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AGS proteins: receptor-independent activators of G-protein signaling
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TRENDS in Pharmacological Sciences
Vol.26 No.9 September 2005
AGS proteins: receptor-independent activators of G-protein signaling Joe B. Blumer1, Mary J. Cismowski2, Motohiko Sato1 and Stephen M. Lanier1 1
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA 2 Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272, USA
The identification of AGS proteins as receptorindependent activators of G-protein signaling reveals unexpected mechanisms for the regulation of heterotrimeric G-protein activation and has opened up new areas of research related to the role of G proteins as signal transducers. In addition to their obvious interest associated with G-protein-coupled receptor signaling, AGS proteins might provide alternative binding partners for G-protein subunits that enable them to serve unexpected functions related to cell division, differentiation and organelle structure that might operate independently of a GPCR. Thus, these proteins and the concepts advanced with their discovery highlight the diversity associated with G-protein signaling and present new avenues for the development of therapeutics that target G-protein signaling.
Factors that impact on the activation state of G proteins Signal processing through heterotrimeric G proteins represents one of the most widely used systems for information transfer across the cell membrane. The initial signal, sensed by a cell-surface G-protein-coupled receptor (GPCR), is transferred to a heterotrimeric G protein, which results in the exchange of GTP for GDP on the G protein and subunit dissociation or conformational subunit rearrangement with both the GaGTP and the Gbg subunits regulating effector molecules and various aspects of receptor regulation. These events are regulated tightly to maximize signal efficiency, optimize signal specificity and integrate cellular responses to diverse stimuli. Recent surprising discoveries regarding receptorindependent activation of G proteins have opened up new areas of research in this field and provide a glimpse of G-protein biology that is generating new concepts. Two general types of G-protein regulators have been identified that either accelerate the GTPase activity of Ga [regulators of G-protein signaling (RGS)] or alter G-protein signaling by influencing nucleotide exchange or G-protein subunit interactions [1–4]. Several proteins that belong to the second type of G-protein regulators were identified in functional screens for receptor-independent activators of G-protein signaling [2,3,5–7]. These proteins have been divided into three groups based on the postulated Corresponding author: Lanier, S.M. ([email protected]). Available online 9 August 2005
mechanism by which they activated G-protein signaling in the functional screens (Table 1). Proteins encoded by cDNAs isolated in these screens were termed activators of G-protein signaling (AGS) proteins and numbered according to the order in which they were isolated. In this article, we focus specifically on AGS proteins (Box 1). The discovery platform used for identifying GPCRindependent entities that activate G-protein signaling was based on the pheromone response pathway in Saccharomyces cerevisiae, which incorporates a GPCR, a heterotrimeric G protein and a mitogen-activated protein kinase (MAPK) cascade that regulates mating and growth [8]. This system enables rapid screening of mammalian cDNAs for their ability to activate the pheromone response pathway in the absence of a receptor. The proteins encoded by cDNAs isolated using this expression cloning system and determined to require the presence of heterotrimeric G protein for bioactivity were termed AGS proteins (Table 1) and thus are functionally defined rather than defined based on conserved protein sequences. Although this strategy focused on signaling through Gia, it can probably be expanded to identify signal regulators of other classes of G proteins and to screen cDNA libraries generated from tissues in various stages of disease to isolate disease-specific post-receptor regulators of G proteins. Additional studies from several laboratories, involving protein-interaction screens [9–12], the subcellular distribution of G proteins [13–15], the analysis of conserved protein motifs [16,17] and functional studies in the model organisms Drosophila melanogaster and Caenorhabditis elegans (for review see [18]), have all contributed to the initial appreciation of alternative binding partners for G-protein subunits and the role of G proteins in cellular functions that do not apparently involve a GPCR (Figure 1). From a therapeutic perspective, studies with AGS proteins have revealed unexpected modes of G-protein regulation that might be able to be manipulated to therapeutic advantage to target heterotrimeric G proteins directly and bypass a cell-surface receptor, as suggested for RGS proteins [19–21]. Direct inhibition of G proteins is of potential value in situations where multiple receptor types are activated in the same cell and feed signals into a specific class of G proteins (e.g. hyperplasia associated with vascular remodeling, cardiac hypertrophy and smallcell lung carcinoma). In addition, selected individual AGS
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Table 1. AGS proteins isolated in the yeast-expression cloning systema Proteins encoded by cDNAb Group I (GEF) AGS1 (RASD1, DEXRAS1) Group II (GDI) AGS3 (GPSM1) AGS4 (GPSM3) AGS5 (GPSM2, LGN) AGS6 (RGS12) Group III AGS2 (TCTEX1) AGS7 AGS8
cDNA library
Ga-protein selectivity Gi2 Gi3
Gs
G16
Properties
Human liver
C
C
K
K
Ras-related
NG108-15 cells Leiomyosarcoma Leiomyosarcoma Leiomyosarcoma
C C ND ND
C C C C
K K ND ND
K K ND ND
Four GPR motifs Three GPR motifs Three GPR motifs One GPR motif
NG108-15 cells Leiomyosarcoma Rat heart
C C C
C C C
C C C
C C C
Binds Gbg Binds Gbg Binds Gbg
a Abbreviations: GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; GPSM, G-protein signal modulator; ND, not determined; C, functions in yeast expressing a given G protein; K, does not function in yeast expressing a given G protein. b AGS refers to proteins encoded by DNAs isolated in the functional screens and are numbered according to the order in which they were isolated [5–7]c. Other names for the proteins are given in parenthesis. GPSM is the name of the individual gene encoding the AGS protein as assigned by the Human Genome Nomenclature Committee. c Sato, M. et al. (2003) Identification of non-receptor activators of G-protein signaling in a functional screen of human heart and prostate leiomyosarcoma cDNAs. American Heart Association Scientific Sessions, Orlando, FL, USA; Sato, M. et al. (2004) Identification of activator of G-protein signaling 8 (AGS8) using a functional genomics screen for post-receptor signal regulators in a rat heart model of repetitive ischemia and coronary collateral growth. American Heart Association Scientific Sessions, New Orleans, LA, USA.
