MINIREVIEW:
RGS Proteins and Signaling by Heterotrimeric G Proteins*

Henrik G. Dohlman Dagger and Jeremy Thorner §

From the Dagger  Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06520-8066 and § Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202

INTRODUCTION
The Cycle of G Protein Activation and Inactivation
Establishing a Paradigm: Identification of Yeast SST2
RGS Proteins in Other Model Organisms
A Superfamily of RGS Proteins
Evidence for Bifunctional RGS Proteins
RGS Proteins Act as GAPs for Galpha Subunits
RGS Proteins and Ras-GAP: Similarities and Differences
Why So Many RGS Family Members?
FOOTNOTES
Acknowledgments
REFERENCES


INTRODUCTION

A ubiquitously employed mechanism for signal transduction involves ligand binding to a cell surface receptor coupled to a heterotrimeric guanine nucleotide-binding protein (G protein). Receptor activation stimulates nucleotide exchange and dissociation of the G protein, releasing the Galpha subunit in its GTP-bound state from the Gbeta gamma complex. The released subunits can stimulate a variety of target (effector) enzymes (1), thereby eliciting biochemical responses and changes in cellular physiology. Hundreds of G protein-coupled receptors have been identified (2, 3). These receptors share a common architecture containing seven membrane-spanning segments (4, 5). G proteins also comprise a superfamily that includes at least 17 distinct Galpha (6), 5 Gbeta , and 6 Ggamma isoforms (1), allowing many combinatorial possibilities. Three-dimensional structures of several Galpha subunits and two different Galpha beta gamma heterotrimers (7, 8) have been determined, providing insights about how these molecular "switches" operate.

How are the strength and duration of signaling adjusted to achieve an appropriate response? Attention in this regard has been devoted primarily to receptors, where phosphorylation by protein kinases (9) and receptor-binding proteins, like arrestins (10, 11), contribute to signal desensitization. However, additional proteins participate in signal attenuation at other levels, including phosducins (which act on Gbeta gamma ) (12) and recoverins (13, 14). Here we focus on discovery of another superfamily of evolutionarily conserved proteins, dubbed RGS proteins, for "regulators of G protein signaling." RGS proteins act as negative regulators of G protein-dependent signaling, at least in part, because they stimulate hydrolysis of the GTP bound to activated Galpha subunits.


The Cycle of G Protein Activation and Inactivation

Activation of a G protein is initiated by agonist binding to a receptor, eliciting conformational change that is transmitted to the G protein, causing the Galpha subunit to release GDP and to bind GTP (Fig. 1). GTP binding alters the conformation of three "switch" regions in Galpha that are its primary contact sites with Gbeta gamma , promoting subunit dissociation (7, 8). Guanine nucleotide exchange can occur spontaneously, but is accelerated by agonist-activated receptor and retarded by Gbeta gamma binding. Thus, the receptor acts as a GDS,1 whereas Gbeta gamma acts as a GDI (15).


Fig. 1. The cycle of G protein activation and inactivation. Top panel, when GDP-bound, Galpha is inactive and associated with Gbeta gamma . Agonist binding to a receptor promotes guanine nucleotide exchange; Galpha releases GDP, binds GTP, and dissociates from Gbeta gamma . Dissociated subunits activate target proteins (effectors). When GTP is hydrolyzed, subunits reassociate. Gbeta gamma antagonizes receptor action by inhibiting guanine nucleotide exchange. RGS proteins bind to Galpha , stimulate GTP hydrolysis, and thereby reverse G protein activation. Bottom panel, the roles of a receptor, Gbeta gamma , and an RGS are completely analogous to the GDSs, GDIs, and GAPs that regulate small monomeric G proteins like Ras.
[View Larger Version of this Image (17K GIF file)]


Inactivation requires hydrolysis of the GTP bound to Galpha , shifting the equilibrium in favor of subunit reassociation, preventing further signaling. Purified Galpha subunits display a measurable intrinsic rate of GTP hydrolysis, but the turnover number in vitro cannot account, in some systems, for the rate at which signaling is terminated in vivo (16, 17). Other regulatory processes (such as those mediated by arrestin or phosducin) could be rate-limiting. Nonetheless, the importance of GTP hydrolysis provoked a search for factors that accelerate the GTPase activity of Galpha subunits, termed GAPs (for "GTPase-activating proteins") (Fig. 1). Effector enzymes can serve this role: phospholipase Cbeta stimulates the GTPase activity of Gqalpha in vitro (18); and gamma  subunit of cGMP phosphodiesterase stimulates GTP hydrolysis by Gtalpha (19). On the other hand, recent findings have converged on the conclusion that RGS proteins are GAPs for Gialpha and Goalpha . Identification of the RGS proteins provides an instructive example of how model organisms, like the yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans, can reveal new mechanisms of signal regulation applicable to more complex organisms, including humans.


