Stephen M. Lanier

Receptor-independent Activators of Heterotrimeric G-protein Signaling Pathways

Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-proteinsignaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. β-galactosidase reporter assays in yeast strains expressing different Gα subunits (Gpa1, Gsα, Giα2 (Gpa1(1–41)), Giα3(Gpa1(1–41)), Gα16(Gpa1(1–41))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the Giα2 and Giα3genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gβγ, whereas AGS3 bound Gα and exhibited a preference for GαGDP versus GαGTPγS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Gα and Gβγ that allow the subunits to subserve functions that do not require initial heterotrimer formation.

Activator of G protein signaling 3: a gatekeeper of cocaine sensitization and drug seeking.

Chronic cocaine administration reduces G protein signaling efficacy. Here, we report that the expression of AGS3, which binds to GialphaGDP and inhibits GDP dissociation, was upregulated in the prefrontal cortex (PFC) during late withdrawal from repeated cocaine administration. Increased AGS3 was mimicked in the PFC of drug-naive rats by microinjecting a peptide containing the Gialpha binding domain (GPR) of AGS3 fused to the cell permeability domain of HIV-Tat. Infusion of Tat-GPR mimicked the phenotype of chronic cocaine-treated rats by manifesting sensitized locomotor behavior and drug seeking and by increasing glutamate transmission in nucleus accumbens. By preventing cocaine withdrawal-induced AGS3 expression with antisense oligonucleotides, signaling through Gialpha was normalized, and both cocaine-induced relapse to drug seeking and locomotor sensitization were prevented. When antisense oligonucleotide infusion was discontinued, drug seeking and sensitization were restored. It is proposed that AGS3 gates the expression of cocaine-induced plasticity by regulating G protein signaling in the PFC.

Asymmetrically distributed C. elegans homologs of AGS3/PINS control Spindle position in the early embryo

BACKGROUND Spindle positioning during an asymmetric cell division is of fundamental importance to ensure correct size of daughter cells and segregation of determinants. In the C. elegans embryo, the first spindle is asymmetrically positioned, and this asymmetry is controlled redundantly by two heterotrimeric Galpha subunits, GOA-1 and GPA-16. The Galpha subunits act downstream of the PAR polarity proteins, which control the relative pulling forces acting on the poles. How these heterotrimeric G proteins are regulated and how they control spindle position is still unknown. RESULTS Here we show that the Galpha subunits are regulated by a receptor-independent mechanism. RNAi depletion of gpr-1 and gpr-2, homologs of mammalian AGS3 and Drosophila PINS (receptor-independent G protein regulators), results in a phenotype identical to that of embryos depleted of both GPA-16 and GOA-1; the first cleavage is symmetric, but polarity is not affected. The loss of spindle asymmetry after RNAi of gpr-1 and gpr-2 appears to be the result of weakened pulling forces acting on the poles. The GPR protein(s) localize around the cortex of one-cell embryos and are enriched at the posterior. Thus, asymmetric G protein regulation could explain the posterior displacement of the spindle. Posterior enrichment is abolished in the absence of the PAR polarity proteins PAR-2 or PAR-3. In addition, LIN-5, a coiled-coil protein also required for spindle positioning, binds to and is required for cortical association of the GPR protein(s). Finally, we show that the GPR domain of GPR-1 and GPR-2 behaves as a GDP dissociation inhibitor for GOA-1, and its activity is thus similar to that of mammalian AGS3. CONCLUSIONS Our results suggest that GPR-1 and/or GPR-2 control an asymmetry in forces exerted on the spindle poles by asymmetrically modulating the activity of the heterotrimeric G protein in response to a signal from the PAR proteins.

Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.

We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways. Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Gα hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified. One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Gα. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.

Interaction of Arrestins with Intracellular Domains of Muscarinic and α2-Adrenergic Receptors

The intracellular domains of G-protein-coupled receptors provide sites for interaction with key proteins involved in signal initiation and termination. As an initial approach to identify proteins interacting with these receptors and the receptor motifs required for such interactions, we used intracellular subdomains of G-protein-coupled receptors as probes to screen brain cytosol proteins. Peptides from the third intracellular loop (i3) of the M2-muscarinic receptor (MR) (His208–Arg387), M3-MR (Gly308–Leu497), or α2A/D-adrenergic receptor (AR) (Lys224–Phe374) were generated in bacteria as glutathione S-transferase (GST) fusion proteins, bound to glutathione-Sepharose and used as affinity matrices to detect interacting proteins in fractionated bovine brain cytosol. Bound proteins were identified by immunoblotting following SDS-polyacrylamide gel electrophoresis. Brain arrestins bound to the GST-M3fusion protein, but not to the control GST peptide or i3 peptides derived from the α2A/D-AR and M2-MR. However, each of the receptor subdomains bound purified β-arrestin and arrestin-3. The interaction of the M3-MR and M2-MR i3 peptides with arrestins was further investigated. The M3-MR i3 peptide bound in vitro translated [3H]β-arrestin and [3H]arrestin-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction ofin vitro translated [3H]β-arrestin, [3H]visual arrestin, and [3H]β-arrestin/visual arrestin chimeras with the M2-MR i3 peptide were similar to those observed with the intact purified M2-MR that was phosphorylated and/or activated by agonist. Subsequent binding site localization studies indicated that the interaction of β-arrestin with the M3-MR peptide required both the amino (Gly308–Leu368) and carboxyl portions (Lys425–Leu497) of the receptor subdomain. In contrast, the carboxyl region of the M3-MR i3 peptide was sufficient for its interaction with arrestin-3.

