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J. Biol. Chem., Vol. 275, Issue 31, 23421-23424, August 4, 2000

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ACCELERATED PUBLICATION
Activation of Heterotrimeric G-protein Signaling by a Ras-related Protein

IMPLICATIONS FOR SIGNAL INTEGRATION^*

Mary J. Cismowski^§, Chienling Ma, Catalina Ribas^¶, Xiaobing Xie, Michael Spruyt, Jeffrey S. Lizano, Stephen M. Lanier^¶, and Emir Duzic^**

From  OSI Pharmaceuticals, Tarrytown, New York 10591, the ^¶ Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425, and  Cadus Pharmaceutical Corporation, Tarrytown, New York 10591

Received for publication, May 12, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Utilizing a functional screen in the yeast Saccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion. One of the identified activators, termed AGS1 (for activator of G-protein signaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily. Expression of AGS1 in yeast and in mammalian cells results in specific activation of G_i/G_o heterotrimeric signaling pathways. In addition, the in vivo and in vitro properties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for G_i/G_o. AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

GPCR¹ signaling pathways represent one of the most widely used mechanisms in nature for transducing signals from the extracellular to the intracellular environment. Each step in the activated GPCR signaling cascade presents a potential regulatory checkpoint for fine-tuning and directing the signal. Although a number of regulatory molecules affecting GPCR signaling have been identified (1-8), evidence suggests the presence of additional pathway modulators (8-10). To isolate such modulators, we developed a series of functional screens in the yeast Saccharomyces cerevisiae designed to detect mammalian proteins that either activate or inactivate the pheromone response pathway, a G-protein coupled pathway in which G acts as the positive signal transducer (11, 12). Genetic manipulation of the yeast strains allowed detection of mammalian modulators through simple growth screens, and the functional redundancy between the pheromone response pathway and mammalian GPCR pathways (13-16) allowed us to replace the yeast G with human G_i2, thereby biasing the screens toward the non-yeast component of the pathway. From these screens we identified three mammalian proteins that appeared to activate signaling by distinct mechanisms (11, 12). As expression of these proteins did not alter G-protein expression levels in yeast, we termed these proteins AGS for activators of G-protein signaling. This report describes the functional characterization of AGS1, a Ras-related protein isolated from a screen of human liver cDNA.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Strains and Plasmids-- Plasmid constructions, except as indicated below, have been described previously (11). Plasmid pSV-gal was purchased from Promega; pYES2, pCEP4, pcDNA3.1(+), pcDNA3.1-His-lacZ, and pcDNA3.1-HisC were from Invitrogen; pYEX4T1 was from Amrad Biotech and pFA2-cJun, pFA2-Elk1, pFA2-CREB, pFA-CHOP, pFR-Luc, pFC-MEK1, and pBluescriptSK(+) were from Stratagene. A plasmid carrying human transducin- (GNAZ) cDNA sequences in pBluescriptSK(+) was a gift from M. Simon. AGS1 and AGS1-G31V (11) were amplified from pYES2 plasmids and ligated into pcDNA3.1-HisC and pYEX4T1, placing the AGS1 coding sequences in-frame with, respectively, an N-terminal His₆ tag sequence and an N-terminal GST sequence. In a similar fashion, yeast CDC42 coding sequences were amplified from yeast genomic DNA and ligated into pYEX4T1. The human nociceptin (ORL1) receptor was amplified from brain poly(A)-RNA by reverse-transcriptase polymerase chain reaction and ligated to pCEP4. Transducin- was excised from pBluescriptSK(+) and ligated to pcDNA3.1(+). Automated dideoxy sequencing was used to verify the correct construction of all plasmids.