Establishing a Paradigm: Identification of Yeast SST2

In yeast, signaling processes can be dissected genetically. Because the cells can grow as haploids, recessive mutations can be isolated and characterized. Haploid cells undergo mating in response to a pheromone signal by transcribing new genes, by changing morphology, and by transiently arresting in the G1 phase of the cell cycle. Genes necessary for pheromone response were identified through isolation of mutations that prevent mating, so-called sterile (ste) mutations (20). If normal haploids fail to mate, they become refractory to pheromone and resume cell division. Thus, yeast cells display adaptation and recovery, as observed in mammalian desensitization. In 1982, to identify gene products involved in this down-regulation, Chan and Otte (21, 22) screened for mutant haploids hypersensitive to pheromone-induced cell cycle arrest. These supersensitive (sst) mutations fell into two classes. One (sst1) was allelic to a gene (BAR1) encoding a secreted protease responsible for inactivation of pheromone (alpha -factor). The sst2 mutations defined a novel gene and the first RGS family member.

In addition to responding to doses of pheromone 2 orders of magnitude lower than normal cells (23), sst2 mutants also fail to emerge from pheromone-imposed cell cycle arrest (22). The SST2 gene was isolated (24) by selecting for genomic DNA clones that allowed sst2 mutants to recover from pheromone-induced growth arrest. At the time, however, knowledge about G protein-dependent signaling was in its infancy, sequence data bases were rudimentary, and relevance of signaling events in a unicellular eukaryote to those in humans was not widely appreciated. By the late 1980s, however, it became clear that signaling pathways in yeast and mammalian cells bear considerable similarity (20). For example, the yeast pheromone receptors have a topology resembling other G protein-coupled receptors (2, 3), and the yeast Galpha subunit shares 45% amino acid identity with mammalian Gialpha (6). This evolutionary conservation even allows some mammalian receptors and G proteins to function in yeast (25, 26).

Because high-copy vectors were used to isolate SST2, other genes that could rescue, when overexpressed, the sst2 mutation were also obtained. One such dosage suppressor was the KSS1 gene (for "kinase suppressor of sst2") (27), the first MAPK cloned from any organism, thus indicating a connection between G protein signaling and MAPKs. Another dosage suppressor was the GPA1 gene (28, 29), which encodes the alpha  subunit of the pheromone receptor-coupled G protein. Cells lacking GPA1 display constitutive activation of the pheromone response pathway (28, 29), suggesting that Gbeta gamma transmits the signal and providing the first in vivo evidence that a Gbeta gamma complex has a positive and direct role in regulating downstream effectors.

The fact that overproduction of GPA1 (Galpha ) overcame the need for functional SST2 implied, in a formal genetic sense, that Galpha operates in the same pathway, but downstream of, SST2. Galpha overexpression helps attenuate signaling through sequestration of Gbeta gamma . Other genetic observations suggested a direct interaction between SST2 and GPA1. First, certain mutations in the Galpha gene could bypass the requirement for a fully active SST2 gene (30, 31). Second, the recovery-promoting effect of Galpha overexpression was more efficient if residual SST2 function was present (32, 33). Third, dominant gain-of-function mutations in SST2 confer resistance to receptor-mediated activation of the pathway but not to activation by mutations that permit constitutive release of Gbeta gamma (33). Fourth, stimulating the pheromone response pathway induces SST2 production; yet, resumption of growth does not occur in cells that lack Galpha , indicating that SST2 cannot stimulate adaptation when Galpha is absent (34). Indeed, it was proposed in 1992 that Sst2 serves as a GAP for Galpha (20). An alternative model, that Sst2 promotes Galpha degradation (35), is inconsistent with the genetic findings and with a subsequent study showing that GPA1 half-life is the same in sst2Delta and SST2 cells (33).

