Modulation of receptor dynamics by the regulator of G protein signaling Sst2

G protein–coupled receptor (GPCR) signaling is fundamental to physiological processes such as vision, the immune response, and wound healing. In the budding yeast Saccharomyces cerevisiae, GPCRs detect and respond to gradients of pheromone during mating. After pheromone stimulation, the GPCR Ste2 is removed from the cell membrane, and new receptors are delivered to the growing edge. The regulator of G protein signaling (RGS) protein Sst2 acts by accelerating GTP hydrolysis and facilitating pathway desensitization. Sst2 is also known to interact with the receptor Ste2. Here we show that Sst2 is required for proper receptor recovery at the growing edge of pheromone-stimulated cells. Mathematical modeling suggested pheromone-induced synthesis of Sst2 together with its interaction with the receptor function to reestablish a receptor pool at the site of polarized growth. To validate the model, we used targeted genetic perturbations to selectively disrupt key properties of Sst2 and its induction by pheromone. Together our results reveal that a regulator of G protein signaling can also regulate the G protein–coupled receptor. Whereas Sst2 negatively regulates G protein signaling, it acts in a positive manner to promote receptor retention at the growing edge.     

Genome-scale analysis reveals Sst2 as the principal regulator of mating pheromone signaling in the yeast Saccharomyces cerevisiae

A common property of G protein-coupled receptors is that they become less responsive with prolonged stimulation. Regulators of G protein signaling (RGS proteins) are well known to accelerate G protein GTPase activity and do so by stabilizing the transition state conformation of the G protein alpha subunit. In the yeast Saccharomyces cerevisiae there are four RGS-homologous proteins (Sst2, Rgs2, Rax1, and Mdm1) and two Galpha proteins (Gpa1 and Gpa2). We show that Sst2 is the only RGS protein that binds selectively to the transition state conformation of Gpa1. The other RGS proteins also bind Gpa1 and modulate pheromone signaling, but to a lesser extent and in a manner clearly distinct from Sst2. To identify other candidate pathway regulators, we compared pheromone responses in 4,349 gene deletion mutants representing nearly all nonessential genes in yeast. A number of mutants produced an increase (sst2, bar1, asc1, and ygl024w) or decrease (cla4) in pheromone sensitivity or resulted in pheromone-independent signaling (sst2, pbs2, gas1, and ygl024w). These findings suggest that Sst2 is the principal regulator of Gpa1-mediated signaling in vivo but that other proteins also contribute in distinct ways to pathway regulation.     

DEP-domain-mediated regulation of GPCR signaling responses

G protein-coupled receptors (GPCRs) mediate cellular responses to a variety of stimuli, but how specific responses are regulated has been elusive, as the types of GPCRs vastly outnumber the classes of G protein heterotrimers available to initiate downstream signaling. In our analysis of signaling proteins containing DEP domains ( approximately 90 residue sequence motifs first recognized in fly Dishevelled, worm EGL-10, and mammalian Pleckstrin), we find that DEP domains are responsible for specific recognition of GPCRs. We examined the yeast regulator of G protein signaling (RGS) protein Sst2 and demonstrate that the DEP domains in Sst2 mediate binding to its cognate GPCR (Ste2). DEP-domain-mediated tethering promotes downregulation by placing the RGS protein in proximity to its substrate (receptor-activated Galpha subunit). Sst2 docks to the Ste2 cytosolic tail, but only its unphosphorylated state, allowing for release and recycling of this regulator upon receptor desensitization and internalization. DEP-domain-mediated targeting of effectors and regulators to specific GPCRs provides a means to dictate the nature, duration, and specificity of the response.     

Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit).

Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST2 gene product fail to resume growth after exposure to pheromone. Conversely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detectably dampened signaling in a short-term assay of pheromone response (phosphorylation of Ste4, Gbeta subunit). When the GPA1 gene product (Galpha subunit) is absent, the pheromone response pathway is constitutively active and, consequently, growth ceases. Despite sustained induction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1delta mutants remain growth arrested, indicating that the action of Sst2 requires the presence of Gpa1. The N-terminal domain (residues 3 to 307) of Sst2 (698 residues) has sequence similarity to the catalytic regions of bovine GTPase-activating protein and human neurofibromatosis tumor suppressor protein; segments in the C-terminal domain of Sst2 (between residues 417 and 685) are homologous to other RGS proteins. Both the N- and C-terminal domains were required for Sst2 function in vivo. Consistent with a role for Sst2 in binding to and affecting the activity of Gpa1, the majority of Sst2 was membrane associated and colocalized with Gpa1 at the plasma membrane, as judged by sucrose density gradient fractionation. Moreover, from cell extracts, Sst2 could be isolated in a complex with Gpa1 (expressed as a glutathione S-transferase fusion); this association withstood the detergent and salt conditions required for extraction of these proteins from cell membranes. Also, SST2+ cells expressing a GTPase-defective GPA1 mutant displayed an increased sensitivity to pheromone, whereas sst2 cells did not. These results demonstrate that Sst2 and Gpa1 interact physically and suggest that Sst2 is a direct negative regulator of Gpa1.     

Yeast GPCR signaling reflects the fraction of occupied receptors,not the number

According to receptor theory, the effect of a ligand depends on the amount of agonist–receptor complex. Therefore, changes in receptor abundance should have quantitative effects. However, the response to pheromone in Saccharomyces cerevisiae is robust (unaltered) to increases or reductions in the abundance of the G‐protein‐coupled receptor (GPCR), Ste2, responding instead to the fraction of occupied receptor. We found experimentally that this robustness originates during G‐protein activation. We developed a complete mathematical model of this step, which suggested the ability to compute fractional occupancy depends on the physical interaction between the inhibitory regulator of G‐protein signaling (RGS), Sst2, and the receptor. Accordingly, replacing Sst2 by the heterologous hsRGS4, incapable of interacting with the receptor, abolished robustness. Conversely, forcing hsRGS4:Ste2 interaction restored robustness. Taken together with other results of our work, we conclude that this GPCR pathway computes fractional occupancy because ligand‐bound GPCR–RGS complexes stimulate signaling while unoccupied complexes actively inhibit it. In eukaryotes, many RGSs bind to specific GPCRs, suggesting these complexes with opposing activities also detect fraction occupancy by a ratiometric measurement. Such complexes operate as push‐pull devices, which we have recently described.     

The N terminus of Saccharomyces cerevisiae Sst2p plays an RGS-domain-independent, Mpt5p-dependent role in recovery from pheromone arrest.

The Saccharomyces cerevisiae RGS protein Sst2p is involved in desensitization to pheromone and acts as a GTPase-activating protein for the Galpha subunit Gpa1p. Other results indicate that Sst2p acts through Mpt5p and that this action occurs downstream of Fus3p and through Cln3p/Cdc28p. Our results indicate that the interaction of Sst2p with Mpt5p requires the N-terminal MPI (Mpt5p-interacting) domain of Sst2p and is independent of the C-terminal RGS domain. Overexpression of the MPI domain results in an Mpt5p-dependent increase in recovery from pheromone arrest. Overexpression of either intact Sst2p or the MPI domain leads to partial suppression of a gpa1 growth defect, and this suppression is dependent on Mpt5p, indicating that MPI function occurs downstream of Gpa1p and through Mpt5p. Combination of an mpt5 mutation with the GPA1(G302S) mutation, which uncouples Gpa1p from Sst2p, results in pheromone supersensitivity similar to the sst2 mutant, and promotion of recovery by overexpression of Sst2p is dependent on both Mpt5p and the Gpa1p interaction. These results indicate that Sst2p is a bifunctional protein and that the MPI domain acts through Mpt5p independently of the RGS domain. RGS family members from other fungi contain N-terminal domains with sequence similarity to the Sst2p MPI domain, suggesting that MPI function may be conserved.     

Interaction with the SH3 domain protein Bem1 regulates signaling by the Saccharomyces cerevisiae p21-activated kinase Ste20