Mammalian Transfection and PathDetect Assays-- COS-7 cells (ATCC 1651) were maintained in DMEM (Life Technologies, Inc.) supplemented with 10% enhanced calf serum (Gemini Bio-Products), 50 µg/ml penicillin, 50 µg/ml streptomycin, and 100 µg/ml neomycin, pH 7.4. For immunoblot analysis, 1 × 10⁷ cells were transfected with 1 µg of pcDNA3.1-lacZ, pcDNA3.1His-AGS1, or pcDNA3.1His-AGS1-G31V and grown 72 h prior to harvesting. Cells were resuspended in 5 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA, containing a protease inhibitor mixture (Roche Molecular Biochemicals), lysed with a Dounce homogenizer, and centrifuged at 100,000 × g for 30 min at 4 °C. Soluble protein was removed and crude membrane pellets resuspended in 50 mM Tris-HCl, pH 7.4, 0.6 mM EDTA, 5 mM MgCl₂ containing protease inhibitors. PathDetect transfections and luciferase assays were performed in triplicate using 5 × 10⁵ cells/well and the protocols provided by Stratagene. Cells were transfected as indicated with 50 ng of pcDNA3.1-HisC, pcDNA3.1His-AGS1, or pcDNA3.1His-AGS1-G31V, 100 ng of pCEP4 or pCEP4-hNocR, 500 ng of pcDNA3.1 or pcDNA3.1-transducin-. Each transfection included 50 ng of pFA2-Elk1, pFA2-cJun, pFA2-CREB, or pFA2-CHOP, 1 µg of pFR-Luc and 1 µg of pSV-gal. For the ERK1/2 positive control, 50 ng of pFC-MEK1 was used in place of pcDNA3.1His-AGS1. Total DNA in each transfection was normalized to 3 µg with pBluescriptSK(+). Where indicated, pertussis toxin (100 ng/ml) was added 18 h prior to harvesting cells, and nociceptin (final concentration 100 nM) or vehicle was added 4 h prior to harvesting cells. -Galactosidase activity was measured at 37 °C on 100 µl of total protein extracts in 96-well microtiter plates by adding 20 µl of freshly prepared 360 mM Na₂HPO₄, 240 mM NaH₂PO₄, 60 mM KCl, 6 mM MgSO₄, 2.5% Triton X-100, 16 µl/ml -mercaptoethanol, 10 mM chlorophenol red -D-galactopyranoside. Reactions were terminated by addition of 60 µl of 1.5 M Tris-HCl, pH 8.8, and absorbance at 575 nm was read with a Beckman Biomek plate reader. Luciferase activities were normalized to the levels of -galactosidase expression, and data represent the average of three to five independent experiments.

GTP Hydrolysis Assays-- Yeast strains were grown in liquid synthetic medium, and GST, GST-AGS1, and GST-Cdc42 purified as described previously (11). Purified proteins were diluted to 500 nM in 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM MgCl₂, 0.01% Thesit^TM, 1 mM DTT and maintained at 25 °C. [-³²P]GTP was added to 5 µM (8 µCi/ml) and, at the indicated times after addition, 50-µl aliquots were removed and added to 750 µl of an ice-cold 5% activated charcoal solution in 50 mM NaH₂PO₄. Samples were mixed and centrifuged 5 min at 4 °C. Aliquots of each supernatant (400 µl) were removed and free [³²P]phosphate quantitated by scintillation counting.

In Vivo Labeling of AGS1-- Log-phase cultures of yeast strain CY4600 (11) transformed with pYEX4T1, pYEX4T1-AGS1, or pYEX4T1-CDC42 were grown in phosphate-depleted medium (17) for 30 min at 30° C, and 0.2 mM CuSO₄ was added. Thirty min after adding CuSO₄, 0.5 mCi/ml H₃³²PO₄ (HCl-free; ICN) was added, and cultures were labeled for 5 h. GST fusion proteins were purified essentially as described (11), except proteins were eluted from glutathione-Sepharose at 65 °C in 50 mM Tris, pH 8.8, 150 mM NaCl, 1% SDS, 20 mM EDTA, 20 mM glutathione, 2 mM GDP, 2 mM GTP. Protein purification required approximately 1 h. Equivalent amounts of each protein extract, as judged by SDS-polyacrylamide gel analysis, were spotted onto polyethyleneimine cellulose plates (J. T. Baker) along with [-³²P]GTP and [-³²P]GDP standards. After allowing samples to dry, plates were washed extensively with distilled water followed by methanol, then air-dried. Nucleotides were resolved in 1 M KH₂PO₄, pH 3.4, and detected using x-ray film and intensifying screens at 80 °C. The area of sample application (ori) was covered with a lead shield to impede radioactive signal from labeled phosphoproteins and/or phospholipids.