Our recent biochemical and cytological studies (34) demonstrate that SST2 and Galpha colocalize in the cell. Both proteins are associated with the plasma membrane and, to a lesser extent, with the Golgi body (34). Membrane localization of SST2 is due, at least in part, to direct binding to Galpha because SST2 can be isolated from cell lysates in a complex with a GPA1-glutathione S-transferase fusion, even after detergent and salt extraction of membranes (34), but binding seems unaffected by the nature of the nucleotide added (GDP or GTPgamma S). Membrane association of Sst2 is striking because its deduced sequence is hydrophilic, lacks any significant stretches of hydrophobic residues, and is devoid of myristoylation or isoprenylation sites. Overexpression of a GTPase-deficient allele of Galpha (gpa1R297H) in wild-type cells enhanced their pheromone sensitivity, resembling the loss of SST2 (34). This Galpha mutant presumably remains in the GTP-bound state, cannot bind Gbeta gamma , but still forms a complex with SST2. By sequestering SST2, less is available to act on the normal Galpha , thereby explaining the observed enhancement in pheromone sensitivity.


RGS Proteins in Other Model Organisms

The closest counterpart to SST2 in another organism is the flbA gene product of the filamentous fungus, A. nidulans (36). Under conditions that should cause sporulation, flbA mutants proliferate, remain undifferentiated, and eventually lyse, a phenotype called "fluffy autolysis." Conversely, overexpression of normal flbA permits sporulation under conditions that would otherwise prevent it. A dominant fluffy autolysis mutation is a substitution (G42R) in a Galpha gene, fadA (37). Alteration of the analogous Gly in mammalian Gsalpha slows its rate of GTP hydrolysis and impairs its ability to release Gbeta gamma (38). On the other hand, certain other fadA (Galpha ) mutations, including a substitution predicted to block Gbeta gamma dissociation, actually suppress a flbA mutation (37). One interpretation of these findings is that free Gbeta gamma somehow blocks sporulation and that flbA is required for sporulation because it promotes the ability of Galpha to remain associated with Gbeta gamma .

Another SST2-related gene was identified in the nematode, C. elegans, during a genetic screen for mutants that alter certain neuronal activities (39). When placed on a lawn of bacterial prey, C. elegans adjusts several of its behaviors, including frequency of egg laying. Egg laying is controlled by serotonergic motor neurons that innervate the vulval and uterine muscle cells and is suited to genetic analysis because the number of laid and unlaid eggs (which are clearly visible inside the adult) can be readily compared. A mutation (egl-10) that decreased the frequency of egg laying was identified and the corresponding gene cloned (39). The C-terminal portion of the 555-residue EGL-10 product bore similarity to the C-terminal segment of the 698-residue SST2 protein (Fig. 2). While egl-10 mutants rarely lay eggs, overexpression of normal EGL-10 causes animals to lay eggs more frequently (39). These phenotypes suggest that EGL-10 is required for serotonin-stimulated egg laying. In other animals, serotonin acts through G protein-coupled receptors (2, 3). Indeed, the goa-1 mutation, which resides in a gene homologous to mammalian Goalpha (40, 41), results in elevated egg laying, and conversely, overexpression of normal GOA-1 causes reduced egg laying (40, 41). The fact that egl-10 and goa-1 mutations have related (but opposite) phenotypes and the fact that an egl-10 mutation has no effect if a goa-1 mutation is present suggest that the normal role of EGL-10 is to down-modulate the activity of GOA-1.


Fig. 2. Sequence alignment of known and presumed RGS proteins. Conserved regions (originally termed GAIP or GOS8 homology ("GH") domains) of the indicated 14 RGS proteins were aligned essentially as described by Siderovski et al. (48). For accession numbers, see references cited in the text. Identities and conventional conservative substitutions (D/E; K/R; E/Q; D/N; N/Q; I/L/M/V; S/T; A/C/S; P/G; and F/Y/W) shared between five or more members are given as white-on-black letters. Dashes indicate single-residue gaps; larger gaps or C-terminal extensions are given as numbers in parentheses. Numbers to the left and right of each line indicate the position of the residue at the beginning and end of each sequence segment shown; an apostrophe indicates tentative numbering derived from cDNAs known to be incomplete. Sc, S. cerevisiae; An, A. nidulans; Ce, C. elegans; and Hs, Homo sapiens.
[View Larger Version of this Image (115K GIF file)]



