TR: . S :N: .EPT : . CFDEAQK I : LM.KDS: RFLKS..Y YNEFISVQATKEVNLDSCTREETSRNMLEPTITCFDEAQKKIFNLMEKDSYRRFLKSRFY

KKLVN 132

.L.N LDLTN 129

Genewise prediction of exon-intron structure using genomic sequence AL390957.10 with human RGS2

Score 160.21 bits over entire alignment

RGS2 HUMAN 69 FIKPSPEEAQLWSEAFDELLASK F + E WSE +D LLA++

FYRSPTAETMTWSENMDTLLANQ Y:A[gct]

AL390957.10 -160657 ttatcaggaaattgaagactgacGGTAAGAT Intron 1 TAGCT tagccccactcgcaatacttcaa 1 [160587 : 15593-1> ccgaatgaagagtatgcgtacca

RGS2 HUMAN 93 GLAAFRAFLKSEFCEENIEF LACEDFKKTKSPQKLSSKARKIYTDFIE GL AFR FLKSEF EEN+EFWLACEDFKKTK+ K++SKA+ IY++FIE GLDAFRIFLKSEFSEENVEFWLACEDFKKTKNADKIASKAKMIYSEFIE

AL390957.10 -155928 gcggtcatcatgtaggaggttcgtggtaaaaaggaagtagaaattgtag gtactgtttacatgaaatatgtcgaataacaacaatccacattacatta tatttaataaagttaattgcgtctactgagatacattcacggtttacta

RGS2 HUMAN 142 KEAPKE INIDFQTKTLIAQNIQEATS

+APKE INIDF T+ LI++NI E T

ADAPKE INIDFGTRDLISKNIAEPTL

AL390957.10 -155781 gggcagGTGAGTG Intron 2 CAGaaagtgaagcataaaggcac caccaaO [155763 : 14205-0>tatatgcgattcaatcacct ttatag tctcctcacccagtttaaac

RGS2 HUMAN 168 GCFTTAQKRVYSLMENNSYPRFLESEFYQDLCKKPQI

CF AQK +Y LM +S+PRFL+SE+Y+ L Q+

KCFDEAQKLIYCLMAKDSFPRFLKSEIYKKLVNSQQV

AL390957.10 -141996 attgggcatattcagagttcctcatgataacgaaccg agtaacaattagttcaactcgttacataaattagaat acttgtgaacttcgcgttctatggagttaagatcagt

FIG. 9 (cont'd)

/ /

>AL390957.10. [160657:141884] .sp.tr

FYRSPTAETMTWSENMDTLLANQAGLDAFRIFLKSEFSEENVEFWLACEDFKKTKNADKI ASKAKMIYSEFIEADAPKEINIDFGTRDLISKNIAEPTLKCFDEAQKLIYCLMAKDSFPR FLKSEI KKLVNSQQV