Activation Assays-- Activation assays using purified, recombinant His₆-G_i2 (11), myristoylated G_i1 (a gift from E. Ross), or purified brain heterotrimer (a gift from J. D. Hildebrandt) were performed as described previously (8). Briefly, 1.2 µM G or 31.2 nM brain heterotrimer were incubated for 30 min at 25° alone, with GST or with GST-AGS1 (at 3 µM for G and 1.2 µM for brain heterotrimer) in the presence of 5 µM GDP in assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.01% Thesit^TM). A control sample with 3 µM GST-AGS1 alone was incubated in assay buffer with 5 µM GDP. Samples were diluted 5-fold into assay buffer containing a final concentration of 2 nM [³⁵S]GTPS (1.3 × 10⁶ cpm/pmol) and 50-µl aliquots removed at the indicated times for filtration onto nitrocellulose membranes. Filters were washed twice with 2 ml of ice-cold 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 25 mM MgCl₂, air-dried, and bound counts determined in the presence of scintillation fluid.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

In yeast screens designed to identify receptor-independent activators of heterotrimeric G-protein signaling we obtained multiple isolates of a single human liver cDNA. Sequence analysis of this cDNA, termed AGS1, revealed it to encode a member of the Ras superfamily (Fig. 1). AGS1 possesses all of the consensus guanine nucleotide binding regions of Ras proteins (18) and shares an overall identity of approximately 35% with each of the major Ras subfamilies. AGS1 also contains internal cationic insert regions (amino acids 123-130 and 193-250) not seen in canonical Ras proteins and amino acid variations at amino acids 33, 80, and 82 similar to ones conferring constitutive activity to RhoE (19). A search of the National Center for Biotechnology Information data base revealed AGS1 to be part of a distinct family of eukaryotic Ras-related proteins possessing both these insert regions and variations (Fig. 1). Putative orthologs with 97% identity to AGS1 have been identified in mouse (GenBank^TM accession number AF009246; Ref. 20) and rat (GenBank^TM accession number AF239157). Closely related human (GenBank^TM accession number AL022334) and rat (GenBank^TM accession number AF134409; Ref. 21) homologs, each with approximately 60% identity to AGS1, have also been identified. In addition, the C-terminal region of a putative Drosophila gene product (GenBank^TM accession number AE003560) shares 49% identity with AGS1.

View larger version (68K): [in this window] [in a new window]   Fig. 1.   AGS1 defines a new family of Ras-related proteins. AGS1 (GenBankTM accession number AAD34206) was aligned with putative human and Drosophila family members and with human c-Ki-Ras. Sequence alignments were performed using the CLUSTALW 1.8 multiple sequence alignment program at the Baylor College of Medicine Search Launcher with default program settings. Identities (asterisks) and similarities (dots) between AGS1 family members and Ras are indicated, as are regions of similarity unique to AGS1 family members (dashes). Ras consensus guanine nucleotide binding regions and the C-terminal CAAX (where A indicates aliphatic acid) motif are shaded. AL022334, putative human protein encoded on chromosome 22; AE003560, putative D. melanogaster gene product CG8641 (amino acids 159-434).

AGS1 function in yeast was specific for the G_i subclass of heterotrimers and was attenuated in strains either lacking G or expressing a mutated AGS1 carrying a glycine 31 to valine substitution in PM1 (11), a region important for guanine nucleotide binding and hydrolysis. AGS1 function was also attenuated in yeast strains rendered incapable of heterotrimer activation either by mutation of G_i2 or by co-expression of RGS4 (11). These initial observations suggested that AGS1 functioned in yeast to facilitate GTP exchange on the engineered heterotrimer and that AGS1 function required guanine nucleotide binding and/or hydrolysis.

