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Activator of G-protein Signaling 3 (AGS3), a receptor independent activator of G-protein signaling, oscillates among different subcellular compartments in a regulated manner including punctate entities referred to as biomolecular condensates (BMCs). The dynamics of the AGS3 oscillation and the specific subcompartment within the cell is intimately related to the functional diversity of the protein. To further address the properties and regulation of AGS3 BMCs, we asked initial questions regarding a) the distribution of AGS3 across the broader BMC landscape with and without cellular stress, and b) the core material properties of these punctate structures. Cellular stress (oxidative, pHi, thermal) induced the formation of AGS3 BMCs in two cell lines (Hela, COS7) as determined by fluorescent microscopy. The AGS3-BMCs generated in response to oxidative stress were distinct from stress granules (SG) as defined by the SG marker protein G3BP1 and RNA processing BMCs defined by the P-body protein Dcp1a. Immunoblots of fractionated cell lysates indicated that cellular stress shifted AGS3 to the membrane pellet fraction, whereas the protein markers for stress granules (G3BP1) SG- BMCs remained in the supernatant. We next asked if the formation of the stress-induced AGS3 BMCs was regulated by protein binding partners involved with signal processing. The stress-induced generation of AGS3 BMCs was regulated by the signaling protein Gαi3, but not by the AGS3 binding partner DVL2. Finally, we addressed the fluidity or rigidity of the stress-induced AGS3-BMCs using fluorescent recovery following photobleaching of individual AGS3-BMCs. The AGS3-BMCs indicated distinct diffusion kinetics that were consistent with restricted mobility of AGS3 within the stress-induced AGS3-BMCs. These data suggest that AGS3 BMCs represents a distinct class of stress granules that define a new type of BMC that may serve as previously unappreciated signal processing nodes.Summary statementAGS3 assembles into distinct biomolecular condensates in response to cell stress and this assembly is selectively regulated by AGS3 binding partners involved in signal transduction within the cell.

Activator of G-protein signaling 3 (AGS3, encoded by GPSM1) was discovered as a one of several receptor-independent activators of G-protein signaling, which are postulated to provide a platform for divergence between canonical and noncanonical G-protein signaling pathways. Similarly, Dishevelled (DVL) proteins serve as a point of divergence for β-catenin-dependent and -independent signaling pathways involving the family of Frizzled (FZD) ligands and cell-surface WNT receptors. We recently discovered the apparent regulated localization of dishevelled-2 (DVL2) and AGS3 to distinct cellular puncta, suggesting that the two proteins interact as part of various cell signaling systems. To address this hypothesis, we asked the following questions: (1) do AGS3 signaling pathways influence the activation of β-catenin (CTNNB1)-regulated transcription through the WNT–Frizzled–Dishevelled axis, and (2) is the AGS3 and DVL2 interaction regulated? The interaction of AGS3 and DVL2 was regulated by protein phosphorylation, subcellular distribution, and a cell-surface G-protein-coupled receptor. These data, and the commonality of functional system impacts observed for AGS3 and DVL2, suggest that the AGS3–DVL2 complex presents an unexpected path for functional integration within the cell. This article has an associated First Person interview with the first author of the paper.

Activator of G-protein signaling 3 (AGS3, also known as GPSM1) exhibits broad functional diversity and oscillates among different subcellular compartments in a regulated manner. AGS3 consists of a tetratricopeptide repeat (TPR) domain and a G-protein regulatory (GPR) domain. Here, we tested the hypothesis that phosphorylation of the AGS3 GPR domain regulates its subcellular distribution and functionality. In contrast to the cortical and/or diffuse non-homogeneous distribution of wild-type (WT) AGS3, an AGS3 construct lacking all 24 potential phosphorylation sites in the GPR domain localized to cytosolic puncta. This change in localization was revealed to be dependent upon phosphorylation of a single threonine amino acid (T602). The punctate distribution of AGS3-T602A was rescued by co-expression of Gα(i) and Gα(o) but not Gα(s) or Gα(q). Following treatment with alkaline phosphatase, both AGS3-T602A and WT AGS3 exhibited a gel shift in SDS-PAGE as compared to untreated WT AGS3, consistent with a loss of protein phosphorylation. The punctate distribution of AGS3-T602A was lost in an AGS3-A602T conversion mutant, but was still present upon T602 mutation to glutamate or aspartate. These results implicate dynamic phosphorylation as a discrete mechanism to regulate the subcellular distribution of AGS3 and associated functionality.

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Activator of G-protein signaling 3 (AGS3, also known as GPSM1) exhibits broad functional diversity and oscillates among different subcellular compartments in a regulated manner. AGS3 consists of a tetratricopeptide repeat (TPR) domain and a G-protein regulatory (GPR) domain. Here, we tested the hypothesis that phosphorylation of the AGS3 GPR domain regulates its subcellular distribution and functionality. In contrast to the cortical and/or diffuse non-homogeneous distribution of wild-type (WT) AGS3, an AGS3 construct lacking all 24 potential phosphorylation sites in the GPR domain localized to cytosolic puncta. This change in localization was revealed to be dependent upon phosphorylation of a single threonine amino acid (T602). The punctate distribution of AGS3-T602A was rescued by co-expression of Gαi and Gαo but not Gαs or Gαq Following treatment with alkaline phosphatase, both AGS3-T602A and WT AGS3 exhibited a gel shift in SDS-PAGE as compared to untreated WT AGS3, consistent with a loss of protein phosphorylation. The punctate distribution of AGS3-T602A was lost in an AGS3-A602T conversion mutant, but was still present upon T602 mutation to glutamate or aspartate. These results implicate dynamic phosphorylation as a discrete mechanism to regulate the subcellular distribution of AGS3 and associated functionality.