The elusive family of imidazoline binding sites

Various imidazoline and guanidinium derivatives elicit diverse cellular responses in peripheral and nervous tissues that are often difficult to attribute to known receptor signalling systems. Biochemical, functional and clinical evidence suggests that some activities of these compounds may be related to their action on defined imidazoline binding sites, which have been recently characterized. Unexpectedly, and of particular significance, recent data indicate that two members of the family of imidazoline binding sites are identical to the A and B isoforms of monoamine oxidase. In this article, Angelo Parini and colleagues summarize the evidence for the characterization and location of imidazoline binding sites, and speculate on the clinical implications of compounds acting on these sites.

Activation of heterotrimeric G-protein signaling by a ras-related protein. Implications for signal integration.

Utilizing a functional screen in the yeastSaccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion. One of the identified activators, termed AGS1 (for activator of G-proteinsignaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily. Expression of AGS1 in yeast and in mammalian cells results in specific activation of Gαi/Gαo heterotrimeric signaling pathways. In addition, the in vivo and in vitroproperties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for Gαi/Gαo. AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways.

Stabilization of the GDP-bound conformation of Gialpha by a peptide derived from the G-protein regulatory motif of AGS3

The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein α subunits. To define the properties of this ∼20-amino acid motif, we designed a GPR consensus peptide and determined its influence on the activation state of G-protein and receptor coupling to G-protein. The GPR peptide sequence (28 amino acids) encompassed the consensus sequence defined by the four GPR motifs conserved in the family of AGS3 proteins. The GPR consensus peptide effectively prevented the binding of AGS3 to Giα1,2 in protein interaction assays, inhibited guanosine 5′-O-(3-thiotriphosphate) binding to Giα, and stabilized the GDP-bound conformation of Giα. The GPR peptide had little effect on nucleotide binding to Goα and brain G-protein indicating selective regulation of Giα. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Giα. The GPR consensus peptide also blocked receptor coupling to Giαβγ indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Giα, this conformation of GiαGDPwas not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Giα- and Gβγ−regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems.

AGS3 Inhibits GDP Dissociation from Gα Subunits of the Gi Family and Rhodopsin-dependent Activation of Transducin

A number of recently discovered proteins that interact with the α subunits of Gi-like G proteins contain homologous repeated sequences named G protein regulatory (GPR) motifs. Activator of G protein signaling 3 (AGS3), identified as an activator of the yeast pheromone pathway in the absence of the pheromone receptor, has a domain with four such repeats. To elucidate the potential mechanisms of regulation of G protein signaling by proteins containing GPR motifs, we examined the effects of the AGS3 GPR domain on the kinetics of guanine nucleotide exchange and GTP hydrolysis by Giα1 and transducin-α (Gtα). The AGS3 GPR domain markedly inhibited the rates of spontaneous guanosine 5′-O-(3-thiotriphosphate) (GTPγS) binding to Giα and rhodopsin-stimulated GTPγS binding to Gtα. The full-length AGS3 GPR domain, AGS3-(463–650), was ∼30-fold more potent than AGS3-(572–629), containing two AGS3 GPR motifs. The IC50values for the AGS3-(463–650) inhibitory effects on Giα and transducin were 0.12 and 0.15 μm, respectively. Furthermore, AGS3-(463–650) and AGS3-(572–629) effectively blocked the GDP release from Giα and rhodopsin-induced dissociation of GDP from Gtα. The potencies of AGS3-(572–629) and AGS3-(463–650) to suppress the GDP dissociation rates correlated with their ability to inhibit the rates of GTPγS binding. Consistent with the inhibition of nucleotide exchange, the AGS3 GPR domain slowed the rate of steady-state GTP hydrolysis by Giα. The catalytic rate of Gtα GTP hydrolysis, measured under single turnover conditions, remained unchanged with the addition of AGS3-(463–650). Altogether, our results suggest that proteins containing GPR motifs, in addition to their potential role as G protein-coupled receptor-independent activators of Gβγ signaling pathways, act as GDP dissociation inhibitors and negatively regulate the activation of a G protein by a G protein-coupled receptor.

Receptor-independent activators of heterotrimeric G-proteins

Heterotrimeric G-protein signalling systems are primarily activated via cell surface receptors possessing the seven membrane span motif. Several observations suggest the existence of other modes of input to such signalling systems either downstream of effectors or at the level of G-proteins themselves. Using a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae, we identified three proteins, AGS1-3 (for Activators of G-protein Signalling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor. AGS1 defines a distinct member of the super family of ras related proteins. AGS2 is identical to mouse Tctex1, a protein that exists as a light chain component of the cytoplasmic motor protein dynein and subserves as yet undefined functions in cell signalling pathways. AGS3 possesses a series of tetratrico repeat motifs and a series of four amino acid repeats termed G-protein regulatory motifs. The GPR motifs are found in a number of proteins that interact with and regulate Galpha. Although each AGS protein activates G-protein signaling, they do so by different mechanisms within the context of the G-protein activation/deactivation cycle. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signalling pathways.