A Superfamily of RGS Proteins

Multiple homologs of SST2 and EGL-10 are present in higher eukaryotes. Using the yeast two-hybrid system, a human protein, GAIP, that interacts with human Gi3alpha was identified (42). Gi3alpha (prepared by in vitro translation) binds to a GAIP-glutathione S-transferase fusion protein. GAIP is expressed in many tissues and is similar over most of its 217-residue length to the C-terminal portions of SST2, FlbA, and EGL-10. GAIP is more related to products of two other short mammalian cDNAs, GOS8 and BL34/1R20 (42). In these proteins, similarity to EGL-10 extends over ~130 contiguous amino acids, whereas in SST2 and FlbA, similarity is divided into three discontinuous blocks (Fig. 2). This ~130-residue core domain defines the RGS superfamily and, in GAIP, is both necessary and sufficient for interaction with Gi3alpha (42). GAIP also interacts more weakly with Gi2alpha , but not with Gqalpha .

BL34/1R20 cDNA was identified because the corresponding mRNA is elevated in chronic lymphocytic leukemia (43). Expression is specific to B lymphocytes and induced by mitogenic stimuli (44). The 196-residue BL34/1R20 product has been renamed RGS1, in light of its presumed function. Similarly, GOS8 cDNA, encoding a 211-residue protein (RGS2), was isolated because expression of its corresponding mRNA in human blood lymphocytes is induced by concanavalin A (a T-cell mitogen) in combination with cycloheximide (45). However, GOS8 mRNA is induced by cycloheximide alone (46). Yet another homolog, RGS3, was identified by screening a B-cell cDNA library with an oligonucleotide corresponding to sequences conserved between RGS1 and RGS2 (47). RGS3 (519 residues) is much longer than RGS1 or RGS2.

Because of the evidence that yeast SST2 and nematode EGL-10 regulate G protein signaling, it was presumed that mammalian homologs would act analogously. Indeed, RGS4 was identified by screening for rat brain cDNAs that, when expressed in a yeast sst2Delta mutant, could stimulate recovery from pheromone-induced growth arrest and partially block pheromone-induced gene transcription (47). In an independent study, RGS2 was also able to confer pheromone resistance to yeast sst2 cells (48). The 205-residue RGS4 product is expressed exclusively in the brain (47).2

RGS1, RGS2, RGS3, and RGS4 regulate G protein signaling in mammalian cells. Elevation of RGS1 by transient transfection attenuated MAPK activation in response to PAF and diminished the Ca2+ flux provoked by either PAF or lysophosphatidic acid; similarly, expression of each of the four RGS proteins attenuated MAPK activation by interleukin 8 (47). Likewise, RGS4 expression attenuates MAPK activation in response to agonist stimulation of M2 muscarinic acetylcholine receptors.2 In contrast, RGS3 had no effect on MAPK stimulation by two post-G protein activators, phorbol ester and activated Raf-1 kinase (47).

Searches of expressed sequence tag (EST) data bases and screening by polymerase chain reaction amplification (39, 47, 48) have revealed more RGS homologs in mammals and in the C. elegans genome, including one with two tandem RGS domains (Fig. 2).


Evidence for Bifunctional RGS Proteins

Some RGS proteins (including SST2, FlbA, and RGS3) possess long N-terminal extensions. This number may increase as RGS clones identified only as EST fragments are fully characterized. In this regard, it is striking that the identity between RGS7 and EGL-10 (39) is 75% across their N-termini versus 46% within their RGS cores, suggesting that the N-terminal segment is needed for function. Yeast SST2 provides a precedent for such a situation.

Whereas the C-terminal 230 residues of SST2 constitute its RGS domain, the N-terminal 300 residues bear weak similarity to the catalytic domain of mammalian Ras-GAP (34). Both the GAP-like and RGS segments are required, since truncations at either the N- or C-terminal ends completely eliminate biological activity (34). A dominant gain-of-function allele, SST2(P20L), maps within the GAP-like region, supporting its functional importance (33). The GAP-like segment could stimulate the GTPase activity of Galpha (in the manner that GAP acts on Ras) after SST2 associates (via its RGS domain) with Galpha . However, as discussed above, RGS proteins lacking a GAP-like domain can partially substitute for loss of SST2 in yeast and, as discussed below, are presumably able to stimulate the GTPase activity of Galpha subunits in situ. Hence, the GAP-like domain of SST2 may contribute to adaptation through another mechanism.