Group II activator of G-protein signaling (AGS) proteins contain one or more G-protein regulatory motifs (GPR), which serve as docking sites for GαiGDP independent of Gβγ and stabilize the GDP-bound conformation of Gαi, acting as guanine nucleotide dissociation inhibitors. The GαGPR interaction is regulated by seven-transmembrane-spanning (7TM) receptors in the intact cell as determined by bioluminescence resonance energy transfer (BRET). It is hypothesized that a 7TM receptor directly couples to the GαGPR complex in a manner analogous to receptor coupling to the Gαβγ heterotrimer. As an initial approach to test this hypothesis, we used BRET to examine 7TM receptor-mediated regulation of GαGPR in the intact cell when Gαi2 yellow fluorescent protein (YFP) was tethered to the carboxyl terminus of the α2A adrenergic receptor (α2AAR-Gαi2YFP). AGS3- and AGS4-Renilla luciferase (Rluc) exhibited robust BRET with the tethered GαiYFP, and this interaction was regulated by receptor activation localizing the regulation to the receptor microenvironment. Agonist regulation of the receptor-Gαi-GPR complex was also confirmed by coimmunoprecipitation and cell fractionation. The tethered Gαi2 was rendered pertussis toxin-insensitive by a C352I mutation, and receptor coupling to endogenous Gαi/oβγ was subsequently eliminated by cell treatment with pertussis toxin (PT). Basal and agonist-induced regulation of α2AAR-Gαi2YFP(C352I):AGS3Rluc and α2AAR-Gαi2YFP(C352I):AGS4Rluc BRET was not altered by PT treatment or Gβγ antagonists. Thus, the localized regulation of GαGPR by receptor activation appears independent of endogenous Gαi/oβγ, suggesting that GαiAGS3 and GαiAGS4 directly sense agonist-induced conformational changes in the receptor, as is the case for 7TM receptor coupling to the Gαβγ heterotrimer. The direct coupling of a receptor to the GαiGPR complex provides an unexpected platform for signal propagation with broad implications.

Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.

Activators of G protein signaling (AGS), initially discovered in the search for receptor-independent activators of G protein signaling, define a broad panel of biologic regulators that influence signal transfer from receptor to G-protein, guanine nucleotide binding and hydrolysis, G protein subunit interactions, and/or serve as alternative binding partners for Gα and Gβγ independently of the classic heterotrimeric Gαβγ. AGS proteins generally fall into three groups based upon their interaction with and regulation of G protein subunits: group I, guanine nucleotide exchange factors (GEF); group II, guanine nucleotide dissociation inhibitors; and group III, entities that bind to Gβγ. Group I AGS proteins can engage all subclasses of G proteins, whereas group II AGS proteins primarily engage the Gi/Go/transducin family of G proteins. A fourth group of AGS proteins with selectivity for Gα16 may be defined by the Mitf-Tfe family of transcription factors. Groups I-III may act in concert, generating a core signaling triad analogous to the core triad for heterotrimeric G proteins (GEF + G proteins + effector). These two core triads may function independently of each other or actually cross-integrate for additional signal processing. AGS proteins have broad functional roles, and their discovery has advanced new concepts in signal processing, cell and tissue biology, receptor pharmacology, and system adaptation, providing unexpected platforms for therapeutic and diagnostic development.

The proto-oncogene and inhibitor of protein phosphatase 2A (PP2A), SET, interacts with the third intracellular loop of the M3 muscarinic receptor (M3-MR), and SET knockdown with small interfering RNA (siRNA) in Chinese hamster ovary (CHO) cells augments M3-MR signaling. However, the mechanism of this action of SET on receptor signaling has not been defined, and we initiated studies to address this question. Knockdown of SET by siRNA in CHO cells stably expressing the M3-MR did not alter agonist-induced receptor phosphorylation or receptor internalization. Instead, it increased the extent of receptor dephosphorylation after agonist removal by ∼60%. In competition binding assays, SET knockdown increased high-affinity binding of agonist in intact cells and membrane preparations. Glutathione transferase pull-down assays and site-directed mutagenesis revealed a SET binding site adjacent to and perhaps overlapping the G protein-binding site within the third intracellular loop of the receptor. Mutation of this region in the M3-MR altered receptor coupling to G protein. These data indicate that SET decreases M3-MR dephosphorylation and regulates receptor engagement with G protein, both of which may contribute to the inhibitory action of SET on M3-MR signaling.

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.

Receptor-independent G-protein regulators provide diverse mechanisms for signal input to G-protein-based signaling systems, revealing unexpected functional roles for G-proteins. As part of a broader effort to identify disease-specific regulators for heterotrimeric G-proteins, we screened for such proteins in cardiac hypertrophy using a yeast-based functional screen of mammalian cDNAs as a discovery platform. We report the identification of three transcription factors belonging to the same family, transcription factor E3 (TFE3), microphthalmia-associated transcription factor, and transcription factor EB, as novel receptor-independent activators of G-protein signaling selective for Gα16. TFE3 and Gα16 were both up-regulated in cardiac hypertrophy initiated by transverse aortic constriction. In protein interaction studies in vitro, TFE3 formed a complex with Gα16 but not with Gαi3 or Gαs. Although increased expression of TFE3 in heterologous systems had no influence on receptor-mediated Gα16 signaling at the plasma membrane, TFE3 actually translocated Gα16 to the nucleus, leading to the induction of claudin 14 expression, a key component of membrane structure in cardiomyocytes. The induction of claudin 14 was dependent on both the accumulation and activation of Gα16 by TFE3 in the nucleus. These findings indicate that TFE3 and Gα16 are up-regulated under pathologic conditions and are involved in a novel mechanism of transcriptional regulation via the relocalization and activation of Gα16.

