Perspectives in Pharmacology Regulator of G Protein Signaling Proteins: Novel Multifunctional Drug Targets

G protein-coupled receptors (GPCRs) play a major role in signal transduction and are targets of many therapeutic drugs. The regulator of G protein signaling (RGS) proteins form a recently identified protein family, and they strongly modulate the activity of G proteins. Their best known function is to inhibit G protein signaling by accelerating GTP hydrolysis [GTPase activating protein (GAP)] thus turning off G protein signals. RGS proteins also possess non-GAP functions, through both their RGS domains and various non-RGS domains and motifs (e.g., GGL, DEP, DH/PH, PDZ domains and a cysteine string motif). They are a highly diverse protein family, have unique tissue distributions, are strongly regulated by signal transduction events, and will likely play diverse functional roles in living cells. Thus they represent intriguing, novel pharmacological/therapeutic targets. Drugs targeting RGS proteins can be divided into five groups: 1) potentiators of endogenous agonist function, 2) potentiators/desensitization blockers of exogenous GPCR agonists, 3) specificity enhancers of exogenous agonists, 4) antagonists of effector signaling by an RGS protein, and 5) RGS agonists. In addition, a novel subsite distinction within the RGS domain has been proposed with significant functional implications and defined herein as “A-site” and “B-site”. Therefore, RGS proteins should provide exciting new opportunities for drug development. G protein-coupled receptors (GPCRs) play a major role in signal transduction and are the targets of a large number of therapeutic drugs. Just as our understanding of receptor, G protein, and effector function seemed nearly complete, a new kid appeared on the scene injecting fresh life into the field. The regulator of G protein signaling (RGS) proteins modulate the activity of G proteins in vitro, and evidence is beginning to emerge on their role in vivo as well. Their best known function is to inhibit G protein signaling by accelerating GTP hydrolysis thus turning off G protein signals (Berman et al., 1996a). They are a highly diverse protein family, have unique tissue distributions, and are strongly regulated by signal transduction events. Also, evidence is emerging that besides G protein inhibition, they can enhance G protein activation, serve as effectors, and act as scaffold proteins to gather receptors, G proteins, effectors, and other regulatory molecules together. There have been several excellent reviews on RGS proteins recently (Hepler, 1999; Siderovski et al., 1999; De Vries et al., 2000; Ross and Wilkie, 2000), so we will focus on known or predicted physiological functions of RGS proteins and on concepts related to RGS proteins as potential drug targets (see also Jones et al., 2000 and Dohlman, 2001). A Brief History of Regulators of G Protein Signaling The RGS proteins were discovered in genetic studies of GPCR signaling pathways in model organisms (Dohlman and Thorner, 1997). The scope and significance of RGS proteins were recognized in 1996 when ;20 mammalian members of the RGS protein family were identified based on sequence 1 Many important articles could not be cited in this paper due to the journal’s policies limiting the number of references. We apologize to our colleagues whose papers could not be included due to these limitations. This work was supported by National Institutes of Health Grant GM 39561. ABBREVIATIONS: GPCR, G protein-coupled receptor; RGS, regulator of G protein signaling; GAP, GTPase activating protein; GIRK, G protein-coupled inwardly rectifying potassium channel; GRK, G protein-coupled receptor kinase; DEP, disheveled, egl-10, and pleckstrin; GGL, G protein g-subunit-like; DH/PH, Dbl/pleckstrin homology; GSK3, glycogen synthase kinase 3; DIX, disheveled homology; PDZ, PSD-95,disclarge, and ZO-1; PLC, phospholipase C; AKAP, A-kinase anchoring protein; IL, interleukin; SH, Src homology; APC, adenomatous polyposis coli protein; PIP3, phosphatidylinositol 1,4,5-trisphosphate; GABA, g-aminobutyric acid; Glut, glucose transporter. 