Sukru Sadik Oner

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.

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.

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.

Regulation of the G-protein regulatory-Gαi signaling complex by nonreceptor guanine nucleotide exchange factors.

Background: The GPR-Gαi complex has diverse functional roles, but regulatory mechanisms are not defined. Results: The GPR-Gαi complex is regulated by Ric-8A but not by increased expression of AGS1 or GIV/Girdin. Conclusion: The GPR-Gαi complex is differentially regulated by specific guanine nucleotide exchange factors. Significance: The GPR proteins, Gαi1 and Ric-8A, exhibit dynamic interactions in the cell that influence their subcellular localization and regulate complex formation. Group II activators of G-protein signaling (AGS) serve as binding partners for Gαi/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 Gαil in the intact cell by bioluminescence resonance energy transfer (BRET) in human embryonic kidney 293 cells. AGS3-Rluc-Gαi1-YFP and AGS4-Rluc-Gαi1-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 Gαi1-YFP. The inhibitory regulation of GPR-Gαi1 BRET by Ric-8A was blocked by pertussis toxin. The enhancement of GPR-Gαi1 BRET observed with Ric-8A was further augmented by pertussis toxin treatment. The regulation of GPR-Gαi interaction by Ric-8A was not altered by RGS4. AGS3-Rluc-Gαi1-YFP and AGS4-Rluc-G-Gαi1-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-Gαi1 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, Gαi1 and Ric-8A, in the cell that influences subcellular localization of the three proteins and regulates complex formation.

Translocation of Activator of G-protein Signaling 3 to the Golgi Apparatus in Response to Receptor Activation and Its Effect on the trans-Golgi Network

Background: The AGS3·Gαi complex is regulated by a GPCR, but downstream signaling events are unknown. Results: Upon receptor activation, AGS3 translocates to the Golgi apparatus, where it regulates events at the TGN. Conclusion: The AGS3·Gαi complex serves as a signal transducer for GPCRs. Significance: The regulated translocation of AGS3 offers unexpected mechanisms for modulating protein secretion and/or endosome recycling events at the TGN. 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.

Influence of the Accessory Protein SET on M3 Muscarinic Receptor Phosphorylation and G Protein Coupling

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.

Direct Coupling of a Seven-Transmembrane-Span Receptor to a Gαi G-Protein Regulatory Motif Complex

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-Gαi2YFPC352I:AGS3Rluc and α2AAR-Gαi2YFPC352I: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.

Group II activators of G-protein signaling: monitoring the interaction of Gα with the G-protein regulatory motif in the intact cell.

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.