proteins that exhibit a restricted expression profile might be candidate drug targets for tissue-specific modulation of G-protein signaling systems. Such diverse modes of G-protein regulation also might enable subtle tuning of the system to modulate tissue responses to agonists and/or inverse agonists. Mechanisms of G-protein signal activation by AGS proteins Three groups of AGS proteins have been defined based on their mechanism of action in the yeast-based functional screen and biochemical studies. The mechanism of G-protein activation by AGS1 (Group I) is similar to that of a GPCR in terms of its ability to function as a guanine nucleotide exchange factor (GEF) for Gia/Goa, increasing GTPgS binding to free Gia2 and purified brain G protein [5,22]. AGS2–8 appear to activate heterotrimeric G-protein signaling by influencing G-protein subunit interactions independently of nucleotide exchange, in contrast to the action of AGS1, which is dependent on GDP dissociation and is antagonized by the GTPase-activating protein RGS4 [5–8]. AGS2–8 can be subgrouped based on their binding to Ga or Gbg. G-protein regulatory (GPR) motifs within AGS3–6 (Group II) preferentially bind to GiaGDP [6,7,9,23–26] whereas AGS2, 7 and 8 (Group III) bind directly to Gbg. Both of these mechanisms apparently result in an increase in the levels of free Gbg for activation of the downstream MAPK cascade and growth promotion in the modified yeast strains used for screening. AGS3–6 are characterized by the presence of one or more 20–25-amino-acid repeats termed GPR [6] or GoLoco motifs [17], which bind Gia and transducin to a greater degree than Goa, but do not bind Gsa. This motif is required for bioactivity in the yeast-based functional Box 1. Characteristics of AGS proteins † Interact with different G-protein subunits and/or conformations † Exhibit selectivity for different G proteins † Use different mechanisms to regulate the G-protein activation– deactivation cycle www.sciencedirect.com
screen. The GPR motif stabilizes the GDP-bound conformation of Ga, can promote subunit dissociation from preformed G-protein heterotrimers and competes with Gbg for binding to Ga such that a GPR protein can be complexed with free GiaGDP (i.e. dissociated from Gbg) [23–32]. Thus, the GPR motif behaves as a guanine nucleotide dissociation inhibitor (GDI) with nanomolar binding affinities [23,27,30]. In the context of the heterotrimeric Gabg activation cycle, the impact of GPR proteins on subunit interactions is probably directly related to its ability to bind preferentially and stabilize the GDP-bound conformation of Ga. The stabilization of
(b)
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(c) Diverse G-protein signals • Mediators of GPCR signaling • Vesicle trafficking and exocytosis • Golgi structure • Protein trafficking • Cell polarity • Cytokinesis? TRENDS in Pharmacological Sciences
Figure 1. Diversification of G-protein signaling. In addition to the role of G proteins in GPCR-mediated signal processing, recent studies indicate that G proteins can be activated by AGS proteins, independently of GPCR activation. This suggests that G proteins can mediate a diverse range of signals within the cell. Both types of signal can be regulated by non-receptor accessory proteins.
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the GDP-bound conformation of Ga by the GPR motif also enables the generation of a GPR–GaGDP complex that is free of Gbg, thus diversifying signal processing events regulated by the guanine nucleotide binding protein. AGS3 and AGS5 (also known as LGN) both have four GPR motifs, whereas AGS4 has three GPR motifs. The AGS6 cDNA isolated in the screen encodes a fragment of the GTPase-activating protein RGS12 that contains a single GPR motif but lacks the RGS motif. Additional GPR-containing proteins are shown in Figure 2. One interesting speculation is that AGS proteins are complexed in the cell with Ga or Gbg independently of heterotrimer formation and that Ga and Gbg might function independently of each other within the cell with AGS-like proteins serving as regulatory binding partners. Only a subpopulation of AGS and G proteins might be complexed at any given time and this association is probably compartmentalized in discrete areas of the cell [18,25,33,34]. Such complexes could target G-protein subunits to specific subcellular domains and/or cellular compartments, control the basal activity of the system or await an activating signal. These ideas will evolve as we learn more regarding the binding affinities of AGS proteins for G-protein subunits [30] and the relative amounts and subcellular location of the proteins in different tissues (Box 2). AGS proteins and signal processing Group I AGS proteins AGS1 (also known as RASD1 and DEXRAS1) is a member of the Ras superfamily of small G proteins with C-terminal and N-terminal extensions beyond the Ras core. RHES {Ras homolog enriched in striatum [also known as tumour endothelial marker (TEM2) or RASD2]} has similar extensions to AGS1 and exhibits 60% amino acid sequence identity to AGS1, defining a distinct branch within the Ras subgrouping of small G proteins. AGS1 mRNA is distributed widely in human and rat [5,35], although AGS1 protein appears to be particularly enriched in rat brain [35]. AGS1 increases GTPgS binding to purified Gi and Go. Although not yet identified in the yeast-based functional screens, additional non-receptor GEFs include
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Box 2. Characteristics of AGS and related proteins † Can influence the population of activated G protein within the cell independently of receptor activation † Provide a cell-specific mechanism for signal amplification or duration following receptor activation † Suggest unexpected modes of regulation of G-protein activation and deactivation † Provide alternative binding partners for G-protein subunits that are independent of heterotrimer formation, suggesting unexpected functions of G proteins
GAP-43 (growth-associated protein 43), Ric-8A (resistant to inhibitors of cholinesterase 8A) and the NG108-15 G-protein activator [36–38]. AGS1 was identified initially as a dexamethasoneinducible cDNA (DEXRAS1) in mouse AtT-20 corticotroph cells [39], where it is postulated to regulate peptide hormone secretion [40]. AGS1 was also identified as a binding partner of the adaptor protein carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON), which is involved in NMDA signaling by complexing with neuronal nitric oxide synthase (nNOS) [41]. Indeed, nNOS appears to activate AGS1 through S-nitrosylation of AGS1 at Cys11 [41]. This suggests a role for AGS1 in NMDA signaling and further provides a mechanism by which AGS1 links NMDA signaling pathways to heterotrimeric G proteins. AGS1 was also identified as a cycling mRNA in the suprachiasmatic nuclei of mice during the circadian rhythm [42] where it has a role in sensory processing [43]. AGS1 specifically activates the extracellular signalregulated kinase 1,2 (ERK1,2) pathway in mammalian cells and this activation is blocked by pertussis toxin pretreatment or cotransfection with Gta, which binds released Gbg [22,44]. AGS1 also inhibits cAMP accumulation in response to constitutively active Gsa or the adenylyl cyclase activator forskolin in human 293T cells [45]. A particularly interesting and surprising observation is that AGS1 blocks G-protein activation by GPCRs coupled to Gi [44,46]. A similar disruption of GPCR activation of a G-protein mediated event has been observed for RHES, with the exception that RHES modifies GPCR coupling to Gsa and not Gia [47]. AGS1
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Rap1GAP TRENDS in Pharmacological Sciences
Figure 2. Mammalian Group II AGS proteins. Domain organization of mammalian Group II AGS proteins that contain GPR motifs. GPR motifs are depicted as red boxes whereas tetrotricopeptide repeat (TPR) domains in AGS3 and AGS5 (LGN) are depicted as black boxes. Purkinje cell protein 2 (PCP2) might have one or two GPR motifs, depending on gene processing [56]. The underlined portion of AGS3 corresponds to the coding region of AGS3-short [71]. Abbreviations: PDZ, PSD95/Discs Large/ZO-1 domain; PTB, phosphotyrosine binding domain; RBD, Ras binding domain; RGS, regulator of G-protein signaling domain. www.sciencedirect.com
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TRENDS in Pharmacological Sciences