The intracellular mechanisms underlying renal tubular epithelial cell proliferation and tubular repair following ischemia-reperfusion injury (IRI) remain poorly understood. In this report, we demonstrate that activator of G-protein signaling 3 (AGS3), an unconventional receptor-independent regulator of heterotrimeric G-protein function, influences renal tubular regeneration following IRI. In rat kidneys exposed to IRI, there was a temporal induction in renal AGS3 protein expression that peaked 72 h after reperfusion and corresponded to the repair and recovery phase following ischemic injury. Renal AGS3 expression was localized predominantly to the recovering outer medullary proximal tubular cells and was highly coexpressed with Ki-67, a marker of cell proliferation. Kidneys from mice deficient in the expression of AGS3 exhibited impaired renal tubular recovery 7 d following IRI compared to wild-type AGS3-expressing mice. Mechanistically, genetic knockdown of endogenous AGS3 mRNA and protein in renal tubular epithelial cells reduced cell proliferation in vitro. Similar reductions in renal tubular epithelial cell proliferation were observed following incubation with gallein, a selective inhibitor of Gβγ subunit activity, and lentiviral overexpression of the carboxyl-terminus of G-protein-coupled receptor kinase 2 (GRK2ct), a scavenger of Gβγ subunits. In summary, these data suggest that AGS3 acts through a novel receptor-independent mechanism to facilitate renal tubular epithelial cell proliferation and renal tubular regeneration.—Regner, K. R., Nozu, K., Lanier, S. M., Blumer, J. B., Avner, E. D., Sweeney, Jr., W. E., Park, F. Loss of activator of G-protein signaling 3 impairs renal tubular regeneration following acute kidney injury in rodents.

Ric-8A and Ric-8B are nonreceptor G protein guanine nucleotide exchange factors that collectively bind the four subfamilies of G protein α subunits. Co-expression of Gα subunits with Ric-8A or Ric-8B in HEK293 cells or insect cells greatly promoted Gα protein expression. We exploited these characteristics of Ric-8 proteins to develop a simplified method for recombinant G protein α subunit purification that was applicable to all Gα subunit classes. The method allowed production of the olfactory adenylyl cyclase stimulatory protein Gαolf for the first time and unprecedented yield of Gαq and Gα13. Gα subunits were co-expressed with GST-tagged Ric-8A or Ric-8B in insect cells. GST-Ric-8·Gα complexes were isolated from whole cell detergent lysates with glutathione-Sepharose. Gα subunits were dissociated from GST-Ric-8 with GDP-AlF4− (GTP mimicry) and found to be >80% pure, bind guanosine 5′-[γ-thio]triphosphate (GTPγS), and stimulate appropriate G protein effector enzymes. A primary characterization of Gαolf showed that it binds GTPγS at a rate marginally slower than Gαs short and directly activates adenylyl cyclase isoforms 3, 5, and 6 with less efficacy than Gαs short.

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 Gα(i) free of Gβγ 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 AGS3·Gα(i) signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Gα(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 α(2)-adrenergic receptors or μ-opioid receptors reduced AGS3-RLuc·Gα(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 Gβγ sequestration with the carboxyl terminus of GRK2. Gα(i)-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to Gα(i) and/or Gα(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 Gαβγ and GPR-Gα(i) offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

AGS3, a receptor-independent activator of G-protein signaling, is involved in unexpected functional diversity for G-protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G-protein regulatory (GPR) motifs that serve as docking sites for Gialpha-GDP. The positioning of AGS3 within the cell and the intramolecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G-protein signaling. We report that AGS3 enters into the aggresome pathway and that distribution of the protein is regulated by the AGS3 binding partners Gialpha and mammalian Inscuteable (mInsc). Gialpha rescues AGS3 from the aggresome, whereas mInsc augments the aggresome-like distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain, and it is accelerated by disruption of the TPR organizational structure or introduction of a nonsynonymous single-nucleotide polymorphism. These data present AGS3, G-proteins, and mInsc as candidate proteins involved in regulating cellular stress associated with protein-processing pathologies.

Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein system. Here, we report a role of the ischemia-inducible regulator of Gβγ subunit, AGS8, in survival of cardiomyocytes under hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia or hypoxia/reoxygenation following transfection of AGS8siRNA or pcDNA::AGS8. Hypoxia-induced apoptosis of NCM was completely blocked by AGS8siRNA, whereas overexpression of AGS8 increased apoptosis. AGS8 formed complexes with G-proteins and channel protein connexin 43 (CX43), which regulates the permeability of small molecules under hypoxic stress. AGS8 initiated CX43 phosphorylation in a Gβγ-dependent manner by providing a scaffold composed of Gβγ and CX43. AGS8siRNA blocked internalization of CX43 following exposure of NCM to repetitive hypoxia; however it did not influence epidermal growth factor-mediated internalization of CX43. The decreased dye flux through CX43 that occurred with hypoxic stress was also prevented by AGS8siRNA. Interestingly, the Gβγ inhibitor Gallein mimicked the effect of AGS8 knockdown on both the CX43 internalization and the changes in cell permeability elicited by hypoxic stress. These data indicate that AGS8 is required for hypoxia-induced apoptosis of NCM, and that AGS8-Gβγ signal input increased the sensitivity of cells to hypoxic stress by influencing CX43 regulation and associated cell permeability. Under hypoxic stress, this unrecognized response program plays a critical role in the fate of NCM.