The Saccharomyces cerevisiae PAK (p21-activated kinase) family kinase Ste20 functions in several signal transduction pathways, including pheromone response, filamentous growth, and hyperosmotic resistance. The GTPase Cdc42 localizes and activates Ste20 by binding to an autoinhibitory motif within Ste20 called the CRIB domain. Another factor that functions with Ste20 and Cdc42 is the protein Bem1. Bem1 has two SH3 domains, but target ligands for these domains have not been described. Here we identify an evolutionarily conserved binding site for Bem1 between the CRIB and kinase domains of Ste20. Mutation of tandem proline-rich (PxxP) motifs in this region disrupts Bem1 binding, suggesting that it serves as a ligand for a Bem1 SH3 domain. These PxxP motif mutations affect signaling additively with CRIB domain mutations, indicating that Bem1 and Cdc42 make separable contributions to Ste20 function, which cooperate to promote optimal signaling. This PxxP region also binds another SH3 domain protein, Nbp2, but analysis of bem1Delta versus nbp2Delta strains shows that the signaling defects of PxxP mutants result from impaired binding to Bem1 rather than from impaired binding to Nbp2. Finally, the PxxP mutations also reduce signaling by constitutively active Ste20, suggesting that postactivation functions of PAKs can be promoted by SH3 domain proteins, possibly by colocalizing PAKs with their substrates. The overall results also illustrate how the final signaling function of a protein can be governed by combinatorial addition of multiple, independent protein-protein interaction modules.     

A novel regulator of G protein signalling in yeast, Rgs2, downregulates glucose-activation of the cAMP pathway through direct inhibition of Gpa2.

We have characterized a novel member of the recently identified family of regulators of heterotrimeric G protein signalling (RGS) in the yeast Saccharomyces cerevisiae. The YOR107w/RGS2 gene was isolated as a multi-copy suppressor of glucose-induced loss of heat resistance in stationary phase cells. The N-terminal half of the Rgs2 protein consists of a typical RGS domain. Deletion and overexpression of Rgs2, respectively, enhances and reduces glucose-induced accumulation of cAMP. Overexpression of RGS2 generates phenotypes consistent with low activity of cAMP-dependent protein kinase A (PKA), such as enhanced accumulation of trehalose and glycogen, enhanced heat resistance and elevated expression of STRE-controlled genes. Deletion of RGS2 causes opposite phenotypes. We demonstrate that Rgs2 functions as a negative regulator of glucose-induced cAMP signalling through direct GTPase activation of the Gs-alpha protein Gpa2. Rgs2 and Gpa2 constitute the second cognate RGS-G-alpha protein pair identified in yeast, in addition to the mating pheromone pathway regulators Sst2 and Gpa1. Moreover, Rgs2 and Sst2 exert specific, non-overlapping functions, and deletion mutants in Rgs2 and Sst2 are complemented to some extent by different mammalian RGS proteins.     

Regulation of G protein-mediated signal transduction by RGS proteins

RGS proteins form a new family of regulatory proteins of G protein signaling. They contain homologous core domains (RGS domains) of about 120 amino acids. RGS domains interact with activated Galpha subunits. Several RGS proteins have been shown biochemically to act as GTPase activating proteins (GAPs) for their interacting Galpha subunits. Other than RGS domains, RGS proteins differ significantly in size, amino acid sequences, and tissue distribution. In addition, many RGS proteins have other protein-protein interaction motifs involved in cell signaling. We have shown that p115RhoGEF, a newly identified GEF(guanine nucleotide exchange factor) for RhoGTPase, has a RGS domain at its N-terminal region and this domain acts as a specific GAP for Galpha12 and Galpha13. Furthermore, binding of activated Galpha13 to this RGS domain stimulated GEF activity of p115RhoGEF. Activated Galpha12 inhibited Galpha13-stimulated GEF activity. Thus p115RhoGEF is a direct link between heterotrimeric G protein and RhoGTPase and it functions as an effector for Galpha12 and Galpha13 in addition to acting as their GAP. We also found that RGS domain at N-terminal regions of G protein receptor kinase 2 (GRK2) specifically interacts with Galphaq/11 and inhibits Galphaq-mediated activation of PLC-beta, apparently through sequestration of activated Galphaq. However, unlike other RGS proteins, this RGS domain did not show significant GAP activity to Galphaq. These results indicate that RGS proteins have far more diverse functions than acting simply as GAPs and the characterization of function of each RGS protein is crucial to understand the G protein signaling network in cells.     

Cdc42 regulation of kinase activity and signaling by the yeast p21-activated kinase Ste20

The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated G beta gamma complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway.     