and a GPCR might compete for the available pool of G proteins or an AGS1-initiated signal might alter receptor–G-protein coupling. Group II AGS proteins Studies with the group of AGS proteins defined by the GPR motif have provided several exciting observations regarding both the biochemistry of G proteins and the expanding functional roles for G proteins. GPR-motifcontaining proteins have been isolated both from the functional yeast screen described earlier (AGS3–6) and by virtue of their ability to interact physically with Gia2 [AGS5 (LGN)] [9], Goa [PCP2-L7 (purkinje cell protein 2-L7) and Rap1GAP (Rap1 GTPase-activating protein)] or Gza (Rap1GAP) [10–12] (Figure 2). The general consensus is that the GPR motifs inhibit exchange of GDP for GTP on Gia/Goa as discussed earlier [23–31]. The majority of data indicates a clear selectivity of known GPR proteins for Gia versus Goa, although the relative affinities for Gia and Goa might differ among individual GPR proteins and additional sequences outside of the core GPR motif in the context of the full-length proteins can influence G-protein selectivity by interactions with the helical domain of Ga [26,27,48–51]. It is not known whether there is an additional ‘GPR-like’ motif in other proteins that interact with G proteins other than Gi and Go. Because GPR motifs behave as GDIs that stabilize the GDP-bound conformation of Ga, this provides an unexpected platform for signal input to G-protein signaling systems in the context of both classical GPCR signaling and alternative, receptor-independent G-protein signals. This can best be visualized in the context of the G-protein activation–deactivation cycle. First, GPR proteins could bind to G-protein heterotrimers (Gabg) and actively promote subunit dissociation, while maintaining Ga in the GDP-bound state [25,31,32,49,52]. Second, during ‘basal’ cycling of Ga through its various states of activation and inactivation or following G-protein activation by a GPCR, there is a period when GaGDP is free of Gbg enabling a GPR protein to bind GaGDP and exclude rebinding of Gbg to Ga. Both possibilities could increase the basal activity of Gbg-regulated effectors and block signaling via GaGTP. If a GPCR cannot couple to a GPR–Gia complex [49], then one would predict that GPR proteins block GPCR signaling through Gia by interfering with the availability of heterotrimeric Gabg for receptor coupling. An additional point of interest is the presence of both a GPR and a RGS (GTPase-activating protein) motif in RGS12 (AGS6) and RGS14, which provides an intramolecular mechanism for accelerating GTP hydrolysis and stabilizing GaGDP [27,53]. The degree to which either of these regulatory steps operate in mammalian cells is not yet resolved [7,53–56] and our current understanding is limited by the use of transfected systems that might not enable GPR proteins to function in their natural context. A third scenario postulates a role for GPR–Gia complexes independent of the classical heterotrimer and suggests that there is a population of Gia in the cell that exists free of Gbg. In such a case, GPR proteins could then regulate the activation state of Ga in a manner analogous to Gbg. Such GPR–Ga complexes could be regulated by www.sciencedirect.com
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entities that promote nucleotide exchange on the GPR–GaGDP complex and release ‘activated’ or ‘functional’ GiaGTP and GPR. The presence of multiple GPR motifs in AGS3, AGS4 and AGS5 (LGN) would enable the generation of a scaffold containing multiple Gia subunits free of Gbg [25,30]. Other motifs present in GPR-containing proteins [e.g. tetrotricopeptide repeat (TPR), phosphotyrosine binding (PTB) or PSD95/Discs Large/ZO-1 (PDZ) domains] (Figure 2) might function as binding scaffolds for nucleotide exchange factors or other postulated regulatory proteins such as LKB1 [also known as serine/threonine kinase 11 (STK11)], nuclear mitotic apparatus protein (NuMA) and lethal giant larvae (LGL) [33,57–59]. Group III AGS proteins In contrast to Group I and II AGS proteins, each member of the third group of AGS proteins binds to mammalian Gbg but not Ga. As such, these proteins are active in yeast strains expressing Gsa, Gia and G16a, consistent with a postulated interaction with yeast Gbg. AGS2 is identical to mouse TCTEX1, a light chain component of the cytoplasmic motor protein dynein and ciliary dynein, providing a potential regulatory role of Gbg in dynein function. AGS7 was isolated from a prostate leiomyosarcoma whereas AGS8 was isolated from a rat heart model of transient ischemia. The role of these proteins in mammalian G-protein signaling is not established, but these proteins might also serve as binding partners for a population of Gbg functioning independently of Ga. AGS proteins: function and therapeutics Although the discovery of AGS and related proteins has provided an exciting and fascinating story, the convergence of these studies with ongoing work in various model organisms exemplifies the rare occasion when research in different areas combine to provide new basic concepts in a given field. Studies with respect to G-protein signaling have involved a particularly dynamic interplay between molecular pharmacology, cell biology and in vivo function. These points are illustrated by the following: (i) AGS1 and NMDA signaling; (ii) AGS3 and the neurobiology of ‘desire’; and (iii) GPR proteins and asymmetric cell division. Initially, AGS1 emerged as a particularly intriguing signal regulator, given its upregulation in response to various challenges or in discrete physiological events, in addition to its dual function as a Ras-like protein with GEF activity for heterotrimeric G proteins. mRNA encoding AGS1 is one of a few mRNAs in suprachiasmatic nuclei that cycle with the circadian rhythm [42]. Loss of AGS1 results in dysregulation of both photic and nonphotic stimuli that involves the neurotransmitters glutamate and neuropeptide Y [43]. This provides potential context to NMDA-mediated activation of AGS1 by S-nitrosylation [41]. Of note is that this signaling cascade in the suprachiasmatic nuclei probably involves AGS1 input to Gi/Go with subsequent activation of ERK1,2, which is consistent with the earlier study demonstrating pertussis-toxin-sensitive activation of ERK1,2 by NMDA [60]. In addition to their ability to regulate heterotrimeric G proteins, AGS1 and RHES probably have functions that
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are independent of G proteins and involve effector systems that are analogous to those regulated by other Ras-related proteins. For example, AGS1 inhibits growth of NIH-3T3 cells, the MCF7 breast cancer cell line and the lung carcinoma epithelial cell line A549 and prevents the development of tumors in athymic nude mice [35]. The gene encoding AGS1 was also identified as one of 19 genes found to be downregulated in primary invasive breast ductal carcinoma and metastatic breast carcinoma isolated from a lymph node [61]. The anti-growth action of AGS1 in NIH-3T3 cells might involve the regulation of signaling pathways by AGS1 independently of its action as a GEF for heterotrimeric G proteins. Strategies directed to restore or augment AGS1 function in cancer tissue might be useful therapeutic approaches to suppress cell growth. Another surprising twist has been provided by the role of AGS3 and G-protein signaling in drug-seeking behavior [62,63]. AGS3 is upregulated in the prefrontal cortex (PFC) during withdrawal from repeated cocaine administration in rats. Subsequent studies with a membranepermeable consensus GPR peptide [62] and AGS3 antisense oligonucleotides [62,63] indicate that AGS3 has a key role in the regulation of the reinstatement of drugseeking behavior and locomotor sensitization following cocaine and heroin withdrawal. Although not completely understood, this action of AGS3 might involve modulation of a GPCR signal in the adaptation process [62,63]. The initial discovery of AGS3 also meshed with a series of elegant studies focused on asymmetric cell division in the model organisms Drosophila and C. elegans [18,64–69]. C. elegans and Drosophila each possess a gene encoding a protein with structural domains similar to that of mammalian AGS3 and AGS5 (LGN). C. elegans has two additional, nearly identical GPR proteins (GPR1 and GPR2) that have one GPR motif. These studies pointed to a role for GPR proteins and G proteins in asymmetric cell division involving an intrinsic cue that does not involve signal input through a cell-surface GPCR. In Drosophila this functionality is imparted by the partner of inscuteable (PINS), the fly ortholog of mammalian AGS3 and AGS5 (LGN). A similar ortholog exists in C. elegans but it is the GPR1 and GPR2 proteins that subserve a functional role in asymmetric cell division. In the latter case, GPR proteins and Gi/oa subunits, together with a GEF (Ric8) and a GAP (RGS7), coordinate signaling cascade(s) that differentially control pulling forces on the mitotic spindle, enabling the generation of daughter cells of a different size [64–69]. Current efforts in this field are addressing the nature of the upstream signal input and how this signaling cascade controls spindle-pulling forces. In the meantime, information obtained in studies of mammalian cells has indicated a role in microtubule dynamics for GPR proteins in symmetrically dividing cells. AGS5 (LGN) localizes to spindle poles and the midbody in non-asymmetrically dividing cells [33,34], which suggests that AGS5 (LGN) and G proteins control crucial aspects of mitosis and cytokinesis. AGS5 (LGN) is apparently targeted to the spindle poles by interaction with the NuMA and this interaction also influences the interaction of the GPR domains with G proteins [58]. The GPR motif presents a potentially interesting therapeutic target for the development of entities that www.sciencedirect.com
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mimic the effect of the GPR peptide on Ga (i.e. GPR agonists) or block the action of the GPR input without having any effect itself (GPR antagonists). Whereas the former strategy might be restricted by a requirement for localized delivery as a result of the ubiquitous expression of G proteins, the latter could offer fairly discrete intervention in G-protein signaling by virtue of the restricted expression of GPR proteins. AGS5 (LGN) appears to be expressed ubiquitously, whereas other members of this group are expressed in a cell-specific and developmentally regulated manner [6,7,9,34,70]. There is a fairly good structural foundation for understanding how the core GPR motif interacts with Gia [23,25,29,71,72], providing fertile ground for a rational approach to lead identification. Based on studies with G-protein signaling in the biology of craving, such compounds might be useful in the control of behaviors that involve excessive or diminished ‘desire’. The studies regarding GPR proteins and cell division suggest new applications for GPR ligands that inhibit cell division in the treatment of various cancers or for the control of asymmetric cell division in stem cell propagation.