The activity of AGS1 in yeast presents a totally unexpected paradigm for signal processing in which a monomeric G-protein provides direct input into a heterotrimeric G-protein signaling pathway. To further define its function we determined how AGS1 integrates into GPCR-regulated signaling in mammalian cells. We used a transient expression system in COS-7 cells and the PathDetect luciferase reporter system to evaluate the effect of AGS1 expression on the basal activity of c-Jun N-terminal kinase (JNK), protein kinase A (PKA), p38, and ERK1/2 signaling pathways (Fig. 2). Only the ERK1/2 system was significantly activated by AGS1 expression, with a magnitude of activation of 2.6 ± 0.4-fold (Fig. 2B). This activation was comparable with that of the G_i-coupled human nociceptin receptor following maximal agonist stimulation (4.4 ± 0.1-fold, Fig. 2C). As in the yeast system, mutation of glycine 31 to valine rendered AGS1 inactive, indicating functional AGS1 is required for Elk1 stimulation.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Functional analysis of AGS1 in mammalian cells. A, immunoblot analysis of AGS1 protein expression in transfected COS-7 cells. Supernatant (S) and membrane (M) fractions (200 µg each) from cells expressing pcDNA3.1His-lacZ (lacZ), pcDNA3.1His-AGS1 (AGS1), or pcDNA3.1His-AGS1-G31V (G31V) were analyzed by immunoblotting with antiserum raised against the hexahistidine tag sequence (11). Molecular mass markers (in kilodaltons) are indicated. B, AGS1 specifically activates an ERK1/2 signaling pathway in COS-7 cells. Plasmids pcDNA3.1HisC (solid bars) or pcDNA3.1His-AGS1 (open bars) were transfected into COS-7 cells along with pFR-Luc, pSV-gal, and either pFA2-cJun (cJun), pFA2-CHOP (CHOP), pFA2-CREB (CREB), or pFA2-Elk1 (Elk1) and relative luciferase activities determined. Basal luciferase activities (in relative luciferase units) were 36,600 ± 3,800, 8,980,000 ± 1,010,000, 78,100 ± 6,700 and 82,500 ± 8,600 for cJun, CHOP, CREB, and Elk1, respectively. C, AGS1 functions in a manner analogous to that of a GPCR. Plasmids pcDNA3.1His-AGS1 (AGS1), pcDNA3.1His-AGS1-G31V (G31V), or pCEP4-hNocR (encoding the human nociceptin receptor; hNocR) were transfected into COS-7 cells with plasmids pFA2-Elk1, pFR-Luc, and pSV-gal and luciferase activity relative to vector controls determined (open bars). Cells were pretreated with pertussis toxin (shaded bars), co-transfected with transducin- (solid bars), or stimulated with nociceptin (+ noc) or vehicle () as indicated. Basal luciferase activities (in relative luciferase units) were 85,100 ± 11,200 and 89,900 ± 3,400 for HisC and pCEP4 vectors, respectively, and luciferase activity upon direct activation of Elk1 by transfection with a MEK1-encoding plasmid was 4.6 × 106 ± 0.2 × 106.

This selective activation of the ERK1/2 pathway by AGS1 mirrors the G_i selectivity previously seen in the yeast system (11). Furthermore, as many G_i-coupled receptors (including the nociceptin receptor) utilize free G to transduce signals through mitogen-activated protein kinase cascades (22-25), this suggests that AGS1 functions in mammalian cells by enhancing G release from G_i. Indeed, Elk1 activation by both AGS1 and the activated nociceptin receptor was blocked by cell pretreatment with pertussis toxin, which ADP-ribosylates G_i/G_o and effectively uncouples it from receptor (26), as well as by co-transfection with transducin-, which attenuates signaling by sequestering free G (Ref. 22; Fig. 2C). In contrast, direct activation of the ERK1/2 signaling pathway by transfection with a plasmid encoding MEK1 was unaffected by either pertussis toxin pretreatment or co-transfection with transducin- (data not shown). Thus, AGS1 function in mammalian cells appeared mechanistically indistinguishable from that of an agonist-stimulated receptor.