Another potentially multifunctional RGS is the mouse fused gene product (49), which contains an N-terminal extension homologous to proteins that bind to phosphoprotein phosphatase, PP2A.3 Establishing the functions of other domains is as important as sorting out specificity determinants in the core RGS domain.


RGS Proteins Act as GAPs for Galpha Subunits

Effects of purified GAIP and RGS4 on purified Gi1alpha , Gi2alpha , Gi3alpha , Goalpha , and Gsalpha have been examined in vitro (50). Neither affected the steady-state rate of GTP hydrolysis by these Galpha subtypes. Under such conditions, however, GTP hydrolysis is limited by GDP dissociation. Hence, the steady-state assay actually measures the rate of guanine nucleotide exchange and suggests that RGS proteins do not function as either GDIs or GDSs. When a single round of GTP hydrolysis was measured, either RGS4 or GAIP stimulated the rate of hydrolysis more than 40-fold for all of the Galpha subtypes, except Gsalpha . Thus, RGS proteins act as GAPs (at least for the Gialpha subfamily). RGS4 partially restored GTPase activity to Gi1alpha (R178C) and to Gi1alpha (S47N) but not to Gi1alpha (Q204L) (50). Arg-178 stabilizes the developing negative charge on the gamma -phosphate leaving group in the transition state during hydrolysis; Ser-47 contributes to Mg2+ binding, which is required for nucleotide hydrolysis and subunit dissociation; and Gln-204 is essential for orienting the attacking water molecule and for transition-state stabilization (51, 52). Restoration of function by RGS4 suggests that RGS proteins may accelerate GTP hydrolysis by stabilizing Galpha proteins in their active conformation. Indeed, RGS1 (53) (also RGS44) interact weakly with Goalpha in the presence of GDP or GTPgamma S but strongly in the presence of both GDP and AlF4-, a combination that mimics the transition-state of the nucleotide during hydrolysis.

Selective binding to the transition-state conformation of Galpha may not be a general feature of RGS proteins, however. A human 173-residue RGS10, isolated via its interaction with a GTPase-deficient Galpha mutant, Gi3alpha (Q204L) (54), can co-immunoprecipitate with either Gi3alpha (Q204L) or Gzalpha (Q204L), both presumably GTP-bound, but not with wild-type (presumably GDP-bound) Gi3alpha or Gzalpha or with either wild-type or mutationally activated Gsalpha . Like RGS4 (50), GAIP (50), and RGS1 (53), RGS10 (54) stimulates the GTPase activity of several members of the Gialpha subfamily but is ineffective against Gsalpha .


RGS Proteins and Ras-GAP: Similarities and Differences

Crystal structures of Ras (55, 56) and Galpha (7, 8, 57, 58) in their GTP- and GDP-bound forms have been solved. Ras and Galpha likely hydrolyze GTP by similar catalytic mechanisms. Nonetheless, by itself, Ras hydrolyzes GTP at a rate about 100-fold slower than Gsalpha (15, 59). In the presence of Ras-GAP, however, Ras hydrolyzes GTP at least 100-fold faster than Gsalpha (60, 61). One model to explain this difference (62) is that Ras-GAP resembles the so-called "helical domain" that is present in Gsalpha , but absent in Ras, and that Ras-GAP and the helical domain both introduce into the catalytic cleft an Arg residue that helps to stabilize the transition state. Indeed, Ras-GAP permits Ras to bind GDP and AlF4- (63) supporting a common mechanism for catalysis.

If Galpha subunits have a "tethered GAP", how does an RGS stimulate GTPase activity? Ras-GAP interaction with Ras may provide a clue. Ras-GAP contacts the GTP-binding pocket and the "effector domain" of Ras, a loop that undergoes significant conformational change upon GTP hydrolysis. In Galpha , the helical domain interacts with the GTP-binding pocket, but not with the "switch" regions that undergo conformational change upon GTP hydrolysis. Hence, an RGS protein could accelerate GTP hydrolysis by binding to one or more of the switch elements, so that the conformation of the RGS-Galpha -GTP complex approximates that of Ras-GAP bound to Ras-GTP. Alternatively, an RGS could introduce an Arg into the catalytic center. In any event, >40-fold stimulation of Galpha GTPase by RGS proteins brings the rate of GTP hydrolysis in line with that of Ras stimulated by Ras-GAP.


Why So Many RGS Family Members?