Acknowledgements This work was supported by MH90531 (S.M.L.), NS24821 (S.M.L.) and F32MH65092 (J.B.B.). S.M.L. is greatly appreciative for this support and that provided by the David R. Bethune/Lederle Laboratories Professorship in Pharmacology and the Research Scholar Award from Yamanouchi Pharmaceutical Company. We acknowledge the ongoing discussions with Emir Duzic (Cephalon) and the continuous input provided during the years by the many fellows and students who have spent time in the laboratory and the many colleagues with whom we have had the pleasure of working and publishing.
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36 Strittmatter, S.M. et al. (1991) An intracellular guanine nucleotide release protein for G0. GAP-43 stimulates isolated alpha subunits by a novel mechanism. J. Biol. Chem. 266, 22465–22471 37 Tall, G.G. et al. (2003) Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor. J. Biol. Chem. 278, 8356–8362 38 Ribas, C. et al. (2002) Pertussis toxin-insensitive activation of the heterotrimeric G-proteins Gi/Go by the NG-10815 G-protein activator. J. Biol. Chem. 277, 50233–50225 39 Kemppainen, R.J. and Behrend, E.N. (1998) Dexamethasone rapidly induces a novel ras superfamily member-related gene in AtT-20 cells. J. Biol. Chem. 273, 3129–3131 40 Graham, T.E. et al. (2001) Dexras1/AGS-1, a steroid hormone-induced guanosine triphosphate-binding protein, inhibits 3 0 ,5 0 -cyclic adenosine monophosphate-stimulated secretion in AtT-20 corticotroph cells. Endocrinology 142, 2631–2640 41 Fang, M. et al. (2000) Dexras1: a G protein specifically coupled to neuronal nitric oxide synthase via CAPON. Neuron 28, 183–193 42 Takahashi, H. et al. (2003) Mouse dexamethasone-induced RAS protein 1 gene is expressed in a circadian rhythmic manner in the suprachiasmatic nucleus. Brain Res. Mol. Brain Res. 110, 1–6 43 Cheng, H.Y. et al. (2004) Dexras1 potentiates photic and suppresses nonphotic responses of the circadian clock. Neuron 43, 715–728 44 Graham, T.E. (2001) Dexras1/AGS-1 inhibits signal transduction from the Gi-coupled formyl peptide receptor to Erk-1/2 MAP kinases. J. Biol. Chem. 277, 10876–10882 45 Graham, T.E. et al. (2004) Dexras1 inhibits adenylyl cyclase. Biochem. Biophys. Res. Commun. 316, 307–312 46 Takesono, A. et al. (2002) Activator of G-protein signaling 1 blocks GIRK channel activation by a G-protein coupled receptor: Apparent disruption of receptor signaling complexes. J. Biol. Chem. 277, 13827–13830 47 Vargiu, P. et al. (2004) The small GTP-binding protein, Rhes, regulates signal transduction from G protein-coupled receptors. Oncogene 23, 559–568 48 Mittal, V. and Linder, M.E. (2004) The RGS14 GoLoco domain discriminates among Galphai isoforms. J. Biol. Chem. 279, 46772–46778 49 Ma, H. et al. (2003) Influence of cytosolic AGS3 on Receptor G-protein Coupling. Biochemistry 42, 8085–8093 50 Kimple, R.J. et al. (2004) Guanine nucleotide dissociation inhibitor activity of the triple GoLoco motif protein G18: alanine-to-aspartate mutation restores function to an inactivesecond GoLoco motif. Biochem. J. 378, 801–808 51 McCudden, C.R. et al. Galpha selectivity and inhibitor function of the multiple GoLoco motif protein GPSM2/LGN. Biochim. Biophys. Acta (in press) 52 Yu, F. et al. (2005) Locomotion defects, together with Pins, regulates heterotrimeric G-protein signaling during Drosophila neuroblast asymmetric divisions. Genes Dev. 19, 1341–1353 53 Traver, S. et al. (2004) The RGS (regulator of G-protein signalling) and GoLoco domains of RGS14 co-operate to regulate Gi-mediated signalling. Biochem. J. 379, 627–632 54 Webb, C.K. et al. (2005) D2 dopamine receptor activation of potassium channels is selectively decoupled by Galpha-specific GoLoco motif peptides. J. Neurochem. 92, 1408–1418 55 Sato, M. et al. (2004) AGS3 and signal integration by Gas and Gai coupled receptors: AGS3 blocks the sensitization of adenyly cyclase following prolonged stimulation of a Gai coupled receptor by influencing processing of Gai. J. Biol. Chem. 279, 13375–13382 56 Kinoshita-Kawada, M. et al. (2004) A Purkinje cell specific GoLoco domain protein, L7/Pcp-2, modulates receptor-mediated inhibition of Cav2.1 Ca2C channels in a dose-dependent manner. Brain Res. Mol. Brain Res. 132, 73–86 57 Blumer, J.B. et al. (2003) Interaction of AGS3 with the LKB1 kinase and phosphorylation of the GPR domains of AGS3: Phosphorylation of the GPR-motif as a regulatory mechanism for the interaction of GPR motifs with Gia. J. Biol. Chem. 278, 23217–23220 58 Du, Q. and Macara, I.G. (2004) Mammalian Pins is a conformational switch that links NuMA to heterotrimeric G proteins. Cell 119, 503–516 59 Yasumi, M. et al. (2005) Direct binding of Lgl2 to LGN during mitosis and its requirement for normal cell division. J. Biol. Chem. 280, 6761–6765
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60 Chandler, L.J. et al. (2001) N-methyl D-aspartate receptor-mediated bidirectional control of extracellular signal-regulated kinase activity in cortical neuronal cultures. J. Biol. Chem. 276, 2627–2636 61 Zucchi, I. et al. (2004) Gene expression profiles of epithelial cells microscopically isolated from a breast-invasive ductal carcinoma and a nodal metastasis. Proc. Natl. Acad. Sci. U. S. A. 101, 18147–18152 62 Bowers, M.S. et al. (2004) Activator of G-protein signaling 3: a gatekeeper of cocaine addiction. Neuron 42, 269–281 63 Yao, L. et al. (2005) Activator of G protein signaling 3 regulates opiate activation of protein kinase A signaling and relapse of heroin-seeking behavior. Proc. Natl. Acad. Sci. U. S. A. 102, 8746–8751 64 Srinivasan, D.G. et al. (2003) A complex of LIN-5 and GPR proteins regulates G protein signaling and spindle function in C. elegans. Genes Dev. 17, 1225–1239 65 Gotta, M. et al. (2003) C. elegans homologues of AGS3/PINS control spindle position in the early embryo. Curr. Biol. 3, 1029–1037 66 Colombo, K. et al. (2003) Translation of polarity cues into asymmetric spindle positioning in Caenorhabditis elegans embryos. Science 300, 1957–1961
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67 Couwenbergs, C. et al. (2004) Control of embryonic spindle positioning and Galpha activity by C. elegans RIC-8. Curr. Biol. 14, 1871–1876 68 Afshar, K. et al. (2004) RIC-8 is required for GPR-1/2-dependent Galpha function during asymmetric division of C. elegans embryos. Cell 119, 219–230 69 Hess, H.A. et al. (2004) RGS-7 completes a receptor-independent heterotrimeric G protein cycle to asymmetrically regulate mitotic spindle positioning in C. elegans. Cell 119, 209–218 70 Pizzinat, N. et al. (2001) Identification of a truncated form of the G-protein regulator AGS3 in heart that lacks the tetratricopeptide repeat domains. J. Biol. Chem. 276, 16601–16610 71 Peterson, Y.K. et al. (2002) Identification of structural features in the GPR motif required for regulation of heterotrimeric G-proteins. J. Biol. Chem. 277, 6767–6770 72 Ja, W.W. and Roberts, R.W. (2004) In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display. Biochemistry 43, 9265–9275