Activator of G protein signaling (AGS)-3 plays functional roles in cell division, synaptic plasticity, addictive behavior, and neuronal development. As part of a broad effort to define the extent of functional diversity of AGS3-regulated-events in vivo, we generated AGS3 null mice. Surprisingly, AGS3 null adult mice exhibited unexpected alterations in cardiovascular and metabolic functions without any obvious changes in motor skills, basic behavioral traits, and brain morphology. AGS3 null mice exhibited a lean phenotype, reduced fat mass, and increased nocturnal energy expenditure. AGS3 null mice also exhibited altered blood pressure control mechanisms. These studies expand the functional repertoire for AGS3 and other G protein regulatory proteins providing unexpected mechanisms by which G protein systems may be targeted to influence obesity and cardiovascular function.

Signal processing via heterotrimeric G-proteins in response to cell surface receptors is a central and much investigated aspect of how cells integrate cellular stimuli to produce coordinated biological responses. The system is a target of numerous therapeutic agents and plays an important role in adaptive processes of organs; aberrant processing of signals through these transducing systems is a component of various disease states. In addition to G-protein coupled receptor (GPCR)-mediated activation of G-protein signaling, nature has evolved creative ways to manipulate and utilize the Galphabetagamma heterotrimer or Galpha and Gbetagamma subunits independent of the cell surface receptor stimuli. In such situations, the G-protein subunits (Galpha and Gbetagamma) may actually be complexed with alternative binding partners independent of the typical heterotrimeric Galphabetagamma. Such regulatory accessory proteins include the family of regulator of G-protein signaling (RGS) proteins that accelerate the GTPase activity of Galpha and various entities that influence nucleotide binding properties and/or subunit interaction. The latter group of proteins includes receptor-independent activators of G-protein signaling (AGS) proteins that play surprising roles in signal processing. This review provides an overview of our current knowledge regarding AGS proteins. AGS proteins are indicative of a growing number of accessory proteins that influence signal propagation, facilitate cross talk between various types of signaling pathways, and provide a platform for diverse functions of both the heterotrimeric Galphabetagamma and the individual Galpha and Gbetagamma subunits.

We addressed the role of the G-protein regulatory (GPR) motif-containing Leu-Gly-Asn-enriched protein (LGN) and G-proteins (Gialpha3) in the positioning of the spindle pole during mammalian cell division. Immunocytochemistry indicated that both LGN and Gialpha3 co-localized at the spindle pole and at the midbody and the cell cortex during the different phases of mitosis. In marked contrast to the positioning of the spindle pole at metaphase midway between the cell cortex and the metaphase plate, the spindle pole was juxtaposed with the cell cortex at metaphase following increased expression of Gialpha3 and LGN. This repositioning of the spindle pole required the interaction of LGN with Gialpha. The influence of LGN and Gialpha3 on the cortical positioning of the spindle pole likely reflects either stronger pulling forces on the spindle pole exerted from the cell cortex or increased pushing forces exerted on the spindle pole from the mitotic spindle indicating that these events are regulated by GPR motif-containing proteins and G-proteins independent of asymmetry.

We have recently reported that lipid structure regulates the interaction with membranes, recruitment to membranes, and distribution to membrane domains of heterotrimeric Galphabetagamma proteins, Galpha subunits, and Gbetagamma dimers (J Biol Chem 279:36540-36545, 2004). Here, we demonstrate that modulation of the membrane structure not only determines G protein localization but also regulates the function of G proteins and related signaling proteins. In this context, the antitumor drug daunorubicin (daunomycin) and oleic acid changed the membrane structure and inhibited G protein activity in biological membranes. They also induced marked changes in the activity of the alpha(2A/D)-adrenergic receptor and adenylyl cyclase. In contrast, elaidic and stearic acid did not change the activity of the above-mentioned proteins. These fatty acids are chemical but not structural analogs of oleic acid, supporting the structural basis of the modulation of membrane lipid organization and subsequent regulation of G protein-coupled receptor signaling. In addition, oleic acid (and also daunorubicin) did not alter G protein activity in a membrane-free system, further demonstrating the involvement of membrane structure in this signal modulation. The present work also unravels in part the molecular bases involved in the antihypertensive (Hypertension 43:249-254, 2004) and anticancer (Mol Pharmacol 67:531-540, 2005) activities of synthetic oleic acid derivatives (e.g., 2-hydroxyoleic acid) as well as the molecular bases of the effects of diet fats on human health.

A long term objective of our research effort is to define factors that influence the specificity and efficiency of signal propagation by heterotrimeric G-proteins (G). G-proteins play a central role in cellular communication mediating the cell response to numerous hormones and neurotransmitters. A major determinant of signalling specificity for heterotrimeric G-proteins is the cell specific expression of the subtypes of the primary signalling entities, receptor, G and effector (E). Another major site for regulating signalling specificity lies at the R-G or G-E interface where these interactions are influenced by cell architecture, the stoichiometry of signalling components and accessory proteins that may segregate the receptor to microdomains of the cell, regulate the efficiency and/or specificity of signal transfer and/or influence the activation state of G-protein independent of a classical G-protein coupled receptor. One strategy to address these issues in our laboratory involves the identification of cellular proteins that regulate the transfer of signal from receptor to G or directly influence the activation state of G independent of a classical G-protein coupled receptor. We identified three proteins, AGS1, AGS2 and AGS3 (for Activators of G-protein Signaling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor. AGS1, 2 and 3 interact with different subunits and/or conformations of heterotrimeric G-proteins, selectively activate different G-proteins, provide unexpected mechanisms for regulation of the G-protein activation cycle and have opened up a new area of research related to the cellular role of G-proteins as signal transducers.