The in vivo function of AGS1 poses many interesting questions relative to signal processing and integration within the guanine nucleotide-binding protein family. Clearly AGS1 is a member of the Ras superfamily, and the phenotype of the glycine 31 mutant suggests that guanine nucleotide binding is required for its function. The similarities between AGS1 and RhoE, as well as the cationic insert regions of AGS1, both suggest the potential for unusual nucleotide binding and/or hydrolysis properties. To investigate this, we expressed and purified an N-terminal GST fusion of AGS1. Purified GST-AGS1 had a steady state GTP hydrolysis activity (0.004 min¹) comparable with that of purified GST-Cdc42 (Fig. 3A) and purified Ras (27), making it distinct from the GTPase deficient RhoE (19). However, under a variety of standard conditions, AGS1 failed to bind significant levels of GDP, GTP, or GTPS (data not shown; see Fig. 4). We therefore asked whether we could detect nucleotide binding on newly synthesized GST-AGS1 by performing in vivo labeling with [³²P]orthophosphate. Following purification and thin-layer chromatography, it was apparent that AGS1 preferentially bound GTP rather than GDP, and that the steady state binding of nucleotide to AGS1 was very low relative to GST-Cdc42 isolated under the same conditions. These data indicate that GDP resulting from GTP hydrolysis is not stably bound by purified AGS1 and that re-binding of GTP to nucleotide-free AGS1 is rate-limiting, suggesting that AGS1 may associate in vivo with mammalian regulators of nucleotide exchange and/or nucleotide dissociation. Such regulators may mediate stimulus input to AGS1.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Biochemical properties of purified AGS1. A, GTP hydrolysis activity. Purified GST (), GST-AGS1 (), and GST-Cdc42 () were incubated with [-32P]GTP and aliquots removed at the indicated times to determine free [32P]PO4 released. Hydrolysis rates were determined by linear regression analysis. B, in vivo labeling of AGS1 with [32P]PO4. Yeast cultures expressing GST, GST-AGS1, or GST-Cdc42 were grown in the presence of [32P]PO4, and labeled nucleotides bound to purified GST fusion proteins were detected by thin-layer chromatography and autoradiography. The migration of free [-32P]GDP and [-32P]GTP standards is indicated.

View larger version (28K): [in this window] [in a new window]   Fig. 4.   AGS1 facilitates nucleotide exchange on heterotrimeric Gi/o. A, activation of recombinant Gi1 and His6-Gi2 by purified AGS1. G proteins were incubated either alone (), with GST (), or with GST-AGS1 () prior to the addition of [35S]GTPS. A control sample with AGS1 alone at 30 min () is indicated. Bound nucleotide was determined at the indicated times after addition of label. B, activation of brain heterotrimeric G-protein by AGS1. Purified brain heterotrimer, GST, and GST-AGS1 (AGS1) were incubated either alone or in combination prior to the addition of [35S]GTPS. Bound counts were determined after 30 min. C, [35S]GTPS incorporation is specific for G. Left, parallel [35S]GTPS binding assays were performed in triplicate as described under "Experimental Procedures" with either His6-Gi2 alone (No AGS1) or both His6-Gi2 and GST-AGS1 (+AGS1). Total bound [35S]GTPS (T) was determined at 30 min on one set by filter binding (open bars). Remaining samples were bound to either nickel-nitrilotriacetic acid (Ni) or glutathione-Sepharose (Gt) column matrices for 30 min, washed three times with 1 ml of assay buffer, and bound counts determined. Right, His6-Gi2 (580 nM) was incubated with assay buffer alone () or with 20 µM GTPS at 30° for 90 min, dialyzed twice for 30 min each against 1 liter of ice-cold 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM MgCl2, 0.01% ThesitTM, 1 mM DTT, then added to purified GST-AGS1 in assay buffer with 5 µM GDP. [35S]GTPS binding assays were performed as described under "Experimental Procedures."