Drug Discovery Today: Therapeutic Strategies Editors in Chief: Ray Baker (ex-Chairman, BBSRC) and Eliot Ohlstein (Senior Vice-President, GSK) Drug Discovery Today: Therapeutic Strategies is one of a series of 4 new journals from the Drug Discovery Today stable that provides a resource of review content aligning the key output of human molecular medicine with the specific requirements of the drug discovery process, in a manner that has relevance to those working on the discovery and development of new drugs. Drug Discovery Today: Therapeutic Strategies systematically reviews each of the key disease states, and discusses the biotechnical challenges in ensuring drug action, with regard to both small molecule therapies and biopharmaceuticals (including the use of stem-cell therapy, vaccination, gene therapy, tissue modelling, and tissue and non-tissue implants) and evaluates these therapies as relevant strategies for specific diseases in specific situations. The first 2005 issue of Drug Discovery Today: Therapeutic Strategies includes sections on Genitourinary Diseases, edited by David P. Brooks and Francois Giuliano, and Addiction, edited by Leslie Iversen. The issue includes: Premature ejaculation – dopaminergic control of ejaculation Monica Levy Andersen and Sergio Tufik, pp. 41–46 Overactive bladder: targeting prostaglandins in sensory pathways Alexandra Wibberley, pp. 7–13 Preterm labour: novel treatments Joanna E. Gullam, Jayanta Chatterjee and Steve Thornton, pp. 47–52 Pharmacotherapy of alcohol dependence: targeting a complex disorder George A. Kenna, pp. 71–78 Medications for the treatment of cocaine addiction: emerging candidates Barbara H. Herman, Ahmed Elkashef and Frank Vocci, pp. 87–92 Full-text can be viewed through ScienceDirect (www.sciencedirect.com); alternatively find out more about the journals and how to subscribe at www.drugdiscoverytoday.com www.sciencedirect.com
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AGS proteins: receptor-independent activators of G-protein signaling Joe B. Blumer1, Mary J. Cismowski2, Motohiko Sato1 and Stephen M. Lanier1 1
Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA 2 Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH 44272, USA
The identification of AGS proteins as receptorindependent activators of G-protein signaling reveals unexpected mechanisms for the regulation of heterotrimeric G-protein activation and has opened up new areas of research related to the role of G proteins as signal transducers. In addition to their obvious interest associated with G-protein-coupled receptor signaling, AGS proteins might provide alternative binding partners for G-protein subunits that enable them to serve unexpected functions related to cell division, differentiation and organelle structure that might operate independently of a GPCR. Thus, these proteins and the concepts advanced with their discovery highlight the diversity associated with G-protein signaling and present new avenues for the development of therapeutics that target G-protein signaling.
Factors that impact on the activation state of G proteins Signal processing through heterotrimeric G proteins represents one of the most widely used systems for information transfer across the cell membrane. The initial signal, sensed by a cell-surface G-protein-coupled receptor (GPCR), is transferred to a heterotrimeric G protein, which results in the exchange of GTP for GDP on the G protein and subunit dissociation or conformational subunit rearrangement with both the GaGTP and the Gbg subunits regulating effector molecules and various aspects of receptor regulation. These events are regulated tightly to maximize signal efficiency, optimize signal specificity and integrate cellular responses to diverse stimuli. Recent surprising discoveries regarding receptorindependent activation of G proteins have opened up new areas of research in this field and provide a glimpse of G-protein biology that is generating new concepts. Two general types of G-protein regulators have been identified that either accelerate the GTPase activity of Ga [regulators of G-protein signaling (RGS)] or alter G-protein signaling by influencing nucleotide exchange or G-protein subunit interactions [1–4]. Several proteins that belong to the second type of G-protein regulators were identified in functional screens for receptor-independent activators of G-protein signaling [2,3,5–7]. These proteins have been divided into three groups based on the postulated Corresponding author: Lanier, S.M. ([email protected]). Available online 9 August 2005
mechanism by which they activated G-protein signaling in the functional screens (Table 1). Proteins encoded by cDNAs isolated in these screens were termed activators of G-protein signaling (AGS) proteins and numbered according to the order in which they were isolated. In this article, we focus specifically on AGS proteins (Box 1). The discovery platform used for identifying GPCRindependent entities that activate G-protein signaling was based on the pheromone response pathway in Saccharomyces cerevisiae, which incorporates a GPCR, a heterotrimeric G protein and a mitogen-activated protein kinase (MAPK) cascade that regulates mating and growth [8]. This system enables rapid screening of mammalian cDNAs for their ability to activate the pheromone response pathway in the absence of a receptor. The proteins encoded by cDNAs isolated using this expression cloning system and determined to require the presence of heterotrimeric G protein for bioactivity were termed AGS proteins (Table 1) and thus are functionally defined rather than defined based on conserved protein sequences. Although this strategy focused on signaling through Gia, it can probably be expanded to identify signal regulators of other classes of G proteins and to screen cDNA libraries generated from tissues in various stages of disease to isolate disease-specific post-receptor regulators of G proteins. Additional studies from several laboratories, involving protein-interaction screens [9–12], the subcellular distribution of G proteins [13–15], the analysis of conserved protein motifs [16,17] and functional studies in the model organisms Drosophila melanogaster and Caenorhabditis elegans (for review see [18]), have all contributed to the initial appreciation of alternative binding partners for G-protein subunits and the role of G proteins in cellular functions that do not apparently involve a GPCR (Figure 1). From a therapeutic perspective, studies with AGS proteins have revealed unexpected modes of G-protein regulation that might be able to be manipulated to therapeutic advantage to target heterotrimeric G proteins directly and bypass a cell-surface receptor, as suggested for RGS proteins [19–21]. Direct inhibition of G proteins is of potential value in situations where multiple receptor types are activated in the same cell and feed signals into a specific class of G proteins (e.g. hyperplasia associated with vascular remodeling, cardiac hypertrophy and smallcell lung carcinoma). In addition, selected individual AGS
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Table 1. AGS proteins isolated in the yeast-expression cloning systema Proteins encoded by cDNAb Group I (GEF) AGS1 (RASD1, DEXRAS1) Group II (GDI) AGS3 (GPSM1) AGS4 (GPSM3) AGS5 (GPSM2, LGN) AGS6 (RGS12) Group III AGS2 (TCTEX1) AGS7 AGS8
cDNA library
Ga-protein selectivity Gi2 Gi3
Gs
G16
Properties
Human liver
C
C
K
K
Ras-related
NG108-15 cells Leiomyosarcoma Leiomyosarcoma Leiomyosarcoma
C C ND ND
C C C C
K K ND ND
K K ND ND
Four GPR motifs Three GPR motifs Three GPR motifs One GPR motif
NG108-15 cells Leiomyosarcoma Rat heart
C C C
C C C
C C C
C C C
Binds Gbg Binds Gbg Binds Gbg