Activators of G-protein signaling 1-3 (AGS1-3) were identified in a functional screen of mammalian cDNAs that activated G-protein signaling in the absence of a receptor. We report the isolation and characterization of an additional AGS protein (AGS4) from a human prostate leiomyosarcoma cDNA library. AGS4 is identical to G18.1b, which is encoded by a gene within the major histocompatibility class III region of chromosome 6. The activity of AGS4 in the yeast-based functional screen was selective for Gi2/Gi3 and independent of guanine-nucleotide exchange by Giα. RNA blots indicated enrichment of AGS4/G18.1b mRNA in heart, placenta, lung, and liver. Immunocytochemistry with AGS4/G18.1b-specific antisera indicated a predominant nonhomogeneous, extranuclear distribution within the cell following expression in COS7 or Chinese hamster ovary cells. AGS4/G18.1b contains three G-protein regulatory motifs downstream of an amino terminus domain with multiple prolines. Glutathione S-transferase (GST)-AGS4/G18.1b fusion proteins interacted with purified Giα, and peptides derived from each of the G-protein regulatory motifs inhibited guanosine 5′-3-O-(thio)triphosphate (GTPγS) binding to purified Giα1. AGS4/G18.1b was also complexed with Giα3 in COS7 cell lysates following cell transfection. However, AGS4/G18.1b did not alter the generation of inositol phosphates in COS7 cells cotransfected with the Gβγ-regulated effector phospholipase C-β2. These data suggest either that an additional signal is required to position AGS4/G18.1b in the proper cellular location where it can access heterotrimer and promote subunit dissociation or that AGS4 serves as an alternative binding partner for Giα independent of Gβγ participating in G-protein signaling events that are independent of classical G-protein-coupled receptors at the cell surface.

AGS3-LONG and AGS3-SHORT contain G-protein regulatory motifs that interact with and stabilize the GDP-bound conformation of Gαi > Gαo. AGS3 and related proteins may influence signal strength or duration as well as the adaptation of the signaling system associated with sustained stimulation. To address these issues, we determined the effect of AGS3 on the integration of stimulatory (Gαs-mediated vasoactive intestinal peptide receptor) and inhibitory (Gαi-mediated α2-adrenergic receptor (α2-AR)) signals to adenylyl cyclase in Chinese hamster ovary cells. AGS3-SHORT and AGS3-LONG did not alter the VIP-induced increase in cAMP or the inhibitory effect of α2-AR activation. System adaptation was addressed by determining the influence of AGS3 on the sensitization of adenylyl cyclase that occurs following prolonged activation of a Gαi-coupled receptor. Incubation of cells with the α2-AR agonist UK14304 (1 μm) for 18 h resulted in a ∼1.8-fold increase in the vasoactive intestinal peptide-induced activation of adenylyl cyclase, and this was associated with a decrease in membrane-associated Gαi3. Both effects were blocked by AGS3-SHORT. AGS3-SHORT also decreased the rate of Gαi3 decay. A mutant AGS3-SHORT incapable of binding G-protein was inactive. These data suggest that AGS3 and perhaps other G-protein regulatory motif-containing proteins increase the stability of Gαi in the membrane, which influences the adaptation of the cell to prolonged activation of Gαi-coupled receptors.

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.

Activator of G protein signaling 3 (AGS3) activates the Gbetagamma mating pathway in yeast in a manner that is independent of heptahelical receptors. It competes with Gbetagamma subunits to bind GDP-bound Gi/o(alpha) subunits via four repeated G protein regulatory (GPR) domains in the carboxyl-terminal half of the molecule. However, little is known about the functional role of AGS3 in cellular signaling. Here the effect of AGS3 on receptor-G protein coupling was examined in an Sf9 cell membrane-based reconstitution system. A GST-AGS3-GPR fusion protein containing the four individual AGS3-GPR domains inhibits receptor coupling to Galpha subunits as effectively as native AGS3 and more effectively than GST fusion proteins containing the individual AGS3-GPR domains. While none of the GPR domains distinguished among the three G(i)alpha subunits, both individual and full-length GPR domains interacted more weakly with G(o)alpha than with G(i)alpha. Cytosolic AGS3, but not membrane-associated AGS3, can interact with G(i)alpha subunits and disrupt their receptor coupling. Immunoblotting studies reveal that cytosolic AGS3 can remove G(i)alpha subunits from the membrane and sequester G(i)alpha subunits in the cytosol. These findings suggest that AGS3 may downregulate heterotrimeric G protein signaling by interfering with receptor coupling.

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 Gα subunits, GOA-1 and GPA-16. The Gα 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 Gα 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.