Finally, we asked if AGS1 altered the nucleotide binding properties of heterotrimeric G-proteins in vitro. We first measured the ability of purified GST-AGS1 to enhance binding of GTPS to purified G_i1 and G_i2. Under conditions optimal for monitoring G_i/G_o activation by a GPCR (8), the addition of AGS1 enhanced GTPS binding to purified G_i1 and G_i2 (Fig. 4A) as well as to purified brain heterotrimeric G-protein (Fig. 4B). AGS1 did not effectively bind GTPS under these incubation conditions. After a 45-min incubation, the 3-fold increase in GTPS binding to both free G_i1 and G_i2 in the presence of AGS1 represented 20-30% of the input G. This increase in GTPS binding was not seen when G_i2 was incubated with another purified Ras-related protein, GST-RhoA (data not shown). These observations are consistent with AGS1 functioning in vitro as an exchange factor for G_i/G_o and activating both free and heterotrimeric G.

To confirm that the increase in GTPS binding in these assays reflected nucleotide binding to G proteins and not to AGS1, we used both glutathione and nickel-nitrilotriacetic acid affinity resins to re-isolate GST-AGS1 and His₆-G_i2 following co-incubation. Although we previously used this approach to detect interaction of GST-AGS1 and His₆-G_i2 (11), aggressive washing of the affinity matrices effectively dissociated the two proteins, as determined by immunoblot analysis (data not shown). After washing >75% of the bound [³⁵S]GTPS was associated with the nickel-affinity resin, while <5% was associated with the glutathione affinity resin (Fig. 4C). In addition "preloading" G_i2 with nonradioactive GTPS prior to association with AGS1 significantly inhibited the subsequent increase in [³⁵S]GTPS binding (Fig. 4C). These data, as well as the in vivo data both in yeast and in mammalian cells, support a direct role for AGS1 in enhancing GTPS binding to G_i/G_o.

There are several examples of cross-talk between Ras-related protein and heterotrimeric G-protein signaling pathways (25, 28). However, in every instance identified so far, activated heterotrimeric G-protein subunits either activate small G-proteins or work in concert with activated small G-proteins to transduce signals. AGS1 is the first example of a monomeric G-protein that functions upstream of a heterotrimeric G-protein to activate it. By virtue of its ability to enhance GTP binding to purified G, and by its sensitivity to pertussis toxin treatment in vivo, AGS1 appears to function by a mechanism akin to that of a GPCR. It is possible that the cationic regions in AGS1, like those found in the activation loops of many G_i/G_o-coupled GPCRs (29, 30), function to directly facilitate GDP release on G, and that conformational changes in AGS1 associated with guanine nucleotide binding and/or hydrolysis unmask these regions.

Within the cell AGS1 may work together with activated GPCRs to enhance or prolong signaling or may compete with GPCRs for activation of heterotrimeric G-proteins. Alternatively AGS1 may function in an independent signaling pathway, activating GPCR- or non-GPCR-coupled heterotrimeric G-proteins either intracellularly or at the cell surface. The unusual nucleotide binding properties of purified AGS1, as well as its abundant transcription in a variety of tissues (not shown), suggests the existence of regulators of AGS1 function. The identity of these putative regulators remains to be determined.

	ACKNOWLEDGEMENTS

We thank Drs. Elliott Ross, Henry Bourne, and Benjamin Benton for helpful discussion and comments; Hao Wu for plasmid constructions; Gary Meissner and Ralph Vaccaro for technical support; and Drs. Elliott Ross, John D. Hildebrandt, and Mel Simon for providing materials used in this study.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported in part by National Institutes of Health Grant RO1-NS24821 (to S. M. L.).

^§ To whom correspondence should be addressed. Present address: Neurocrine Biosciences, Inc., 10555 Science Center Dr., San Diego, CA 92121. E-mail: mcismowski@neurocrine.com.

^** Present address: Millenium Pharmaceuticals Inc., 270 Albany St., Cambridge, MA 02139.

Published, JBC Papers in Press, June 5, 2000, DOI 10.1074/jbc.C000322200

	ABBREVIATIONS

The abbreviations used are: GPCR, G-protein-coupled receptor; DMEM, Dulbecco's modified Eagle's medium; GST, glutathione S-transferase; DTT, dithiothreitol; GTPS, guanosine 5'-3-O-(thio)triphosphate; ERK, extracellular regulated kinase; Thesit^TM, polyoxyethylene 9 lauryl ether.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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