Henrik G. Dohlman

RGS Proteins and Signaling by Heterotrimeric G Proteins

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 Ga subunit in its GTP-bound state from the Gbg 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 proteincoupled 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 Ga (6), 5 Gb, and 6 Gg isoforms (1), allowing many combinatorial possibilities. Three-dimensional structures of several Ga subunits and two different Gabg 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 Gbg) (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 proteindependent signaling, at least in part, because they stimulate hydrolysis of the GTP bound to activated Ga subunits.

Regulators of G-protein signaling (RGS) proteins: Region-specific expression of nine subtypes in rat brain

The recently discovered regulators of G-protein signaling (RGS) proteins potently modulate the functioning of heterotrimeric G-proteins by stimulating the GTPase activity of G-protein α subunits. The mRNAs for numerous subtypes of putative RGS proteins have been identified in mammalian tissues, but little is known about their expression in brain. We performed a systematic survey of the localization of mRNAs encoding nine of these RGSs, RGS3–RGS11, in brain by in situhybridization. Striking region-specific patterns of expression were observed. Five subtypes, RGS4, RGS7, RGS8, RGS9, and RGS10 mRNAs, are densely expressed in brain, whereas the other subtypes (RGS3, RGS5, RGS6, and RGS11) are expressed at lower density and in more restricted regions. RGS4 mRNA is notable for its dense expression in neocortex, piriform cortex, caudoputamen, and ventrobasal thalamus. RGS8 mRNA is highly expressed in the cerebellar Purkinje cell layer as well as in several midbrain nuclei. RGS9 mRNA is remarkable for its nearly exclusive enrichment in striatal regions. RGS10 mRNA is densely expressed in dentate gyrus granule cells, superficial layers of neocortex, and dorsal raphe. To assess whether the expression of RGS mRNAs can be regulated, we examined the effect of an acute seizure on levels of RGS7, RGS8, and RGS10 mRNAs in hippocampus. Of the three subtypes, changes in RGS10 levels were most pronounced, decreasing by ∼40% in a time-dependent manner in response to a single seizure. These results, which document highly specific patterns of RGS mRNA expression in brain and their ability to be regulated in a dynamic manner, support the view that RGS proteins may play an important role in determining the intensity and specificity of signaling pathways in brain as well as their adaptations to synaptic activity.

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.

Activation of the Phosphatidylinositol 3-Kinase Vps34 by a G Protein α Subunit at the Endosome

Summary In the yeast Saccharomyces cerevisiae , the G protein βγ subunits are essential for pheromone signaling. The Gα subunit Gpa1 can also promote signaling, but the effectors in this pathway are not well characterized. To identify candidate Gpa1 effectors, we expressed the constitutively active Gpa1 Q323L mutant in each of nearly 5000 gene-deletion strains and measured mating-specific responses. Our analysis reveals a requirement for both the catalytic (Vps34) and regulatory (Vps15) subunits of the sole phosphatidylinositol 3-kinase in yeast. We demonstrate that Gpa1 is present at endosomes, where it interacts directly with both Vps34 and Vps15 and stimulates increased production of phosphatidylinositol 3-phosphate. Notably, Vps15 binds to GDP-bound Gpa1 and is predicted to have a seven-WD repeat structure similar to that of known G protein β subunits. These findings reveal two new components of the pheromone signaling pathway. More remarkably, these proteins appear to comprise a preformed effector-Gβ subunit assembly and function at the endosome rather than at the plasma membrane.

Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae.

Genetic analysis of cell-cell signaling in Saccharomyces cerevisiae has led to the identification of a novel factor, known as Sst2p, that promotes recovery after pheromone-induced growth arrest (R. K. Chan and C. A. Otte, Mol. Cell. Biol. 2:11-20, 1982). Loss-of-function mutations lead to increased pheromone sensitivity, but this phenotype is partially suppressed by overexpression of the G protein alpha subunit gene (GPA1). Suppression is allele specific, however, suggesting that there is direct interaction between the two gene products. To test this model directly, we isolated and characterized several dominant gain-of-function mutants of SST2. These mutations block the normal pheromone response, including a loss of pheromone-stimulated gene transcription, cell cycle growth arrest, and G protein myristoylation. Although the SST2 mutations confer a pheromone-resistant phenotype, they do not prevent downstream activation by overexpression of G beta (STE4), a constitutively active G beta mutation (STE4Hpl), or a disruption of GPA1. None of the SST2 alleles affects the expression or stability of G alpha. These results point to the G protein alpha subunit as being the direct target of Sst2p action and underscore the importance of this novel desensitization factor in G-protein-mediated signaling.