0022-3565/01/2973-837–845$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 297, No. 3 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 900027/904896 JPET 297:837–845, 2001 Printed in U.S.A. 837 at A PE T Jornals on Jne 6, 2017 jpet.asjournals.org D ow nladed from homologies with a conserved 120-amino acid domain in the original yeast and worm RGS proteins, Sst2 and EGL-10, respectively (Druey et al., 1996; Koelle and Horvitz, 1996; Siderovski et al., 1996). Later in 1996, several groups showed that RGS proteins were GTPase accelerating proteins (GAPs) (Berman et al., 1996a). The crystal structure of a Gai1-RGS4 suggested a mechanism for the GAP activity: stabilization of the transition state conformation of Ga (Berman et al., 1996b). The GAP activity explains RGS-mediated inhibition of G protein signaling. It also explains the paradox that some signals, visual responses and cardiac potassium channels (Szabo and Otero, 1989; Arshavsky et al., 1994), turn off much faster than expected given the slow hydrolysis of GTP by purified Ga subunits. More Than Just Ga GAPs Recently, there has been a paradigm shift in thinking about RGS proteins (Hepler, 1999; Siderovski et al., 1999). In addition to GAP activity, RGS proteins also: 1) directly antagonize Ga effectors (Hepler et al., 1997; Tesmer et al., 1997), 2) bind Gb5 (Snow et al., 1998b), 3) potentially target protein kinase A and receptor kinases since AKAP contains an RGS-like domain and is a predicted RGS family member (Koch et al., 1993; Huang et al., 1997), 4) scaffold Wnt signaling proteins (reviewed in Kikuchi, 1999), 5) are Ga13 effectors activating Rho (Hart et al., 1998; Kozasa et al., 1998), and 6) enhance receptor-G protein coupling (see below). Thus, we should think of RGS domains as modular, regulatable, Ga subunit recognition domains along the lines of SH2, SH3, or PDZ domains. As SH2 domains only bind tyrosine-phosphorylated peptides, RGS binding to Ga depends on the Ga functional state. For example, RGS4 binds only to the (AlF4 -bound) transition state of Gai1 subunits, while RGS2 binds both transition state and active (GTPgSbound) Gaq, and neither of them binds to the resting state of Ga (Berman et al., 1996b; Heximer et al., 1997). This produces state-dependent recruitment of the RGS protein to the vicinity of Ga subunits. Effector antagonism uses the RGS domain (Hepler et al., 1997; Tesmer et al., 1997), but most other non-GAP functions involve functional domains in Nand C-terminal extensions. The wide array of domains found in RGS proteins has been reviewed recently (Hepler, 1999; Siderovski et al., 1999; De Vries et al., 2000) and includes GGL (G protein g-subunitlike), DEP (disheveled, egl-10, pleckstrin), DH/PH (Dbl/ pleckstrin homology) and PDZ (PSD-95, disc-large, and ZO-1 homology) domains. These non-RGS components of the RGScontaining proteins serve to link other proteins and signaling pathways (Fig. 1). When RGS binds to Ga, it carries with it other functional units providing a great diversity of proteinprotein interactions as described in the reviews cited above. Recent studies suggest evidence for RGS interactions with receptors, providing additional specificity of RGS actions. A PDZ domain in the amino terminus of RGS12 binds selectively with the carboxyl terminus of the IL-8 receptor (Snow et al., 1998). Also, RGS4, in the context of intact cells, can selectively inhibit calcium signals induced by muscarinic versus cholecystokinin receptors, and this specificity is dependent on the amino-terminal extension of RGS4 (Zeng et al., 1998a). Moreover, the specificity of RGS4 for a-subunits is modified in the context of a receptor-Ga fusion protein (Cavalli et al., 2000). All of these results indicate that RGS specificity will depend on factors outside of the RGS-Ga interface. Furthermore, RGS proteins can accelerate the activation as well as the deactivation of receptor-stimulated G proteincoupled inwardly rectifying potassium (GIRK) currents (Doupnik et al., 1997; Saitoh et al., 1999) (for more references, see Ross and Wilkie, 2000). The GAP function of RGS explains the temporally enhanced deactivation and activation, but the fact that this acceleration is not accompanied by an expected decrease in the amplitude of stimulated currents suggests that RGS may have additional positive functions in receptor signaling. We recently found (H. Zhong, S. M. Wade, 2 The abbreviation GAP (for GTPase accelerating protein) is used in several ways that may not please grammarians but does facilitate discussion of RGS function. The noun form “GAP” is commonly recognized and understood. A corruption that greatly simplifies speaking or writing about RGS proteins is the verb form “to GAP”, which means to accelerate GTP hydrolysis. Also, it is occasionally used as an adjective as in “GAP activities” or “non-GAP activities” meaning, respectively, functions that do or do not depend on acceleration of GTP hydrolysis. Fig. 1. RGS proteins have multiple, independent protein interaction domains that confer unique specificity and functions. In addition to the RGS domain that mediates the interactions with Ga subunits, RGS proteins contain a variety of other protein interaction domains that localize the RGS to specific macromolecular complexes or mediate additional functions. The proteins represented as unfilled shapes illustrate the RGS domain and associated protein interaction motifs for several families of RGS proteins. The characteristics of these RGS families and the individual RGS proteins are outlined in Table 1 and in the text. The proteins shown in the graph are not necessarily oriented from N to C terminus (see Hepler, 1999). (Reprinted from Trends Pharmacol Sci, Vol 20, Hepler, J. R., Emerging roles for RGS proteins in cell signaling, pp 376–382, Copyright (1999), with permission from Elsevier Science.) 838 Zhong and Neubig at A PE T Jornals on Jne 6, 2017 jpet.asjournals.org D ow nladed from and R. R. Neubig, submitted) that RGS4 can enhance a2adrenergic receptor-stimulated GTPgS binding, acting as a positive kinetic modulator in receptor-G protein coupling. Another possibility is that the GGL domain containing RGS proteins may actually act as Gg proteins to allow receptor-G protein coupling. The above information implies a direct or indirect interaction of the RGS with receptor as well as with the G protein and provides further support for receptorspecific actions of RGS proteins. These results show that RGS proteins can also enhance receptor signaling, and the net effect must be determined from studies of intact physiological systems. Physiological Roles of RGS Proteins A large number of studies (reviewed in De Vries et al., 2000) have demonstrated that RGS proteins, when ectopically expressed in mammalian cells, can suppress G protein signaling. In Table 1, we summarize the functional aspects of known RGS proteins. Despite extensive overexpression data, much less is known about the physiological role of endogenous RGS proteins. The model organisms, Saccharomyces cervesiae and Caenorhabditis elegans, have provided the best evidence for functional roles of RGS proteins. In both cases, loss of RGS protein function leads to hyperstimulation of the signaling pathway, consistent with a primary action via the GAP activity of the RGS protein to suppress G protein signaling. In mammalian systems, there is little direct information on the roles of endogenous RGS proteins (Table 1). Recently, Jeong and Ikeda (2000) used RGS-insensitive mutants of Gao (Lan et al., 1998) to show that a2-adrenergic inhibition of N-type calcium currents in rat sympathetic ganglia is markedly inhibited by endogenous RGS proteins. When RGS-insensitive Gao subunits were expressed, the rate of Ca channel recovery from norepinephrine-induced inhibition was much slower (50–60 s versus 10 s) presumably due to the inability of endogenous RGS proteins to GAP the mutant Gao. As a critical proof-of-principle for the use of RGS inhibitor drugs, dose-response curves for norepinephrine were leftshifted 6to 8-fold by expression of the RGS-insensitive Gao subunits. The effect of these RGS-insensitive Ga subunit mutations should be the genetic equivalent of blocking the RGS-Ga interaction pharmacologically. Thus RGS inhibitors should lead to enhanced responses to physiologically released or pharmacologically administered agonists. Thus the endogenous levels of RGS proteins, at least in neurons, appear sufficient to influence steady-state ion channel responses through G protein-coupled receptors. The only reported RGS “knockout” shows that endogenous RGS9-1 (alternative splicing product of