Activator of G-protein signaling 3 (AGS3) has a modular domain structure consisting of seven tetratricopeptide repeats (TPRs) and four G-protein regulatory (GPR) motifs. Each GPR motif binds to the α subunit of Gi/Go (Giα > Goα) stabilizing the GDP-bound conformation of Gα and apparently competing with Gβγ for GαGDP binding. As an initial approach to identify regulatory mechanisms for AGS3-G-protein interactions, a yeast two-hybrid screen was initiated using the TPR and linker region of AGS3 as bait. This screen identified the serine/threonine kinase LKB1, which is involved in the regulation of cell cycle progression and polarity. Protein interaction assays in mammalian systems using transfected cells or brain lysate indicated the regulated formation of a protein complex consisting of LKB1, AGS3, and G-proteins. The interaction between AGS3 and LKB1 was also observed with orthologous proteins in Drosophila where both proteins are involved in cell polarity. LKB1 immunoprecipitates from COS7 cells transfected with LKB1 phosphorylated the GPR domains of AGS3 and the related protein LGN but not the AGS3-TPR domain. GPR domain phosphorylation was completely blocked by a consensus GPR motif peptide, and placement of a phosphate moiety within a consensus GPR motif reduced the ability of the peptide to interact with G-proteins. These data suggest that phosphorylation of GPR domains may be a general mechanism regulating the interaction of GPR-containing proteins with G-proteins. Such a mechanism may be of particular note in regard to localized signal processing in the plasma membrane involving G-protein subunits and/or intracellular functions regulated by heterotrimeric G-proteins that occur independently of a typical G-protein-coupled receptor.

Heterotrimeric G proteins are key transducers for signal transfer from outside of the cell. In addition to their regulation by the superfamily of G protein-coupled receptors, many if not all of the subtypes of heterotrimeric G proteins are also regulated by additional accessory proteins that influence guanine nucleotide binding and/or hydrolysis or subunit interactions. Activators of G protein signaling (AGS1-3) refer to a functionally defined group of proteins that activate G protein-signaling systems in the absence of a classical G protein-coupled receptor. AGS and related proteins provide unexpected insights into the regulation of the G protein activation/deactivation cycle and the functional roles of G proteins. These proteins likely play important roles in the generation of signaling complexes, the positioning of signaling proteins within the cell, and in biological roles of G proteins unrelated to a cell surface receptor. As such, these proteins and the concepts advanced with their discovery provide unexpected avenues for therapeutics and understanding disease mechanisms.

Activator of G-protein signaling 3 (AGS3) and LGN have a similar domain structure and contain four G-protein regulatory motifs that serve as anchors for the binding of the GDP-bound conformation of specific G-protein α subunits. As an initial approach to define further the different functional roles of AGS3 and LGN, we determined their expression profile and subcellular distribution. AGS3- and LGN-specific antisera indicated a widespread tissue distribution of LGN, whereas AGS3 is primarily enriched in brain. Brain punch biopsies of 13 discrete brain regions indicated that both AGS3 and LGN are expressed in all areas tested but are differentially regulated during development. LGN is expressed in neuronal, astroglial, and microglial cultures, whereas AGS3 expression is restricted to neurons. In primary neuronal cultures as well as in dividing cultures of PC12 cells, immunocytochemistry indicated distinct subcellular locations of AGS3 and LGN. The subcellular locations of the two proteins were differentially regulated by external stimuli and the cell cycle. In PC12 and COS7 cells, LGN moves from the nucleus to the midbody structure separating daughter cells during the later stages of mitosis, suggesting a role for G-proteins in cytokinesis. Thus, although AGS3 and LGN share a similar overall motif structure and both bind G-proteins, nature has endowed these proteins with different regulatory elements that allow functional diversity by virtue of tissue-specific expression and subcellular positioning.

The Ras-related protein, activator of G-protein signaling 1 (AGS1) or Dexras1, interacts with G(i)/G(o)alpha and activates heterotrimeric G-protein signaling systems independent of a G-protein-coupled receptor (GPCR). As an initial approach to further define the cellular role of AGS1 in GPCR signaling, we determined the influence of AGS1 on the regulation of G(betagamma)-regulated inwardly rectifying K(+) channel (GIRK) current (I(ACh)) by M(2)-muscarinic receptor (M(2)-MR) in Xenopus oocytes. AGS1 expression inhibited receptor-mediated current activation by >80%. Mutation of a key residue (G31V) within the G(1) domain involved in nucleotide binding for Ras-related proteins eliminated the action of AGS1. The inhibition of I(ACh) was not overcome by increasing concentrations of the muscarinic agonist acetylcholine but was progressively lost upon injection of increasing amounts of M(2)-MR cRNA. These data suggest that AGS1 may antagonize GPCR signaling by altering the pool of heterotrimeric G-proteins available for receptor coupling and/or disruption of a preformed signaling complex. Such regulation would be of particular importance for those receptors that exist precoupled to heterotrimeric G-protein and for receptors operating within signaling complexes.

alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR >> alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).

The G-protein regulatory (GPR) motif in AGS3 was recently identified as a region for protein binding to heterotrimeric G-protein alpha subunits. To define the properties of this approximately 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 Gialpha1,2 in protein interaction assays, inhibited guanosine 5'-O-(3-thiotriphosphate) binding to Gialpha, and stabilized the GDP-bound conformation of Gialpha. The GPR peptide had little effect on nucleotide binding to Goalpha and brain G-protein indicating selective regulation of Gialpha. Thus, the GPR peptide functions as a guanine nucleotide dissociation inhibitor for Gialpha. The GPR consensus peptide also blocked receptor coupling to Gialphabetagamma indicating that although the AGS3-GPR peptide stabilized the GDP-bound conformation of Gialpha, this conformation of Gialpha(GDP) was not recognized by a G-protein coupled receptor. The AGS3-GPR motif presents an opportunity for selective control of Gialpha- and Gbetagamma-regulated effector systems, and the GPR motif allows for alternative modes of signal input to G-protein signaling systems.