//

>AL390957.10. [160657:141884] . sp

TTCTACAGGTCACCAACTGCGGAAACAATGACATGGTCTGAAAATATGGACACGCTTTTA

GCCAACCAAGCTGGTCTAGATGCTTTTCGAATATTTCTAAAATCAGAGTTTAGTGAAGAA

AATGTTGAGTTCTGGCTTGCCTGTGAAGACTTTAAGAAAACGAAAAATGCAGACAAAATT

GCTTCCAAAGCCAAGATGATTTATTCTGAATTCATTGAAGCTGATGCACCTAAAGAGATT

AACATTGACTTCGGTACCAGAGACCTCATCTCAAAGAATATTGCTGAACCAACACTCAAA

TGCTTTGATGAGGCTCAGAAATTAATCTATTGTCTCATGGCCAAGGATTCTTTCCCTCGA

TTTCTGAAGTCAGAGATTTATAAAAAACTGGTAAATAGCCAACAGGTT

/ /

AL390957.10 match 141884 160657 160.21 - . RGS2 HUMAN

AL390957.10 cds 160656 160586 0.00 - 0

AL390957.10 intron 160587 155931 0.00 - . RGS2 HUMAN

AL390957.10 cds 155929 155762 0.00 - 2 ^_

AL390957.10 intron 155763 142057 0.00 - . RGS2 HUMAN

AL390957.10 cds 142055 141884 0.00 - 0

SEQUENCE LISTING

<110> Bayer AG

<120> REGULATION OF HUMAN REGULATOR OF G-PROTEIN SIGNALING

<130> LI0379 Foreign Countries

<150> US 60/312,345 <151> 2001-08-16

<160> 6

<170> Patentln version 3.1

<210> 1

<211> 750

<212> DNA

<213> Homo sapiens

<400> 1 atgaatcctg ttgcattatg gagtatggcc tttagaaagg aaagagaagt tgccaggtca 60 ccaaσtgσgg aaacaatgac atggtctgaa aatatggaca cgcttttagσ caaccaagga 120 aaggagcagc tgcttggagc acatgcaaag ggggσctccσ tggcaggtgg aggagaaaac 180 cacσaacgtt ctggaaggca aatttctctg agtggaaaaa gtgctggcat gttttactαc 240 tcagagatat acattctcaa aaatagagac agccagcgaa aggctggtct agatgctttt 300 cgaatatttc taaaatcaga gtttagtgaa gaaaatgttg agttctggσt tgcctgtgaa 360 gactttaaga aaacgaaaaa tgcagacaaa attgcttcca aagccaagat gatttattct 420 gaattσattg aagctgatgc acc aaagag attaacattg acttcggtac cagagacctσ 480 atctσaaaga atattgctga accaaσaσtc aaatgctttg atgaggctca gaaattaatc 540 tattgtctca tggccaagga ttctttccct cgatttctga agtcagagat ttataaaaaa 600 ctgaaagctg cccaaaagat tggtgtctgt cttgcccaaσ tcaatgggaa actcatttac 660 tttagaagcc σagggagcgc tgacaacaaa agagagccta gtggctcatg ggagaactac 720 agaaσttgta atactgtgga cagaagctga 750

<210> 2

<211> 249

<212> PRT

<213> Homo sapiens

<400> 2

Met Asn Pro Val Ala Leu Trp Ser Met Ala Phe Arg Lys Glu Arg Glu 1 5 10 15

Val Ala Arg Ser Pro Thr Ala Glu Thr Met Thr Trp Ser Glu Asn Met 20 25 30

Asp Thr Leu Leu Ala Asn Gin Gly Lys Glu Gin Leu Leu Gly Ala His 35 40 45

Ala Lys Gly Ala Ser Leu Ala Gly Gly Gly Glu Asn His Gin Arg Ser 50 55 60

Gly Arg Gin lie Ser Leu Ser Gly Lys Ser Ala Gly Met Phe Tyr Ser 65 70 75 80

Ser Glu lie Tyr lie Leu Lys Asn Arg Asp Ser Gin Arg Lys Ala Gly 85 90 95

Leu Asp Ala Phe Arg lie Phe Leu Lys Ser Glu Phe Ser Glu Glu Asn 100 105 110

Val Glu Phe Trp Leu Ala Cys Glu Asp Phe Lys Lys Thr Lys Asn Ala 115 120 125

Asp Lys lie Ala Ser Lys Ala Lys Met lie Tyr Ser Glu Phe lie Glu 130 135 140

Ala Asp Ala Pro Lys Glu lie Asn lie Asp Phe Gly Thr Arg Asp Leu 145 150 155 160 lie Ser Lys Asn lie Ala Glu Pro Thr Leu Lys Cys Phe Asp Glu Ala 165 170 175

Gin Lys Leu lie Tyr Cys Leu Met Ala Lys Asp Ser Phe Pro Arg Phe 180 185 190

Leu Lys Ser Glu lie Tyr Lys Lys Leu Lys Ala Ala Gin Lys lie Gly 195 200 205

Val Cys Leu Ala Gin Leu Asn Gly Lys Leu lie Tyr Phe Arg Ser Pro 210 215 220

Gly Ser Ala Asp Asn Lys Arg Glu Pro Ser Gly Ser Trp Glu Asn Tyr 225 230 235 240

Arg Thr Cys Asn Thr Val Asp Arg Ser 245

<210> 3

<211> 211

<212> PRT

<213> Homo sapiens

<300>

<308> Genbank/P41220

<309> 1995-02-01

<400> 3

Met Gin Ser Ala Met Phe Leu Ala Val Gin His Asp Cys Arg Pro Met 1 5 10 15

Asp Lys Ser Ala Gly Ser Gly His Lys Ser Glu Glu Lys Arg Glu Lys 20 25 30

Met Lys Arg Thr Leu Leu Lys Asp Trp Lys Thr Arg Leu Ser Tyr Phe 35 40 45 Leu Gin Asn Ser Ser Thr Pro Gly Lys Pro Lys Thr Gly Lys Lys Ser 50 55 60

