Joe B. Blumer

Activator of G protein signaling 3 null mice: I. Unexpected alterations in metabolic and cardiovascular function.

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.

Purification of heterotrimeric G protein α subunits by GST-Ric-8 association: Primary characterization of purified Gαolf

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.

Receptor-regulated interaction of activator of G-protein signaling 4 and Gαi

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 Gαi-GDP subunits free of Gβγ. 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 Gαi1 in living cells using bioluminescence resonance energy transfer (BRET). AGS4 and Gαi1 reciprocally tagged with either Renilla luciferase (RLuc) or yellow fluorescent protein (YFP) demonstrated saturable, specific BRET signals. BRET signals observed between AGS4-RLuc and Gαi1-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 α2-adrenergic receptor-Venus, which were Gαi-dependent and reduced by agonist, indicating that AGS4-Gαi complexes are receptor-proximal. These data suggest that AGS4-Gαi complexes directly couple to a G-protein-coupled receptor and may serve as substrates for agonist-induced G-protein activation.

AKAP Signaling in Reinstated Cocaine Seeking Revealed by iTRAQ Proteomic Analysis

To identify candidate proteins in the nucleus accumbens (NAc) as potential pharmacotherapeutic targets for treating cocaine addition, an 8-plex iTRAQ (isobaric tag for relative and absolute quantitation) proteomic screen was performed using NAc tissue obtained from rats trained to self-administer cocaine followed by extinction training. Compared with yoked-saline controls, 42 proteins in a postsynaptic density (PSD)-enriched subfraction of the NAc from cocaine-trained animals were identified as significantly changed. Among proteins of interest whose levels were identified as increased was AKAP79/150, the rat ortholog of human AKAP5, a PSD scaffolding protein that localizes signaling molecules to the synapse. Functional downregulation of AKAP79/150 by microinjecting a cell-permeable synthetic AKAP (A-kinase anchor protein) peptide into the NAc to disrupt AKAP-dependent signaling revealed that inhibition of AKAP signaling impaired the reinstatement of cocaine seeking. Reinstatement of cocaine seeking is thought to require upregulated surface expression of AMPA glutamate receptors, and the inhibitory AKAP peptide reduced the PSD content of protein kinase A (PKA) as well as surface expression of GluR1 in NAc. However, reduced surface expression was not associated with changes in PKA phosphorylation of GluR1. This series of experiments demonstrates that proteomic analysis provides a useful tool for identifying proteins that can regulate cocaine relapse and that AKAP proteins may contribute to relapse vulnerability by promoting increased surface expression of AMPA receptors in the NAc.

Regulation of the AGS3·Gαi Signaling Complex by a Seven-transmembrane Span Receptor

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 Gαi 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·Gαi signaling module by a cell surface, seven-transmembrane receptor. AGS3 and Gαi1 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 α2-adrenergic receptors or μ-opioid receptors reduced AGS3-RLuc·Gαi1-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. Gαi-dependent and agonist-sensitive bioluminescence resonance energy transfer was also observed between AGS3 and cell-surface receptors typically coupled to Gαi and/or Gαo 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-Gαi offer additional flexibility for systems to respond and adapt to challenges and orchestrate complex behaviors.

Loss of activator of G-protein signaling 3 impairs renal tubular regeneration following acute kidney injury in rodents

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 het‐erotrimeric 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 coex‐pressed 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 overex‐pression of the carboxyl‐terminus of G‐protein‐coupled receptor kinase 2 (GRK2ct), a scavenger of Gβγ sub‐units. 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. FASEB J. 25, 1844‐1855 (2011). www.fasebj.org

Activator of G Protein Signaling 3 Promotes Epithelial Cell Proliferation in PKD

The activation of heterotrimeric G protein signaling is a key feature in the pathophysiology of polycystic kidney diseases (PKD). In this study, we report abnormal overexpression of activator of G protein signaling 3 (AGS3), a receptor-independent regulator of heterotrimeric G proteins, in rodents and humans with both autosomal recessive and autosomal dominant PKD. Increased AGS3 expression correlated with kidney size, which is an index of severity of cystic kidney disease. AGS3 expression localized exclusively to distal tubular segments in both normal and cystic kidneys. Short hairpin RNA-induced knockdown of endogenous AGS3 protein significantly reduced proliferation of cystic renal epithelial cells by 26 +/- 2% (P < 0.001) compared with vehicle-treated and control short hairpin RNA-expressing epithelial cells. In summary, this study suggests a relationship between aberrantly increased AGS3 expression in renal tubular epithelia affected by PKD and epithelial cell proliferation. AGS3 may play a receptor-independent role to regulate Galpha subunit function and control epithelial cell function in PKD.

Group II activators of G-protein signalling and proteins containing a G-protein regulatory motif

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.

Activators of G Protein Signaling Exhibit Broad Functionality and Define a Distinct Core Signaling Triad

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 PDZ and Band 4.1 Containing Protein Frmpd1 Regulates the Subcellular Location of Activator of G-protein Signaling 3 and Its Interaction with G-proteins

Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Gαi. Such proteins have revealed unexpected functional diversity for the “G-switch” in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by ∼50% and enhanced the α2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Gαi3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Gαi3 indicating that the interaction of AGS3 with Frmpd1 and Gαi3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Gαi in a regulated manner.