The possibility that an RGS regulates one (or a small subset) of Galpha classes is unlikely since divergent RGS proteins (RGS1, RGS4, RGS10, and GAIP) all stimulate GTPase activity of the Gi subfamily (59, 63). Other RGS members may act on other Galpha classes. Differences among the RGS members may not be apparent under available assay conditions. Association with additional regulatory factors or post-translational modifications may impose specificity on RGS function. RGS action may be restricted by expression pattern or agonist induction. Indeed, SST2 is induced by pheromone as a feedback mechanism to limit the extent of G protein-mediated signaling (34). Similarly, RGS1 expression is induced by PAF, and elevated RGS1 expression blocks PAF-induced MAPK activation (47). Combinatorial, spatial, temporal, and developmental regulation of RGS function clearly could modulate the intensity, duration, localization, and cell-type specificity of G protein signaling.


FOOTNOTES

*   This minireview will be reprinted in the 1997 Minireview Compendium, which will be available in December, 1997. This work was supported, in part, by grants from the American Heart Association and the Donaghue Medical Research Foundation (to H. G. D.) and by National Institutes of Health Grant GM21841 (to J. T.).
   To whom correspondence should be addressed. Tel.: 510-642-2558; Fax: 510-643-5035; E-mail: jthorner{at}mendel.berkeley.edu.
1    The abbreviations used are: GDS, guanine nucleotide dissociation stimulator; GDI, guanine nucleotide dissociation inhibitor; GAP, GTPase-activating protein; MAPK, mitogen-activated protein kinase; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate; PAF, platelet-activating factor.
2    K. Druey and J. Kehrl, personal communication.
3    F. Costantini, personal communication.
4    Berman, D. M., Kozasa, T., and Gilman, A. G. (1996) J. Biol. Chem. 271, 27209-27212.

Acknowledgments

We are grateful to T. Adams, H. Bourne, K. Blumer, P. Casey, A. Gilman, M. Koelle, and E. Neer for helpful comments and communication of unpublished results.


