title: Differential effects of the Gβ5-RGS7 complex on muscarinic M3 receptor-induced Ca2+ influx and release.
abstract: The G protein β subunit Gβ5 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Gγ. Although the Gβ5-RGS7 complex attenuates Ca(2+) signaling mediated by the muscarinic M3 receptor (M3R), the route of Ca(2+) entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here, we show that, in addition to suppressing carbachol-stimulated Ca(2+) release, Gβ5-RGS7 enhanced Ca(2+) influx. This novel effect of Gβ5-RGS7 was blocked by nifedipine and 2-aminoethoxydiphenyl borate. Experiments with pertussis toxin, an RGS domain-deficient mutant of RGS7, and UBO-QIC {L-threonine,(3R)-N-acetyl-3-hydroxy-L-leucyl-(aR)-a-hydroxybenzenepropanoyl-2,3-idehydro-N-methylalanyl-L-alanyl-N-methyl-L-alanyl-(3R)-3-[[(2S,3R)-3-hydroxy-4- methyl-1-oxo-2-[(1-oxopropyl)amino]pentyl]oxy]-L-leucyl-N,O-dimethyl-,(7→1)-lactone (9CI)}, a novel inhibitor of Gq, showed that Gβ5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gβ5-RGS7, independent of RGS7 GTPase-accelerating protein activity, couples M3R to a nifedipine-sensitive Ca(2+) channel. We also compared the action of Gβ5-RGS7 on M3R-induced Ca(2+) influx and release elicited by different muscarinic agonists. Responses to Oxo-M [oxotremorine methiodide N,N,N,-trimethyl-4-(2-oxo-1-pyrrolidinyl)-2-butyn-1-ammonium iodide] were insensitive to Gβ5-RGS7. Pilocarpine responses consisted of a large release and modest influx components, of which the former was strongly inhibited whereas the latter was insensitive to Gβ5-RGS7. McN-A-343 [(4-hydroxy-2-butynyl)-1-trimethylammonium-3-chlorocarbanilate chloride] was the only compound whose total Ca(2+) response was enhanced by Gβ5-RGS7, attributed to, in part, by the relatively small Ca(2+) release this partial agonist stimulated. Together, these results show that distinct agonists not only have differential M3R functional selectivity, but also confer specific sensitivity to the Gβ5-RGS7 complex.
authors.affiliation: Department of Molecular and Cellular Pharmacology (D.K.-S., V.Z.S.) and Center for Therapeutic Innovation, Department of Psychiatry and Behavioral Sciences (C.-H.V., S.P.B.), University of Miami Miller School of Medicine, Miami, Florida.
authors.forename: Darla
authors.initials: D
authors.lastname: Karpinsky-Semper
authors.forename: Claude-Henry
authors.initials: CH
authors.lastname: Volmar
authors.forename: Shaun P
authors.initials: SP
authors.lastname: Brothers
authors.forename: Vladlen Z
authors.initials: VZ
authors.lastname: Slepak
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D054333:Drug Partial Agonism; D044387:GTP-Binding Protein beta Subunits / Q000819:agonists / Q000378:metabolism*; D020710:RGS Proteins / Q000819:agonists / Q000378:metabolism*; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000378:metabolism*
chemical_list: C490611:GNB5 protein, human; D044387:GTP-Binding Protein beta Subunits; D020710:RGS Proteins; C492039:RGS7 protein, human; D043587:Receptor, Muscarinic M3; D002118:Calcium