G-protein signaling modulators (GPSM) play diverse functional roles through their interaction with G-protein subunits. AGS3 (GPSM1) contains four G-protein regulatory motifs (GPR) that directly bind G␣ i free of G␤␥ providing an unusual scaffold for the "G-switch" and signaling complexes, but the mechanism by which signals track into this scaffold are not well understood. We report the regulation of the AGS3⅐ G␣ i signaling module by a cell surface, seven-transmembrane receptor. AGS3 and G␣ i1 tagged with Renilla luciferase or yellow fluorescent protein expressed in mammalian cells exhibited saturable, specific bioluminescence resonance energy transfer indicating complex formation in the cell. Activation of ␣ 2 -adrenergic receptors or -opioid receptors reduced AGS3-RLuc⅐G␣ i1 -YFP energy transfer by over 30%. The agonist-mediated effects were inhibited by pertussis toxin and co-expression of RGS4, but were not altered by G␤␥ sequestration with the carboxyl terminus of GRK2. G␣ i -dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to G␣ i and/or G␣ o indicating that AGS3 is part of a larger signaling complex. Upon receptor activation, AGS3 reversibly dissociates from this complex at the cell cortex. Receptor coupling to both G␣␤␥ and GPR-G␣ i offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

Activator of G-protein signaling-4 (AGS4), via its three G-protein regulatory motifs, is well positioned to modulate G-protein signal processing by virtue of its ability to bind G␣ i -GDP subunits free of G␤␥. Apart from initial observations on the biochemical activity of the G-protein regulatory motifs of AGS4, very little is known about the nature of the AGS4-G-protein interaction, how this interaction is regulated, or where the interaction takes place. As an initial approach to these questions, we evaluated the interaction of AGS4 with G␣ i1 in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and G␣ i1 reciprocally tagged with either Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) demonstrated saturable, specific BRET signals. BRET signals observed between AGS4-RLuc and G␣ i1 -YFP were reduced by G-protein-coupled receptor activation, and this agonist-induced reduction in BRET was blocked by pertussis toxin. In addition, specific BRET signals were observed for AGS4-RLuc and ␣ 2 -adrenergic receptor-Venus, which were G␣ i -dependent and reduced by agonist, indicating that AGS4-G␣ i complexes are receptor-proximal. These data suggest that AGS4-G␣ i complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation. Activators of G-protein signaling (AGS)3 proteins were identified using a yeast-based functional screen of mammalian cDNA libraries for cDNAs that activated G-protein signaling in the absence of a GPCR (1-4). Group II AGS proteins all contain at least one G-protein regulatory (GPR) motif (3, 5) (also termed the GoLoco motif (6)), a 20 -25-amino acid motif that binds and stabilizes the GDP-bound conformation of G␣ i / o / t and competes with G␤␥ for G␣ binding (reviewed in Ref. 5). Proteins with multiple GPR motifs can bind to multiple G␣ subunits simultaneously, which presents a unique opportunity to act as a scaffold to organize a signaling complex (7,8).Functional studies indicate crucial roles for GPR proteins beginning with the original observations in model organisms describing a role for GPR proteins and their interaction with G-proteins in asymmetric cell division (5, 9). Additional functional studies with GPR proteins indicate further functional diversity with roles observed in blood pressure control, fat deposition and energy expenditure, neuronal outgrowth, drug addiction and relapse behavior, autophagy, G-protein-coupled inwardly rectifying potassium channel regulation, and transport of membrane proteins to the cell surface (10 -18). These observations indicate crucial functionality of the GPR motif in biological systems and implicate G␣ i -GPR complexes in the regulation of G-protein signaling in unexpected, albeit poorly understood ways. In the context of the group II AGS proteins, which contain multiple GPR motifs, many outstanding questions remain to be addressed. Chief among them is what regulates the formation and disassembly of GPR-G␣ i complexes? Is their interaction with G-protein in...

Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein ␣ subunits. Co-expression of G␣ subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted G␣ protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein ␣ subunit purification that was applicable to all G␣ subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein G␣ olf for the first time and unprecedented yield of G␣ q and G␣ 13 . G␣ subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8⅐G␣ complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. G␣ subunits were dissociated from GST-Ric-8 with GDP-AlF 4 ؊ (GTP mimicry) and found to be >80% pure, bind guanosine 5-[␥-thio]triphosphate (GTP␥S), and stimulate appropriate G protein effector enzymes. A primary characterization of G␣ olf showed that it binds GTP␥S at a rate marginally slower than G␣ s short and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than G␣ s short .Heterotrimeric G proteins are the foremost signal-transducing molecules used by G protein-coupled-receptors (GPCRs) 3 to regulate sensation and cellular physiology. Agonist-stimulated GPCRs are guanine nucleotide exchange factors that stimulate G protein ␣ subunit (G␣) GDP release. Subsequent GTP binding to G␣ causes heterotrimer dissociation or rearrangement so that G␣-GTP and G␤␥ adopt states for efficient activation of downstream effector enzymes. Purified G protein subunits have been essential reagents used to develop the current understanding of G protein function, structure, and signaling pathways (1, 2). Current knowledge of traditional G protein signaling network complexity is expanding, and G proteins have been assigned new nontraditional signaling roles including regulation of cell division through unique classes of effector and modulatory enzymes (3-5). As cross-disciplinary G protein research proliferates, the need for purified components to elucidate G protein functionality is significant.G protein heterotrimers are classified by the identity of the guanine nucleotide-binding subunit: G␣. There are four classes of G␣ subunits: G␣ s , G␣ i , G␣ q , and G␣ 12/13 . Efficient procedures are in place to produce most G␣ i class subunits and G␣ s from Escherichia coli (6, 7). Members of the G␣ q and G␣ 12/13 classes can be prepared from an insect cell expression system using a G␤␥ co-purification procedure. This method involves tagging the G␥ subunit with a His 6 tag, isolating the trimeric G protein by metal chelate chromatography, and eluting the G␣ with high specificity using GTP mimicry. This method is tried and true but rather laborious and involves extensive steps of cell membrane preparation, washing, and detergent extraction. The procedure also results in low G␣ yields (Յ50 -200 g of protein/liter of cell culture) (8 -11). To ...

Beyond the core triad of receptor, Gαβγ and effector, there are multiple accessory proteins that provide alternative modes of signal input and regulatory adaptability to G-protein signalling systems. Such accessory proteins may segregate a signalling complex to microdomains of the cell, regulate the basal activity, efficiency and specificity of signal propagation and/or serve as alternative binding partners for Gα or Gβγ independent of the classical heterotrimeric Gαβγ complex. The latter concept led to the postulate that Gα and Gβγ regulate intracellular events distinct from their role as transducers for cell surface seven-transmembrane span receptors. One general class of such accessory proteins is defined by AGS proteins or activators of G-protein signalling that refer to mammalian cDNAs identified in a specific yeast-based functional screen. The discovery of AGS proteins and related entities revealed a number of unexpected mechanisms for regulation of G-protein signalling systems and expanded functional roles for this important signalling system.

No abstract