REFERENCES

  1. Neer, E. J. (1995) Cell 80, 249-257 [Medline] [Order article via Infotrieve]
  2. Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1991) Annu. Rev. Biochem. 60, 653-688 [CrossRef][Medline] [Order article via Infotrieve]
  3. Peroutka, S. J. (1994) Handbook of Receptors and Channels: G Protein-coupled Receptors, CRC Press, Inc., Boca Raton, FL
  4. Schertler, G. F., Villa, C., and Henderson, R. (1993) Nature 362, 770-772 [CrossRef][Medline] [Order article via Infotrieve]
  5. Dohlman, H. G., Bouvier, M., Benovic, J. L., Caron, M. G., and Lefkowitz, R. J. (1987) J. Biol. Chem. 262, 14282-14288 [Abstract/Free Full Text]
  6. Wilkie, T. M., and Yokoyama, S. (1994) Soc. Gen. Physiol. Ser. 49, 249-270 [Medline] [Order article via Infotrieve]
  7. Wall, M. A., Coleman, D. E., Lee, E., Iniguez, L. J., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058 [Medline] [Order article via Infotrieve]
  8. Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319 [CrossRef][Medline] [Order article via Infotrieve]
  9. Premont, R. T., Inglese, J., and Lefkowitz, R. J. (1995) FASEB J. 9, 175-182 [Abstract/Free Full Text]
  10. Gurevich, V. V., Dion, S. B., Onorato, J. J., Ptasienski, J., Kim, C. M., Sterne, M. R., Hosey, M. M., and Benovic, J. L. (1995) J. Biol. Chem. 270, 720-731 [Abstract/Free Full Text]
  11. Ferguson, S. S., Downey, W. E., III, Colapietro, A. M., Barak, L. S., Menard, L., and Caron, M. G. (1996) Science 271, 363-366 [Abstract]
  12. Bauer, P. H., Muller, S., Puzicha, M., Pippig, S., Obermaier, B., Helmreich, E. J., and Lohse, M. J. (1992) Nature 358, 73-76 [CrossRef][Medline] [Order article via Infotrieve]
  13. Chen, C. K., Inglese, J., Lefkowitz, R. J., and Hurley, J. B. (1995) J. Biol. Chem. 270, 18060-18066 [Abstract/Free Full Text]
  14. Faurobert, E., Chen, C. K., Hurley, J. B., and Teng, D. H. (1996) J. Biol. Chem. 271, 10256-10262 [Abstract/Free Full Text]
  15. Bourne, H. R., Sanders, D. A., and McCormick, P. (1990) Nature 348, 125-132 [CrossRef][Medline] [Order article via Infotrieve]
  16. Dratz, E. A., Lewis, J. W., Schaechter, L. E., Parker, K. R., and Kliger, D. S. (1987) Biochem. Biophys. Res. Commun. 146, 379-386 [Medline] [Order article via Infotrieve]
  17. Vuong, T. M., and Chabre, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9813-9817 [Abstract]
  18. Biddlecome, G. H., Berstein, G., and Ross, E. M. (1996) J. Biol. Chem. 271, 7999-8007 [Abstract/Free Full Text]
  19. Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417 [CrossRef][Medline] [Order article via Infotrieve]
  20. Sprague, G. F., Jr., and Thorner, J. (1992) in The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression (Broach, J. R., Pringle, J. R., and Jones, E. W., eds), pp. 657-744, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  21. Chan, R. K., and Otte, C. A. (1982) Mol. Cell. Biol. 2, 11-20 [Medline] [Order article via Infotrieve]
  22. Chan, R. K., and Otte, C. A. (1982) Mol. Cell. Biol. 2, 21-29 [Medline] [Order article via Infotrieve]
  23. Chan, R. K., Melnick, L. M., Blair, L. C., and Thorner, J. (1983) J. Bacteriol. 155, 903-906 [Medline] [Order article via Infotrieve]
  24. Dietzel, C., and Kurjan, J. (1987) Mol. Cell. Biol. 7, 4169-4177 [Medline] [Order article via Infotrieve]
  25. King, K., Dohlman, H. G., Thorner, J., Caron, M. G., and Lefkowitz, R. J. (1990) Science 250, 121-123 [Medline] [Order article via Infotrieve]
  26. Price, L. A., Kajkowski, E. M., Hadcock, J. R., Ozenberger, B. A., and Pausch, M. H. (1995) Mol. Cell. Biol. 15, 6188-6195 [Abstract]
  27. Courchesne, W. E., Kunisawa, R., and Thorner, J. (1989) Cell 58, 1107-1119 [Medline] [Order article via Infotrieve]
  28. Dietzel, C., and Kurjan, J. (1987) Cell 50, 1001-1010 [Medline] [Order article via Infotrieve]
  29. Miyajima, I., Nakafuku, M., Nakayama, N., Brenner, C., Miyajima, A., Kaibuchi, K., Arai, K. I., Kaziro, Y., and Matsumoto, K. (1987) Cell 50, 1011-1019 [Medline] [Order article via Infotrieve]
  30. Miyajima, I., Arai, K., and Matsumoto, K. (1989) Mol. Cell. Biol. 9, 2289-2297 [Medline] [Order article via Infotrieve]
  31. Kurjan, J., Hirsch, J. P., and Dietzel, C. (1991) Genes Dev. 5, 475-483 [Abstract]
  32. Hasson, M. S., Blinder, D., Thorner, J., and Jenness, D. D. (1994) Mol. Cell. Biol. 14, 1054-1065 [Abstract]
