Gßγ subunit inhibitor decreases DOM-induced head twitch response via the PLCß/IP3/Ca2+/ERK and cAMP signaling pathways.

AIMS: (-)-2,5-dimethoxy-4-methylamphetamine (DOM) induces the head-twitch response (HTR) primarily by activating the serotonin 5-hydroxytryptamine 2A receptor (5-HT2A receptor) in mice. However, the mechanisms underlying 5-HT2A receptor activation and the HTR remain elusive. Gßγ subunits are a potential treatment target in numerous diseases. The present study investigated the mechanism whereby Gßγ subunits influence DOM-induced HTR. MAIN METHODS: The effects of the Gßγ inhibitor 3',4',5',6'-tetrahydroxyspiro[2-benzofuran-3,9'-xanthene]-1-one (gallein) and antagonistic peptide ßARKct (ß-adrenergic receptor kinase C-terminal fragment) on DOM-induced HTR were studied via an HTR test. The activation of the phospholipase C ß (PLCß)/inositol triphosphate (IP3)/calcium (Ca2+) signaling pathway and extracellular signal-regulated kinase (ERK) following Gßγ subunit inhibition was detected by western blotting, Homogeneous Time-Resolved Fluorescence (HTRF) inositol phosphate (IP1) assay and Fluorometric Imaging Plate Reader (FLIPR) calcium 6 assay. The Gßγ subunit-mediated regulation of cyclic adenosine monophosphate (cAMP) was assessed via a GloSensor™ cAMP assay. KEY FINDINGS: The Gßγ subunit inhibitors gallein and ßARKct reduced DOM-induced HTR in C57BL/6J mice. Like the 5-HT2A receptor-selective antagonist (R)-[2,3-di(methoxy)phenyl]-[1-[2-(4-fluorophenyl)ethyl]piperidin-4-yl]methanol (M100907), gallein inhibited PLCß phosphorylation (pPLCß), IP1 production, Ca2+ transients, ERK1/2 phosphorylation (pERK1/2) and cAMP accumulation induced by DOM in human embryonic kidney (HEK) 293T cells stably or transiently transfected with the human 5-HT2A receptor. Moreover, PLCß protein inhibitor 1-[6-[[(8R,9S,13S,14S,17S)-3-methoxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]amino]hexyl]pyrrole-2,5-dione (U73122) (10 nmol/mouse), intracellular Ca2+ blocker 6-[6-[6-[5-acetamido-4,6-dihydroxy-2-(sulfooxymethyl)oxan-3-yl]oxy-2-carboxy-4-hydroxy-5-sulfooxyoxan-3-yl]oxy-2-(hydroxymethyl)-5-(sulfoamino)-4-sulfooxyoxan-3-yl]oxy-3,4-dihydroxy-5-sulfooxyoxane-2-carboxylic acid (heparin) (5 nmol/mouse), L-type Ca2+ channel blocker 3-O-(2-methoxyethyl) 5-O-propan-2-yl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (nimodipine) (4 mg/kg), mitogen extracellular regulating kinase 1/2 (MEK1/2) inhibitor (Z)-3-amino-3-(4-aminophenyl)sulfanyl-2-[2-(trifluoromethyl)phenyl]prop-2-enenitrile (SL327) (30 mg/kg), and Gαs protein selective antagonist 4,4',4″,4‴-(Carbonylbis-(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakisbenzene-1,3-disulfonic acid (NF449) (10 nmol/mouse) reduced DOM-induced HTR in C57BL/6J mice. SIGNIFICANCE: The Gßγ subunits potentially mediate the HTR after 5-HT2A receptor activation via the PLCß/IP3/Ca2+/ERK1/2 and cAMP signaling pathways. Inhibitors targeting the Gßγ subunits potentially inhibit the hallucinogenic effects of 5-HT2A receptor agonists.

Two naturally occurring mutations of human GPR103 define distinct G protein selection bias.

The neuropeptide 26RFa plays important roles in the regulation of many physiological functions. 26RFa has been recognized as an endogenous ligand for receptor GPR103. In the present study, we demonstrate that GPR103 dually couples to Gαq and Gαi/o proteins. However, two naturally occurring missense mutations were identified from a young male patient. In the first, Y68H, induction of Ca2+ mobilization was noted without detection of ERK1/2 activation. In the second, R371W, the potential to activate ERK1/2 signaling was retained but with failure to evoke Ca2+ mobilization. Further analysis provides evidence that Gαq, L-type Ca2+ channel and PKCßI and ßII are involved in the Y68H-mediated signaling pathway, whereas Gαi/o, Gßγ, and PKCζ are implicated in the R371W-induced signaling. Our results demonstrate that two point mutations, Y68H and R371W, affect the equilibrium between the different receptor conformations, leading to alteration of G protein-coupling preferences. Importantly, these findings provide a foundation for future elucidation of GPCR-mediated biased signaling and the physiological implications of their bias.