A point mutation in Gα(o) and Gα(i1) blocks interaction with regulator of G protein signaling proteins

Regulator of G protein-signaling (RGS) proteins accelerate GTP hydrolysis by Gα subunits and are thought to be responsible for rapid deactivation of enzymes and ion channels controlled by G proteins. We wanted to identify and characterize Gi-family α subunits that were insensitive to RGS action. Based on a glycine to serine mutation in the yeast Gα subunit Gpa1sst that prevents deactivation by Sst2 (DiBello, P. R., Garrison, T. R., Apanovitch, D. M., Hoffman, G., Shuey, D. J., Mason, K., Cockett, M. I., and Dohlman, H. G. (1998) J. Biol. Chem. 273, 5780–5784), site-directed mutagenesis of αo and αi1 was done. G184S αo and G183S αi1 show kinetics of GDP release and GTP hydrolysis similar to wild type. In contrast, GTP hydrolysis by the G → S mutant proteins is not stimulated by RGS4 or by a truncated RGS7. Quantitative flow cytometry binding studies show IC50 values of 30 and 96 nm, respectively, for aluminum fluoride-activated wild type αo and αi1 to compete with fluorescein isothiocyanate-αo binding to glutathioneS-transferase-RGS4. The G → S mutant proteins showed a greater than 30–100-fold lower affinity for RGS4. Thus, we have defined the mechanism of a point mutation in αo and αi1 that prevents RGS binding and GTPase activating activity. These mutant subunits should be useful in biochemical or expression studies to evaluate the role of endogenous RGS proteins in Gi function.

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.

Selective Uncoupling of RGS Action by a Single Point Mutation in the G Protein α-Subunit

Heterotrimeric G proteins function as molecular relays, shuttling between cell surface receptors and intracellular effectors that propagate a signal. G protein signaling is governed by the rates of GTP binding (catalyzed by the receptor) and GTP hydrolysis. RGS proteins (regulators of G protein signaling) were identified as potent negative regulators of G protein signaling pathways in simple eukaryotes and are now known to act as GTPase-activating proteins (GAPs) for G protein α-subunits in vitro. It is not known, however, if Gα GAP activity is responsible for the regulatory action of RGS proteins in vivo. We describe here a Gα mutant in yeast (gpa1 sst ) that phenotypically mimics the loss of its cognate RGS protein (SST2). Thegpa1 sst mutant is resistant to an activated allele of SST2 in vivo and is unresponsive to RGS GAP activityin vitro. The analogous mutation in a mammalian Gqα is also resistant to RGS action in transfected cells. These mutants demonstrate that RGS proteins act through Gα and that RGS-GAP activity is responsible for their desensitizing activity in cells. The Gαsst mutant will be useful for uncoupling RGS-mediated regulation from other modes of signal regulation in whole cells and animals.

Regulation of Cell Signaling Dynamics by the Protein Kinase-Scaffold Ste5

Cell differentiation requires the ability to detect and respond appropriately to a variety of extracellular signals. Here we investigate a differentiation switch induced by changes in the concentration of a single stimulus. Yeast cells exposed to high doses of mating pheromone undergo cell division arrest. Cells at intermediate doses become elongated and divide in the direction of a pheromone gradient (chemotropic growth). Either of the pheromone-responsive MAP kinases, Fus3 and Kss1, promotes cell elongation, but only Fus3 promotes chemotropic growth. Whereas Kss1 is activated rapidly and with a graded dose-response profile, Fus3 is activated slowly and exhibits a steeper dose-response relationship (ultrasensitivity). Fus3 activity requires the scaffold protein Ste5; when binding to Ste5 is abrogated, Fus3 behaves like Kss1, and the cells no longer respond to a gradient or mate efficiently with distant partners. We propose that scaffold proteins serve to modulate the temporal and dose-response behavior of the MAP kinase.

A Systems-Biology Analysis of Feedback Inhibition in the Sho1 Osmotic-Stress-Response Pathway

BACKGROUND A common property of signal transduction systems is that they rapidly lose their ability to respond to a given stimulus. For instance in yeast, the mitogen-activated protein (MAP) kinase Hog1 is activated and inactivated within minutes, even when the osmotic-stress stimulus is sustained. RESULTS Here, we used a combination of experimental and computational analyses to investigate the dynamic behavior of Hog1 activation in vivo. Computational modeling suggested that a negative-feedback loop operates early in the pathway and leads to rapid attenuation of Hog1 signaling. Experimental analysis revealed that the membrane-bound osmosensor Sho1 is phosphorylated by Hog1 and that phosphorylation occurs on Ser-166. Moreover, Sho1 exists in a homo-oligomeric complex, and phosphorylation by Hog1 promotes a transition from the oligomeric to monomeric state. A phosphorylation-site mutation (Sho1(S166E)) diminishes the formation of Sho1-oligomers, dampens activation of the Hog1 kinase, and impairs growth in high-salt or sorbitol conditions. CONCLUSIONS These findings reveal a novel phosphorylation-dependent feedback loop leading to diminished cellular responses to an osmotic-stress stimulus.