RGS9 gene, the other product being RGS9-2; see also Table 1) rapidly deactivates transducin in vivo (Chen et al., 2000). In homozygous RGS9-1 knockout mice, the half-time for single photon responses was greatly prolonged (;3 s versus ;0.5 s). An RGS2 knockout exhibits an “anxious” behavioral phenotype, and an RGS14 knockout shows embryonic lethality at the preimplantation stage demonstrating clear functions for these proteins (D. Siderovski, personal communication). Further genetic studies are likely to provide important insights into the physiological functions of different RGS proteins in the near future. Some additional physiological functions have not yet been proven but could be predicted based on the known biology of RGS protein regulation. The mRNA and/or protein levels of many RGS proteins exhibit rapid induction following physiological signals (for review, see Hepler, 1999; Siderovski et al., 1999; De Vries et al., 2000; Ross and Wilkie, 2000). The yeast RGS protein, Sst2, is up-regulated upon stimulation of yeast by the alpha factor mating pheromone, and this upregulation leads to a rapid inhibition of pheromone signaling (Dohlman et al., 1996). Deletion of the SST2 gene leads to a 100-fold enhancement in sensitivity to alpha factor. The mammalian RGS proteins, RGS1 and RGS2, were originally discovered due to the up-regulation of their mRNA levels in immune cells (De Vries et al., 2000). Several studies have demonstrated enhanced mRNA expression of RGS2 by increased cAMP levels (Pepperl et al., 1998) or angiotensin II receptors (Grant et al., 2000; Table 1). Since RGS2 is relatively selective for Gaq, it will be very interesting to determine the role of RGS2 up-regulation in the commonly observed rapid tachyphylaxis to angiotensin II and other activators of phospholipase C (PLC). Additionally, the nuclear localization of some RGS proteins when overexpressed in cells (RGS2, RGS3T, and RGS10) (Chatterjee and Fisher, 2000; Dulin et al., 2000) may suggest a role in regulating gene activation. RGS proteins may also participate in desensitization or tolerance to opioids (Potenza et al., 1999). Rapid tolerance develops to opioids and many RGS proteins act on the Gai/ Gao family G proteins (the main targets of opioid receptors). Thus it will again be very interesting to determine whether RGS proteins play a significant role in opioid desensitization, tolerance, and dependence. Physiological Relevance of “Positive” Signaling Properties of RGS Proteins. Several RGS proteins (such as p115-RhoGEF and Axin) play active roles in transmitting receptor signals to downstream effectors. Several GPCRs can activate Rho including receptors for thrombin (PAR2), lysophosphatidic acid (edg2 and 4), sphingosine-1-phosphate (edg3 and 5), thromboxane A2, and endothelin (Sah et al., 2000). Interestingly, several guanine nucleotide exchange factors for Rho (p115-RhoGEF, PDZ-RhoGEF, and KIAA380) contain an RGS-like domain that selectively interacts with Ga12 or Ga13 (Kozasa et al., 1998). In particular, purified p115-RhoGEF binds to and stimulates the GTPase activity of both Ga12 and Ga13, and GTPgS-bound Ga13 stimulates activity of purified p115-RhoGEF (Hart et al., 1998; Kozasa et al., 1998). Thus, p115-RhoGEF appears to serve as a direct effector for Ga13, transferring the signal from a GPCR-activated heterotrimeric G protein to the low-molecular weight G protein, Rho. This mechanism fits very well with the large body of literature showing that activation of Ga12 and Ga13 causes Rho-dependent changes in cell shape and growth properties (Sah et al., 2000). Axin, a component of the Wnt signaling system important in embryonic development, organogenesis, and cancer (Wodarz and Nusse, 1998; Peifer and Polakis, 2000), contains a functionally important RGS domain. Signals from the ligand (Wnt) and receptor (Frizzled) to downstream components such as Axin, glycogen synthase kinase 3 (GSK3), adenomatous polyposis coli protein (APC), and b-catenin are poorly understood. APC is a known tumor suppressor, and b-catenin is an oncogene. Axin, in complex with APC and GSK3, negatively regulates the transcription factor b-cateRGS Proteins as Novel Drug Targets 839 at A PE T Jornals on Jne 6, 2017 jpet.asjournals.org D ow nladed from