The G-protein ßγ subunits regulate platelet function.

AIMS: G-protein coupled receptors (GPCRs) tightly regulate platelet function by interacting with various physiological agonists. An essential mediator of GPCR signaling is the G protein αßγ heterotrimers, in which the ßγ subunits are central players in downstream signaling. Herein, we investigated the role of Gßγ subunits in platelet function, hemostasis and thrombogenesis. METHODS: To achieve this goal, platelets from both mice and humans were employed in the context of a small molecule inhibitor of Gßγ, namely gallein. We used an aggregometer to examine aggregation and dense granules secretion. We also used flow cytometry for P-selectin and PAC1 to determine the impact of inhibiting Gßγ on α -granule secretion and αIIbß3 activation. Clot retraction and the platelet spreading assay were used to examine Gßγ role in outside-in platelet signaling, whereas Western blot was employed to examine its role in Akt activation. Finally, we used the bleeding time assay and the FeCl3-induced carotid-artery injury thrombosis model to determine Gßγ contribution to in vivo platelet function. RESULTS: We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbß3 activation, clot retraction, platelet spreading and Akt activation/phosphorylation. Finally, gallein's inhibitory effects manifested in vivo, as documented by its ability to modulate physiological hemostasis and delay thrombus formation. CONCLUSION: Our findings demonstrate, for the first time, that Gßγ subunits directly regulate GPCR-dependent platelet function, in vitro and in vivo. Moreover, these data highlight Gßγ as a novel therapeutic target for managing thrombotic disorders.

Involvement of Gßγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes.

Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear. Here, we show that Rho family GTPases, including Cdc42 and Rac1, are constitutively activated on exosomal MVEs and are regulated by S1P signaling as measured by fluorescence resonance energy transfer (FRET)-based conformational changes. Moreover, we detected S1P signaling-induced filamentous actin (F-actin) formation. A selective inhibitor of Gßγ subunits, M119, strongly inhibited both F-actin formation on MVEs and cargo sorting into exosomal intralumenal vesicles of MVEs, both of which were fully rescued by the simultaneous expression of constitutively active Cdc42 and Rac1. Our results shed light on the mechanism underlying exosomal MVE maturation and inform the understanding of the physiological relevance of continuous activation of the S1P receptor and subsequent downstream G protein signaling to Gßγ subunits/Rho family GTPases-regulated F-actin formation on MVEs for cargo sorting into exosomal intralumenal vesicles.

On the selectivity of the Gαq inhibitor UBO-QIC: A comparison with the Gαi inhibitor pertussis toxin.

Gαq inhibitor UBO-QIC (FR900359) is becoming an important pharmacological tool, but its selectivity against other G proteins and their subunits, especially ßγ, has not been well characterized. We examined UBO-QIC's effect on diverse signaling pathways mediated via various G protein-coupled receptors (GPCRs) and G protein subunits by comparison with known Gαi inhibitor pertussis toxin. As expected, UBO-QIC inhibited Gαq signaling in all assay systems examined. However, other non-Gαq-events, e.g. Gßγ-mediated intracellular calcium release and inositol phosphate production, following activation of Gi-coupled A1 adenosine and M2 muscarinic acetylcholine receptors, were also blocked by low concentrations of UBO-QIC, indicating that its effect is not limited to Gαq. Thus, UBO-QIC also inhibits Gßγ-mediated signaling similarly to pertussis toxin, although UBO-QIC does not affect Gαi-mediated inhibition or Gαs-mediated stimulation of adenylyl cyclase activity. However, the blockade by UBO-QIC of GPCR signaling, such as carbachol- or adenosine-mediated calcium or inositol phosphate increases, does not always indicate inhibition of Gαq-mediated events, as the ßγ subunits released from Gi proteins following the activation of Gi-coupled receptors, e.g. M2 and A1Rs, may produce similar signaling events. Furthermore, UBO-QIC completely inhibited Akt signaling, but only partially blocked ERK1/2 activity stimulated by the Gq-coupled P2Y1R. Thus, we have revealed new aspects of the pharmacological interactions of UBO-QIC.

Kir3 channel ontogeny - the role of Gßγ subunits in channel assembly and trafficking.

The role of Gßγ subunits in Kir3 channel gating is well characterized. Here, we have studied the role of Gßγ dimers during their initial contact with Kir3 channels, prior to their insertion into the plasma membrane. We show that distinct Gßγ subunits play an important role in orchestrating and fine-tuning parts of the Kir3 channel life cycle. Gß1γ2, apart from its role in channel opening that it shares with other Gßγ subunit combinations, may play a unique role in protecting maturing channels from degradation as they transit to the cell surface. Taken together, our data suggest that Gß1γ2 prolongs the lifetime of the Kir3.1/Kir3.2 heterotetramer, although further studies would be required to shed more light on these early Gßγ effects on Kir3 maturation and trafficking.