Lys Gin Gin Ala Phe lie Lys Pro Ser Pro Glu Glu Ala Gin Leu Trp 65 70 75 80

Ser Glu Ala Phe Asp Glu Leu Leu Ala Ser Lys Tyr Gly Leu Ala Ala 85 90 95

Phe Arg Ala Phe Leu Lys Ser Glu Phe Cys Glu Glu Asn lie Glu Phe 100 105 110

Trp Leu Ala Cys Glu Asp Phe Lys Lys Thr Lys Ser Pro Gin Lys Leu 115 120 125

Ser Ser Lys Ala Arg Lys lie Tyr Thr Asp Phe lie Glu Lys Glu Ala 130 135 140

Pro Lys Glu lie Asn lie Asp Phe Gin Thr Lys Thr Leu lie Ala Gin 145 150 155 160

Asn lie Gin Glu Ala Thr Ser Gly Cys Phe Thr Thr Ala Gin Lys Arg 165 170 175

Val Tyr Ser Leu Met Glu Asn Asn Ser Tyr Pro Arg Phe Leu Glu Ser 180 185 190

Glu Phe Tyr Gin Asp Leu Cys Lys Lys Pro Gin He Thr Thr Glu Pro 195 200 205

His Ala Thr 210

<210> 4

<211> 408

<212> DNA

<213> Homo sapiens

<400> 4 ttctacaggt cacσaactgc ggaaacaatg aσatggtctg aaaatatgga σaσgctttta 60 gccaaccaag ctggtctaga tgcttttσga atatttσtaa aatσagagtt tagtgaagaa 120 aatgttgagt tctggσttgσ σtgtgaagaσ tttaagaaaa cgaaaaatgα agacaaaatt 180 gcttσcaaag cαaagatgat ttattσtgaa ttcattgaag ctgatgcacc taaagagatt 240 aacattgaσt tcggtaccag agaασtαatσ tσaaagaata ttgσtgaaσα aaαaσtcaaa 300 tgctttgatg aggσtcagaa attaatc at tgtctαatgg σcaaggattc tttccctσga 360 tttσtgaagt cagagattta taaaaaactg gtaaatagcc aacaggtt 408

<210> 5

<211> 135

<212> PRT

<213> Homo sapiens

<400> 5

Tyr Arg Ser Pro Thr Ala Glu Thr Met Thr Trp Ser Glu Asn Met Asp 1 5 10 15

Thr Leu Leu Ala Asn Gin Ala Gly Leu Asp Ala Phe Arg He Phe Leu 20 25 30

Lys Ser Glu Phe Ser Glu Glu Asn Val Glu Phe Trp Leu Ala Cys Glu 35 40 45

Asp Phe Lys Lys Thr Lys Asn Ala Asp Lys He Ala Ser Lys Ala Lys 50 55 60

Met He Tyr Ser Glu Phe He Glu Ala Asp Ala Pro Lys Glu He Asn 65 70 75 80

He Asp Phe Gly Thr Arg Asp Leu He Ser Lys Asn He Ala Glu Pro 85 90 95

Thr Leu Lys Cys Phe Asp Glu Ala Gin Lys Leu He Tyr Cys Leu Met 100 105 HO

Ala Lys Asp Ser Phe Pro Arg Phe Leu Lys Ser Glu He Tyr Lys Lys 115 120 125

Leu Val Asn Ser Gin Gin Val 130 135 <210> 6

<211> 24

<212> DNA

<213> Artifiσial sequenσe

<220>

<223> random primer

<400> 6 tσaaσtgact agatgtaσat ggaσ 24