  33. Dohlman, H. G., Apaniesk, D., Chen, Y., Song, J., and Nusskern, D. (1995) Mol. Cell. Biol. 15, 3635-3643 [Abstract]
  34. Dohlman, H. G., Song, J., Ma, D., Courchesne, W. E., and Thorner, J. (1996) Mol. Cell. Biol. 16, 5194-5209 [Abstract]
  35. Madura, K., and Varshavsky, A. (1994) Science 265, 1454-1458 [Medline] [Order article via Infotrieve]
  36. Lee, B. N., and Adams, T. H. (1994) Mol. Microbiol. 14, 323-334 [Medline] [Order article via Infotrieve]
  37. Yu, J. H., Wieser, J., and Adams, T. H. (1996) EMBO J. 15, 5184-5190 [Abstract]
  38. Masters, S. B., Miller, R. T., Chi, M. H., Chang, F. H., Beiderman, B., Lopez, N. G., and Bourne, H. R. (1989) J. Biol. Chem. 264, 15467-15474 [Abstract/Free Full Text]
  39. Koelle, M. R., and Horvitz, H. R. (1996) Cell 84, 115-125 [Medline] [Order article via Infotrieve]
  40. Segalat, L., Elkes, D. A., and Kaplan, J. M. (1995) Science 267, 1648-1651 [Medline] [Order article via Infotrieve]
  41. Mendel, J. E., Korswagen, H. C., Liu, K. S., Hajdu, C. Y., Simon, M. I., Plasterk, R. H., and Sternberg, P. W. (1995) Science 267, 1652-1655 [Medline] [Order article via Infotrieve]
  42. DeVries, L., Mousli, M., Wurmser, A., and Farquhar, M. G. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11916-11920 [Abstract]
  43. Hong, J. X., Wilson, G. L., Fox, C. H., and Kehrl, J. H. (1993) J. Immunol. 150, 3895-3904 [Abstract/Free Full Text]
  44. Newton, J. S., Deed, R. W., Mitchell, E. L., Murphy, J. J., and Norton, J. D. (1993) Biochim. Biophys. Acta 1216, 314-316 [Medline] [Order article via Infotrieve]
  45. Wu, H. K., Heng, H. H., Shi, X. M., Forsdyke, D. R., Tsui, L. C., Mak, T. W., Minden, M. D., and Siderovski, D. P. (1995) Leukemia 9, 1291-1298 [Medline] [Order article via Infotrieve]
  46. Siderovski, D. P., Blum, S., Forsdyke, R. E., and Forsdyke, D. R. (1990) DNA Cell Biol. 9, 579-587 [Medline] [Order article via Infotrieve]
  47. Druey, K. M., Blumer, K. J., Kang, V. H., and Kehrl, J. H. (1996) Nature 379, 742-746 [CrossRef][Medline] [Order article via Infotrieve]
  48. Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212 [Medline] [Order article via Infotrieve]
  49. Perry, W. L., Vasicek, T. J., Lee, J. J., Rossi, J. M., Zeng, L., Zhang, T., Tilghman, S. M., and Costantini, F. (1995) Genetics 141, 321-332 [Abstract/Free Full Text]
  50. Berman, D. M., Wilkie, T. M., and Gilman, A. G. (1996) Cell 86, 445-452 [Medline] [Order article via Infotrieve]
  51. Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Science 265, 1405-1412 [Medline] [Order article via Infotrieve]
  52. Sondek, J., Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 372, 276-279 [CrossRef][Medline] [Order article via Infotrieve]
  53. Watson, N., Linder, M. E., Druey, K. M., Kehrl, J. H., and Blumer, K. J. (1996) Nature 383, 172-175 [CrossRef][Medline] [Order article via Infotrieve]
  54. Hunt, T. W., Fields, T. A., Casey, P. J., and Peralta, E. G. (1996) Nature 383, 175-177 [CrossRef][Medline] [Order article via Infotrieve]
  55. Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsch, W., and Wittinghofer, A. (1990) EMBO J. 9, 2351-2359 [Abstract]
  56. Milburn, M. V., Tong, L., deVos, A. M., Brunger, A., Yamaizumi, Z., Nishimura, S., and Kim, S. H. (1990) Science 247, 939-945 [Medline] [Order article via Infotrieve]
  57. Lambright, D. G., Noel, J. P., Hamm, H. E., and Sigler, P. B. (1994) Nature 369, 621-628 [CrossRef][Medline] [Order article via Infotrieve]
  58. Mixon, M. B., Lee, E., Coleman, D. E., Berghuis, A. M., Gilman, A. G., and Sprang, S. R. (1995) Science 270, 954-960 [Abstract]
  59. Bourne, H. R., Sanders, D. A., and McCormick, F. (1991) Nature 349, 117-127 [CrossRef][Medline] [Order article via Infotrieve]
  60. Gideon, P., John, J., Frech, M., Lautwein, A., Clark, R., Scheffler, J. E., and Wittinghofer, A. (1992) Mol. Cell. Biol. 12, 2050-2056 [Abstract]
  61. Eccleston, J. F., Moore, K. J., Morgan, L., Skinner, R. H., and Lowe, P. N. (1993) J. Biol. Chem. 268, 27012-27019 [Abstract/Free Full Text]
  62. Markby, D. W., Onrust, R., and Bourne, H. R. (1993) Science 262, 1895-1901 [Medline] [Order article via Infotrieve]
  63. Mittal, R., Ahmadian, M. R., Goody, R. S., and Wittinghofer, A. (1996) Science 273, 115-117 [Abstract]

©1997 by The American Society for Biochemistry and Molecular Biology, Inc.