a Abbreviations: GDI, guanine nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange factor; GPSM, G-protein signal modulator; ND, not determined; C, functions in yeast expressing a given G protein; K, does not function in yeast expressing a given G protein. b AGS refers to proteins encoded by DNAs isolated in the functional screens and are numbered according to the order in which they were isolated [5–7]c. Other names for the proteins are given in parenthesis. GPSM is the name of the individual gene encoding the AGS protein as assigned by the Human Genome Nomenclature Committee. c Sato, M. et al. (2003) Identification of non-receptor activators of G-protein signaling in a functional screen of human heart and prostate leiomyosarcoma cDNAs. American Heart Association Scientific Sessions, Orlando, FL, USA; Sato, M. et al. (2004) Identification of activator of G-protein signaling 8 (AGS8) using a functional genomics screen for post-receptor signal regulators in a rat heart model of repetitive ischemia and coronary collateral growth. American Heart Association Scientific Sessions, New Orleans, LA, USA.
proteins that exhibit a restricted expression profile might be candidate drug targets for tissue-specific modulation of G-protein signaling systems. Such diverse modes of G-protein regulation also might enable subtle tuning of the system to modulate tissue responses to agonists and/or inverse agonists. Mechanisms of G-protein signal activation by AGS proteins Three groups of AGS proteins have been defined based on their mechanism of action in the yeast-based functional screen and biochemical studies. The mechanism of G-protein activation by AGS1 (Group I) is similar to that of a GPCR in terms of its ability to function as a guanine nucleotide exchange factor (GEF) for Gia/Goa, increasing GTPgS binding to free Gia2 and purified brain G protein [5,22]. AGS2–8 appear to activate heterotrimeric G-protein signaling by influencing G-protein subunit interactions independently of nucleotide exchange, in contrast to the action of AGS1, which is dependent on GDP dissociation and is antagonized by the GTPase-activating protein RGS4 [5–8]. AGS2–8 can be subgrouped based on their binding to Ga or Gbg. G-protein regulatory (GPR) motifs within AGS3–6 (Group II) preferentially bind to GiaGDP [6,7,9,23–26] whereas AGS2, 7 and 8 (Group III) bind directly to Gbg. Both of these mechanisms apparently result in an increase in the levels of free Gbg for activation of the downstream MAPK cascade and growth promotion in the modified yeast strains used for screening. AGS3–6 are characterized by the presence of one or more 20–25-amino-acid repeats termed GPR [6] or GoLoco motifs [17], which bind Gia and transducin to a greater degree than Goa, but do not bind Gsa. This motif is required for bioactivity in the yeast-based functional Box 1. Characteristics of AGS proteins † Interact with different G-protein subunits and/or conformations † Exhibit selectivity for different G proteins † Use different mechanisms to regulate the G-protein activation– deactivation cycle www.sciencedirect.com
screen. The GPR motif stabilizes the GDP-bound conformation of Ga, can promote subunit dissociation from preformed G-protein heterotrimers and competes with Gbg for binding to Ga such that a GPR protein can be complexed with free GiaGDP (i.e. dissociated from Gbg) [23–32]. Thus, the GPR motif behaves as a guanine nucleotide dissociation inhibitor (GDI) with nanomolar binding affinities [23,27,30]. In the context of the heterotrimeric Gabg activation cycle, the impact of GPR proteins on subunit interactions is probably directly related to its ability to bind preferentially and stabilize the GDP-bound conformation of Ga. The stabilization of
(b)
(a) Classical paradigm of G-protein signaling
Unexpected signal input to G proteins
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Figure 1. Diversification of G-protein signaling. In addition to the role of G proteins in GPCR-mediated signal processing, recent studies indicate that G proteins can be activated by AGS proteins, independently of GPCR activation. This suggests that G proteins can mediate a diverse range of signals within the cell. Both types of signal can be regulated by non-receptor accessory proteins.
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the GDP-bound conformation of Ga by the GPR motif also enables the generation of a GPR–GaGDP complex that is free of Gbg, thus diversifying signal processing events regulated by the guanine nucleotide binding protein. AGS3 and AGS5 (also known as LGN) both have four GPR motifs, whereas AGS4 has three GPR motifs. The AGS6 cDNA isolated in the screen encodes a fragment of the GTPase-activating protein RGS12 that contains a single GPR motif but lacks the RGS motif. Additional GPR-containing proteins are shown in Figure 2. One interesting speculation is that AGS proteins are complexed in the cell with Ga or Gbg independently of heterotrimer formation and that Ga and Gbg might function independently of each other within the cell with AGS-like proteins serving as regulatory binding partners. Only a subpopulation of AGS and G proteins might be complexed at any given time and this association is probably compartmentalized in discrete areas of the cell [18,25,33,34]. Such complexes could target G-protein subunits to specific subcellular domains and/or cellular compartments, control the basal activity of the system or await an activating signal. These ideas will evolve as we learn more regarding the binding affinities of AGS proteins for G-protein subunits [30] and the relative amounts and subcellular location of the proteins in different tissues (Box 2). AGS proteins and signal processing Group I AGS proteins AGS1 (also known as RASD1 and DEXRAS1) is a member of the Ras superfamily of small G proteins with C-terminal and N-terminal extensions beyond the Ras core. RHES {Ras homolog enriched in striatum [also known as tumour endothelial marker (TEM2) or RASD2]} has similar extensions to AGS1 and exhibits 60% amino acid sequence identity to AGS1, defining a distinct branch within the Ras subgrouping of small G proteins. AGS1 mRNA is distributed widely in human and rat [5,35], although AGS1 protein appears to be particularly enriched in rat brain [35]. AGS1 increases GTPgS binding to purified Gi and Go. Although not yet identified in the yeast-based functional screens, additional non-receptor GEFs include
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Box 2. Characteristics of AGS and related proteins † Can influence the population of activated G protein within the cell independently of receptor activation † Provide a cell-specific mechanism for signal amplification or duration following receptor activation † Suggest unexpected modes of regulation of G-protein activation and deactivation † Provide alternative binding partners for G-protein subunits that are independent of heterotrimer formation, suggesting unexpected functions of G proteins
GAP-43 (growth-associated protein 43), Ric-8A (resistant to inhibitors of cholinesterase 8A) and the NG108-15 G-protein activator [36–38]. AGS1 was identified initially as a dexamethasoneinducible cDNA (DEXRAS1) in mouse AtT-20 corticotroph cells [39], where it is postulated to regulate peptide hormone secretion [40]. AGS1 was also identified as a binding partner of the adaptor protein carboxyl-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON), which is involved in NMDA signaling by complexing with neuronal nitric oxide synthase (nNOS) [41]. Indeed, nNOS appears to activate AGS1 through S-nitrosylation of AGS1 at Cys11 [41]. This suggests a role for AGS1 in NMDA signaling and further provides a mechanism by which AGS1 links NMDA signaling pathways to heterotrimeric G proteins. AGS1 was also identified as a cycling mRNA in the suprachiasmatic nuclei of mice during the circadian rhythm [42] where it has a role in sensory processing [43]. AGS1 specifically activates the extracellular signalregulated kinase 1,2 (ERK1,2) pathway in mammalian cells and this activation is blocked by pertussis toxin pretreatment or cotransfection with Gta, which binds released Gbg [22,44]. AGS1 also inhibits cAMP accumulation in response to constitutively active Gsa or the adenylyl cyclase activator forskolin in human 293T cells [45]. A particularly interesting and surprising observation is that AGS1 blocks G-protein activation by GPCRs coupled to Gi [44,46]. A similar disruption of GPCR activation of a G-protein mediated event has been observed for RHES, with the exception that RHES modifies GPCR coupling to Gsa and not Gia [47]. AGS1