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.

An agonist at a specific G protein-coupled receptor may exhibit a range of efficacies for any given response in a cell-specific manner. We report that the relationship between different states of agonism is regulated by the type of G protein expressed in the cell. In NIH-3T3 alpha(2)-adrenergic receptor (AR) transfectants, the alpha(2)-AR agonists clonidine, oxymetazoline, UK-14304, and epinephrine increased [(35)S]guanosine-5'-O-(3-thio)triphosphate binding in a dose-dependent manner from a basal value of 101.2 +/- 6. 5 fmol/mg to a maximal response (100 microM) of 196.6 +/- 9.8, 182.3 +/- 2, 328.1 +/- 11.2, and 340.6 +/- 3 fmol/mg, respectively. Thus, clonidine and oxymetazoline behaved as partial agonists. Receptor-mediated activation of G proteins in membrane preparations was blocked by cell pretreatment with pertussis toxin, indicating receptor coupling to the subgroup of pertussis toxin-sensitive G protein (Gialpha2,3) expressed in NIH-3T3 cells. Ectopic expression of Goalpha1 but not Gialpha1 increased the relative efficacy of clonidine and oxymetazoline such that the two ligands now behaved as close to full agonists in this assay system. The relationship between full and partial agonists in the different genetic backgrounds was not altered by progressive reduction in the amount of G protein available for coupling to receptor. The increased efficacy observed for clonidine in the Goalpha1 transfectants was not due to changes in the relative affinities or amounts of high-affinity, Gpp(NH)p-sensitive binding of agonist. These data suggest that there is little difference in the ability of clonidine to interact with or stabilize alpha(2)-AR-Gialpha2/Gialpha3 versus alpha(2)-AR-Goalpha1 complexes, but that the subsequent step of signal transfer from receptor to G protein is more readily achieved for the clonidine/alpha(2)-AR/Goalpha1 complex. Such observations have important implications for receptor theory and drug development.

The ability of recombinant rat alpha2D-and alpha2B-adrenoceptors expressed in nerve-growth-factor-differentiated pheochromocytoma PC-12 cells to modulate Ca2+ currents, recorded by the whole-cell patch-clamp technique, has been studied. Ca2+ currents in different cells were either reversibly reduced or increased by dexmedetomidine, an alpha2-adrenergic agonist, in a concentration-dependent manner. Pertussis toxin pretreatment reduced the number of cells that showed an inhibitory response and reduced the magnitude of inhibition. In cells expressing the alpha2B-adrenoceptor, pertussis toxin increased the proportion of cells from which a stimulatory effect on Ca2+ currents could be recorded. The magnitude of the inhibitory responses was unaffected but the stimulatory responses were considerably reduced by the dihydropyridine Ca2+ channel blocker nifedipine (5 microM). All effects of dexmedetomidine were reversible upon wash-out and inhibited by the antagonist rauwolscine. The results support the idea that modulation of voltage-dependent Ca2+ channels in transfected PC-12 cells is mediated by activation of recombinant alpha2D- and alpha2B-adrenoceptors. This receptor activation predominantly causes inhibition of dihydropyridine-insensitive Ca2+ channels via pertussis-toxin-sensitive G proteins. Additionally receptor activation can also lead to stimulation of dihydropyridine-sensitive Ca2+ channels via pertussis-toxin-insensitive mechanisms.

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 alpha2A/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-M3 fusion protein, but not to the control GST peptide or i3 peptides derived from the alpha2A/D-AR and M2-MR. However, each of the receptor subdomains bound purified beta-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]beta-arrestin and [3H]arrestin-3, but did not interact with in vitro translated or purified visual arrestin. The properties and specificity of the interaction of in vitro translated [3H]beta-arrestin, [3H]visual arrestin, and [3H]beta-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 beta-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.

To define the integration of multiple signals by different types of adenylyl cyclase (AC) within the cell, we altered the population of enzymes expressed in the cell and determined the subsequent processing of stimulatory and inhibitory input. DDT1-MF2 cells expressed AC VI–IX and were stably transfected with AC II, III, or IV. Enzyme expression was confirmed by RNA blot analysis and functional assays. Basal enzyme activity was only increased in AC II transfectants (6-fold). Maximum stimulation of enzyme activity was increased in each of the AC transfectants to varying extents. α2A/D-AR activation elicited enzyme type-specific responses. α2-AR activation inhibited the effect of isoproterenol in control transfectants, and this action was magnified in AC III transfectants. In AC II and AC IV transfectants, α2-AR activation initiated both positive (Gβγ) and negative signals (Giα) to the Gsα-stimulated enzyme, and both types of signals were blocked by cell pretreatment with pertussis toxin. The negative input to AC II from the α2-AR was blocked by protein kinase C activation in AC II transfectants, but it was the positive input to AC IV that was compromised by protein kinase C activation. These data indicate that the integration of multiple signals by adenylyl cyclases is a dynamic process depending upon the enzyme type and phosphorylation status.