RGS17/RGSZ2, a novel regulator of Gi/o, Gz, and Gq signaling

To identify novel regulators of Galpha(o), the most abundant G-protein in brain, we used yeast two-hybrid screening with constitutively active Galpha(o) as bait and identified a new regulator of G-protein signaling (RGS) protein, RGS17 (RGSZ2), as a novel human member of the RZ (or A) subfamily of RGS proteins. RGS17 contains an amino-terminal cysteine-rich motif and a carboxyl-terminal RGS domain with highest homology to hRGSZ1- and hRGS-Galpha-interacting protein. RGS17 RNA was strongly expressed as multiple species in cerebellum and other brain regions. The interactions between hRGS17 and active forms of Galpha(i1-3), Galpha(o), Galpha(z), or Galpha(q) but not Galpha(s) were detected by yeast two-hybrid assay, in vitro pull-down assay, and co-immunoprecipitation studies. Recombinant RGS17 acted as a GTPase-activating protein (GAP) on free Galpha(i2) and Galpha(o) under pre-steady-state conditions, and on M2-muscarinic receptor-activated Galpha(i1), Galpha(i2), Galpha(i3), Galpha(z), and Galpha(o) in steady-state GTPase assays in vitro. Unlike RGSZ1, which is highly selective for G(z), RGS17 exhibited limited selectivity for G(o) among G(i)/G(o) proteins. All RZ family members reduced dopamine-D2/Galpha(i)-mediated inhibition of cAMP formation and abolished thyrotropin-releasing hormone receptor/Galpha(q)-mediated calcium mobilization. RGS17 is a new RZ member that preferentially inhibits receptor signaling via G(i/o), G(z), and G(q) over G(s) to enhance cAMP-dependent signaling and inhibit calcium signaling. Differences observed between in vitro GAP assays and whole-cell signaling suggest additional determinants of the G-protein specificity of RGS GAP effects that could include receptors and effectors.     

Co-expression of Gbeta5 enhances the function of two Ggamma subunit-like domain-containing regulators of G protein signaling proteins

Regulators of G protein signaling (RGS) stimulate the GTPase activity of G protein Galpha subunits and probably play additional roles. Some RGS proteins contain a Ggamma subunit-like (GGL) domain, which mediates a specific interaction with Gbeta5. The role of such interactions in RGS function is unclear. RGS proteins can accelerate the kinetics of coupling of G protein-coupled receptors to G-protein-gated inwardly rectifying K(+) (GIRK) channels. Therefore, we coupled m2-muscarinic acetylcholine receptors to GIRK channels in Xenopus oocytes to evaluate the effect of Gbeta5 on RGS function. Co-expression of either RGS7 or RGS9 modestly accelerated GIRK channel kinetics. When Gbeta5 was co-expressed with either RGS7 or RGS9, the acceleration of GIRK channel kinetics was strongly increased over that produced by RGS7 or RGS9 alone. RGS function was not enhanced by co-expression of Gbeta1, and co-expression of Gbeta5 alone had no effect on GIRK channel kinetics. Gbeta5 did not modulate the function either of RGS4, an RGS protein that lacks a GGL domain, or of a functional RGS7 construct in which the GGL domain was omitted. Enhancement of RGS7 function by Gbeta5 was not a consequence of an increase in the amount of plasma membrane or cytosolic RGS7 protein.     

Regulators of G protein signaling: a bestiary of modular protein binding domains

Members of the newly discovered regulator of G protein signaling (RGS) families of proteins have a common RGS domain. This RGS domain is necessary for conferring upon RGS proteins the capacity to regulate negatively a variety of Galpha protein subunits. However, RGS proteins are more than simply negative regulators of signaling. RGS proteins can function as effector antagonists, and recent evidence suggests that RGS proteins can have positive effects on signaling as well. Many RGS proteins possess additional C- and N-terminal modular protein-binding domains and motifs. The presence of these additional modules within the RGS proteins provides for multiple novel regulatory interactions performed by these molecules. These regions are involved in conferring regulatory selectivity to specific Galpha-coupled signaling pathways, enhancing the efficacy of the RGS domain, and the translocation or targeting of RGS proteins to intracellular membranes. In other instances, these domains are involved in cross-talk between different Galpha-coupled signaling pathways and, in some cases, likely serve to integrate small GTPases with these G protein signaling pathways. This review discusses these C- and N-terminal domains and their roles in the biology of the brain-enriched RGS proteins. Methods that can be used to investigate the function of these domains are also discussed.