AGS3
TPR
AGS4
GPR
AGS5 (LGN) PCP2 RGS12 RGS14
PDZ RGS
PTB
RGS
RBD
RBD
Rap1GAP TRENDS in Pharmacological Sciences
Figure 2. Mammalian Group II AGS proteins. Domain organization of mammalian Group II AGS proteins that contain GPR motifs. GPR motifs are depicted as red boxes whereas tetrotricopeptide repeat (TPR) domains in AGS3 and AGS5 (LGN) are depicted as black boxes. Purkinje cell protein 2 (PCP2) might have one or two GPR motifs, depending on gene processing [56]. The underlined portion of AGS3 corresponds to the coding region of AGS3-short [71]. Abbreviations: PDZ, PSD95/Discs Large/ZO-1 domain; PTB, phosphotyrosine binding domain; RBD, Ras binding domain; RGS, regulator of G-protein signaling domain. www.sciencedirect.com
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and a GPCR might compete for the available pool of G proteins or an AGS1-initiated signal might alter receptor–G-protein coupling. Group II AGS proteins Studies with the group of AGS proteins defined by the GPR motif have provided several exciting observations regarding both the biochemistry of G proteins and the expanding functional roles for G proteins. GPR-motifcontaining proteins have been isolated both from the functional yeast screen described earlier (AGS3–6) and by virtue of their ability to interact physically with Gia2 [AGS5 (LGN)] [9], Goa [PCP2-L7 (purkinje cell protein 2-L7) and Rap1GAP (Rap1 GTPase-activating protein)] or Gza (Rap1GAP) [10–12] (Figure 2). The general consensus is that the GPR motifs inhibit exchange of GDP for GTP on Gia/Goa as discussed earlier [23–31]. The majority of data indicates a clear selectivity of known GPR proteins for Gia versus Goa, although the relative affinities for Gia and Goa might differ among individual GPR proteins and additional sequences outside of the core GPR motif in the context of the full-length proteins can influence G-protein selectivity by interactions with the helical domain of Ga [26,27,48–51]. It is not known whether there is an additional ‘GPR-like’ motif in other proteins that interact with G proteins other than Gi and Go. Because GPR motifs behave as GDIs that stabilize the GDP-bound conformation of Ga, this provides an unexpected platform for signal input to G-protein signaling systems in the context of both classical GPCR signaling and alternative, receptor-independent G-protein signals. This can best be visualized in the context of the G-protein activation–deactivation cycle. First, GPR proteins could bind to G-protein heterotrimers (Gabg) and actively promote subunit dissociation, while maintaining Ga in the GDP-bound state [25,31,32,49,52]. Second, during ‘basal’ cycling of Ga through its various states of activation and inactivation or following G-protein activation by a GPCR, there is a period when GaGDP is free of Gbg enabling a GPR protein to bind GaGDP and exclude rebinding of Gbg to Ga. Both possibilities could increase the basal activity of Gbg-regulated effectors and block signaling via GaGTP. If a GPCR cannot couple to a GPR–Gia complex [49], then one would predict that GPR proteins block GPCR signaling through Gia by interfering with the availability of heterotrimeric Gabg for receptor coupling. An additional point of interest is the presence of both a GPR and a RGS (GTPase-activating protein) motif in RGS12 (AGS6) and RGS14, which provides an intramolecular mechanism for accelerating GTP hydrolysis and stabilizing GaGDP [27,53]. The degree to which either of these regulatory steps operate in mammalian cells is not yet resolved [7,53–56] and our current understanding is limited by the use of transfected systems that might not enable GPR proteins to function in their natural context. A third scenario postulates a role for GPR–Gia complexes independent of the classical heterotrimer and suggests that there is a population of Gia in the cell that exists free of Gbg. In such a case, GPR proteins could then regulate the activation state of Ga in a manner analogous to Gbg. Such GPR–Ga complexes could be regulated by www.sciencedirect.com
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entities that promote nucleotide exchange on the GPR–GaGDP complex and release ‘activated’ or ‘functional’ GiaGTP and GPR. The presence of multiple GPR motifs in AGS3, AGS4 and AGS5 (LGN) would enable the generation of a scaffold containing multiple Gia subunits free of Gbg [25,30]. Other motifs present in GPR-containing proteins [e.g. tetrotricopeptide repeat (TPR), phosphotyrosine binding (PTB) or PSD95/Discs Large/ZO-1 (PDZ) domains] (Figure 2) might function as binding scaffolds for nucleotide exchange factors or other postulated regulatory proteins such as LKB1 [also known as serine/threonine kinase 11 (STK11)], nuclear mitotic apparatus protein (NuMA) and lethal giant larvae (LGL) [33,57–59]. Group III AGS proteins In contrast to Group I and II AGS proteins, each member of the third group of AGS proteins binds to mammalian Gbg but not Ga. As such, these proteins are active in yeast strains expressing Gsa, Gia and G16a, consistent with a postulated interaction with yeast Gbg. AGS2 is identical to mouse TCTEX1, a light chain component of the cytoplasmic motor protein dynein and ciliary dynein, providing a potential regulatory role of Gbg in dynein function. AGS7 was isolated from a prostate leiomyosarcoma whereas AGS8 was isolated from a rat heart model of transient ischemia. The role of these proteins in mammalian G-protein signaling is not established, but these proteins might also serve as binding partners for a population of Gbg functioning independently of Ga. AGS proteins: function and therapeutics Although the discovery of AGS and related proteins has provided an exciting and fascinating story, the convergence of these studies with ongoing work in various model organisms exemplifies the rare occasion when research in different areas combine to provide new basic concepts in a given field. Studies with respect to G-protein signaling have involved a particularly dynamic interplay between molecular pharmacology, cell biology and in vivo function. These points are illustrated by the following: (i) AGS1 and NMDA signaling; (ii) AGS3 and the neurobiology of ‘desire’; and (iii) GPR proteins and asymmetric cell division. Initially, AGS1 emerged as a particularly intriguing signal regulator, given its upregulation in response to various challenges or in discrete physiological events, in addition to its dual function as a Ras-like protein with GEF activity for heterotrimeric G proteins. mRNA encoding AGS1 is one of a few mRNAs in suprachiasmatic nuclei that cycle with the circadian rhythm [42]. Loss of AGS1 results in dysregulation of both photic and nonphotic stimuli that involves the neurotransmitters glutamate and neuropeptide Y [43]. This provides potential context to NMDA-mediated activation of AGS1 by S-nitrosylation [41]. Of note is that this signaling cascade in the suprachiasmatic nuclei probably involves AGS1 input to Gi/Go with subsequent activation of ERK1,2, which is consistent with the earlier study demonstrating pertussis-toxin-sensitive activation of ERK1,2 by NMDA [60]. In addition to their ability to regulate heterotrimeric G proteins, AGS1 and RHES probably have functions that