Group II activators of G-protein signaling play diverse functional roles through their interaction with Gαi, Gαt, and Gαo via a G-protein regulatory (GPR) motif that serves as a docking site for Gα-GDP. We recently reported the regulation of the AGS3-Gαi signaling module by a cell surface, seven-transmembrane receptor. Upon receptor activation, AGS3 reversibly dissociates from the cell cortex, suggesting that it may function as a signal transducer with downstream signaling implications, and this question is addressed in the current report. In HEK-293 and COS-7 cells expressing the α2A/D-AR and Gαi3, receptor activation resulted in the translocation of endogenous AGS3 and AGS3-GFP from the cell cortex to a juxtanuclear region, where it co-localized with markers of the Golgi apparatus (GA). The agonist-induced translocation of AGS3 was reversed by the α2-AR antagonist rauwolscine. The TPR domain of AGS3 was required for agonist-induced translocation of AGS3 from the cell cortex to the GA, and the translocation was blocked by pertussis toxin pretreatment or by the phospholipase Cβ inhibitor U73122. Agonist-induced translocation of AGS3 to the GA altered the functional organization and protein sorting at the trans-Golgi network. The regulated movement of AGS3 between the cell cortex and the GA offers unexpected mechanisms for modulating protein secretion and/or endosome recycling events at the trans-Golgi network.

The G-protein regulatory (GPR) motif serves as a docking site for Gαi-GDP free of Gβγ. The GPR-Gα complex may function at the cell cortex and/or at intracellular sites. GPR proteins include the Group II Activators of G-protein signaling identified in a functional screen for receptor-independent activators of G-protein signaling (GPSM1-3, RGS12) each of which contain 1-4 GPR motifs. GPR motifs are also found in PCP2/L7(GPSM4), Rap1-Gap1 Transcript Variant 1, and RGS14. While the biochemistry of the interaction of GPR proteins with purified Gα is generally understood, the dynamics of this signaling complex and its regulation within the cell remains undefined. Major questions in the field revolve around the factors that regulate the subcellular location of GPR proteins and their interaction with Gαi and other binding partners in the cell. As an initial approach to this question, we established a platform to monitor the GPR-Gαi complex in intact cells using bioluminescence resonance energy transfer.

Pharmacologically active compounds with an imidazoline and/or guanidinium moiety are recognized with high affinity by a family of membrane-bound proteins collectively known as imidazoline binding sites or imidazoline/guanidinium receptive sites. Two such receptive sites may correspond to imidazoline binding domains identified on the A and B isoforms of monoamine oxidase (MAO), but the detection of monoamine oxidase isoforms in multiple tissues contrasts with the restricted expression of imidazoline-binding proteins. To address these issues, we determined the relationship between monoamine oxidase isoforms and subtypes of imidazoline-binding proteins in human tissues known to express one or both isoforms of MAO. 2-(3-Azido-4-[125I]iodophenoxy)methylimidazoline ([125I]AZIPI), a photoaffinity adduct that selectively labels imidazoline-binding proteins, photolabeled an Mr = ∼59,000 peptide in liver and an Mr = ∼63,000 peptide in placenta, consistent with the Mr of the MAO isoforms identified by immunoblots in these tissues. The photolabeled species in liver was immunoprecipitated with MAO-B selective antibodies, whereas the photolabeled species in placenta was immunoprecipitated by MAO-A selective antibodies consistent with the isoform of MAO predominantly expressed in these tissues. The imidazoline/guanidinium ligands interact with the enzyme at a site distinct from the substrate recognition domain, and the immunoprecipitated peptides in liver and placenta display distinct ligand recognition properties consistent with those reported for subtypes of imidazoline binding sites. However, the imidazoline binding domain was not detected in platelet membrane preparations containing amounts of MAO-B equivalent to those in the photolabeled liver membranes indicating that recognition of this domain is tissue-restricted. Restricted access to the imidazoline binding domain on platelet MAO-B was not altered by membrane washing with 500 mM KCl or by solubilization and partial purification of the enzyme suggesting that there are distinct subpopulations of MAO. Identification of a binding domain on MAO that recognizes this class of pharmacologically active compounds suggests a novel mechanism for regulation of substrate oxidation/selectivity or that the enzyme may subserve an as yet undefined function.

Group II activators of G-protein signaling (AGS) serve as binding partners for Gα(i/o/t) via one or more G-protein regulatory (GPR) motifs. GPR-Gα signaling modules may be differentially regulated by cell surface receptors or by different nonreceptor guanine nucleotide exchange factors. We determined the effect of the nonreceptor guanine nucleotide exchange factors AGS1, GIV/Girdin, and Ric-8A on the interaction of two distinct GPR proteins, AGS3 and AGS4, with Gα(il) in the intact cell by bioluminescence resonance energy transfer (BRET) in human embryonic kidney 293 cells. AGS3-Rluc-Gα(i1)-YFP and AGS4-Rluc-Gα(i1)-YFP BRET were regulated by Ric-8A but not by Gα-interacting vesicle-associated protein (GIV) or AGS1. The Ric-8A regulation was biphasic and dependent upon the amount of Ric-8A and Gα(i1)-YFP. The inhibitory regulation of GPR-Gα(i1) BRET by Ric-8A was blocked by pertussis toxin. The enhancement of GPR-Gα(i1) BRET observed with Ric-8A was further augmented by pertussis toxin treatment. The regulation of GPR-Gα(i) interaction by Ric-8A was not altered by RGS4. AGS3-Rluc-Gα(i1)-YFP and AGS4-Rluc-G-Gα(i1)-YFP BRET were observed in both pellet and supernatant subcellular fractions and were regulated by Ric-8A in both fractions. The regulation of the GPR-Gα(i1) complex by Ric-8A, as well as the ability of Ric-8A to restore Gα expression in Ric8A(-/-) mouse embryonic stem cells, involved two helical domains at the carboxyl terminus of Ric-8A. These data indicate a dynamic interaction between GPR proteins, Gα(i1) and Ric-8A, in the cell that influences subcellular localization of the three proteins and regulates complex formation.