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are independent of G proteins and involve effector systems that are analogous to those regulated by other Ras-related proteins. For example, AGS1 inhibits growth of NIH-3T3 cells, the MCF7 breast cancer cell line and the lung carcinoma epithelial cell line A549 and prevents the development of tumors in athymic nude mice [35]. The gene encoding AGS1 was also identified as one of 19 genes found to be downregulated in primary invasive breast ductal carcinoma and metastatic breast carcinoma isolated from a lymph node [61]. The anti-growth action of AGS1 in NIH-3T3 cells might involve the regulation of signaling pathways by AGS1 independently of its action as a GEF for heterotrimeric G proteins. Strategies directed to restore or augment AGS1 function in cancer tissue might be useful therapeutic approaches to suppress cell growth. Another surprising twist has been provided by the role of AGS3 and G-protein signaling in drug-seeking behavior [62,63]. AGS3 is upregulated in the prefrontal cortex (PFC) during withdrawal from repeated cocaine administration in rats. Subsequent studies with a membranepermeable consensus GPR peptide [62] and AGS3 antisense oligonucleotides [62,63] indicate that AGS3 has a key role in the regulation of the reinstatement of drugseeking behavior and locomotor sensitization following cocaine and heroin withdrawal. Although not completely understood, this action of AGS3 might involve modulation of a GPCR signal in the adaptation process [62,63]. The initial discovery of AGS3 also meshed with a series of elegant studies focused on asymmetric cell division in the model organisms Drosophila and C. elegans [18,64–69]. C. elegans and Drosophila each possess a gene encoding a protein with structural domains similar to that of mammalian AGS3 and AGS5 (LGN). C. elegans has two additional, nearly identical GPR proteins (GPR1 and GPR2) that have one GPR motif. These studies pointed to a role for GPR proteins and G proteins in asymmetric cell division involving an intrinsic cue that does not involve signal input through a cell-surface GPCR. In Drosophila this functionality is imparted by the partner of inscuteable (PINS), the fly ortholog of mammalian AGS3 and AGS5 (LGN). A similar ortholog exists in C. elegans but it is the GPR1 and GPR2 proteins that subserve a functional role in asymmetric cell division. In the latter case, GPR proteins and Gi/oa subunits, together with a GEF (Ric8) and a GAP (RGS7), coordinate signaling cascade(s) that differentially control pulling forces on the mitotic spindle, enabling the generation of daughter cells of a different size [64–69]. Current efforts in this field are addressing the nature of the upstream signal input and how this signaling cascade controls spindle-pulling forces. In the meantime, information obtained in studies of mammalian cells has indicated a role in microtubule dynamics for GPR proteins in symmetrically dividing cells. AGS5 (LGN) localizes to spindle poles and the midbody in non-asymmetrically dividing cells [33,34], which suggests that AGS5 (LGN) and G proteins control crucial aspects of mitosis and cytokinesis. AGS5 (LGN) is apparently targeted to the spindle poles by interaction with the NuMA and this interaction also influences the interaction of the GPR domains with G proteins [58]. The GPR motif presents a potentially interesting therapeutic target for the development of entities that www.sciencedirect.com
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mimic the effect of the GPR peptide on Ga (i.e. GPR agonists) or block the action of the GPR input without having any effect itself (GPR antagonists). Whereas the former strategy might be restricted by a requirement for localized delivery as a result of the ubiquitous expression of G proteins, the latter could offer fairly discrete intervention in G-protein signaling by virtue of the restricted expression of GPR proteins. AGS5 (LGN) appears to be expressed ubiquitously, whereas other members of this group are expressed in a cell-specific and developmentally regulated manner [6,7,9,34,70]. There is a fairly good structural foundation for understanding how the core GPR motif interacts with Gia [23,25,29,71,72], providing fertile ground for a rational approach to lead identification. Based on studies with G-protein signaling in the biology of craving, such compounds might be useful in the control of behaviors that involve excessive or diminished ‘desire’. The studies regarding GPR proteins and cell division suggest new applications for GPR ligands that inhibit cell division in the treatment of various cancers or for the control of asymmetric cell division in stem cell propagation.
Acknowledgements This work was supported by MH90531 (S.M.L.), NS24821 (S.M.L.) and F32MH65092 (J.B.B.). S.M.L. is greatly appreciative for this support and that provided by the David R. Bethune/Lederle Laboratories Professorship in Pharmacology and the Research Scholar Award from Yamanouchi Pharmaceutical Company. We acknowledge the ongoing discussions with Emir Duzic (Cephalon) and the continuous input provided during the years by the many fellows and students who have spent time in the laboratory and the many colleagues with whom we have had the pleasure of working and publishing.
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Drug Discovery Today: Therapeutic Strategies Editors in Chief: Ray Baker (ex-Chairman, BBSRC) and Eliot Ohlstein (Senior Vice-President, GSK) Drug Discovery Today: Therapeutic Strategies is one of a series of 4 new journals from the Drug Discovery Today stable that provides a resource of review content aligning the key output of human molecular medicine with the specific requirements of the drug discovery process, in a manner that has relevance to those working on the discovery and development of new drugs. Drug Discovery Today: Therapeutic Strategies systematically reviews each of the key disease states, and discusses the biotechnical challenges in ensuring drug action, with regard to both small molecule therapies and biopharmaceuticals (including the use of stem-cell therapy, vaccination, gene therapy, tissue modelling, and tissue and non-tissue implants) and evaluates these therapies as relevant strategies for specific diseases in specific situations. The first 2005 issue of Drug Discovery Today: Therapeutic Strategies includes sections on Genitourinary Diseases, edited by David P. Brooks and Francois Giuliano, and Addiction, edited by Leslie Iversen. The issue includes: Premature ejaculation – dopaminergic control of ejaculation Monica Levy Andersen and Sergio Tufik, pp. 41–46 Overactive bladder: targeting prostaglandins in sensory pathways Alexandra Wibberley, pp. 7–13 Preterm labour: novel treatments Joanna E. Gullam, Jayanta Chatterjee and Steve Thornton, pp. 47–52 Pharmacotherapy of alcohol dependence: targeting a complex disorder George A. Kenna, pp. 71–78 Medications for the treatment of cocaine addiction: emerging candidates Barbara H. Herman, Ahmed Elkashef and Frank Vocci, pp. 87–92 Full-text can be viewed through ScienceDirect (www.sciencedirect.com); alternatively find out more about the journals and how to subscribe at www.drugdiscoverytoday.com www.sciencedirect.com