Abstract
The neurotransmitter acetylcholine (Ach) is arguably the most important in the CNS. Two classes of receptors are activated by Ach: nicotinic (ionotropic) and muscarinic (metabotropic). Muscarinic receptor pharmacology is well-defined, but still lacks receptor sub-type specificity seen in other GPCR families like adrenergic receptors. We have found that Gβ5-RGS7, a unique G-protein obligate heterodimer complex, selectively attenuates Ca2+ signaling through the muscarinic M3 receptor (M3R). The G protein beta subunit Gβ5 uniquely forms heterodimers with R7 family regulators of G protein signaling (RGS) proteins (RGS6, RGS7, RGS9, and RGS11) instead of Gγ. While the Gβ5-RGS7 complex attenuates Ca2+ signaling mediated by M3R, the route of Ca2+ entry (i.e., release from intracellular stores and/or influx across the plasma membrane) is unknown. Here I show that in addition to suppressing carbachol-stimulated Ca2+ release, Gβ5-RGS7 enhanced Ca2+ influx. This novel effect of Gβ5-RGS7 was blocked by nifedipine and 2-APB. Experiments with pertussis toxin, RGS domain-deficient mutant of RGS7 and UBO-QIC, a novel inhibitor of Gq, showed that Gβ5-RGS7 modulated a Gq-mediated pathway. These studies indicate that Gβ5-RGS7, independent of RGS7 GAP activity, couples M3R to a nifedipine-sensitive Ca2+ channel. In neurons and glands, muscarinic signaling plays a major role in secretion. The novel finding that Gβ5-RGS7 enhances M3R-stimulated insulin secretion can explain why loss of Gβ5 results in impaired insulin secretion in mice. In insulin secreting cells, I found that the mechanism of Gβ5-RGS7-enhanced Ca2+ signaling is similar to the one identified in CHO-K1 cells as it is sensitive to nifedipine. I also compared the action of Gβ5-RGS7 on M3R-induced Ca2+ influx and release elicited by different muscarinic agonists. Responses to oxotremorine-m 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 was the only compound whose total Ca2+ response was enhanced by Gβ5-RGS7, attributed to, in part, by the relatively small Ca2+ 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. A deeper understanding of the structural basis of this ligand bias towards sensitivity to Gβ5-RGS7 may lead to new strategies for selective therapeutics. My biophysical studies provide additional insights into the structural basis of Gβ5-RGS7 regulation of M3R signaling. Altogether, this dissertation work led to the novel findng that Gβ5-RGS7 has a dual effect on M3R-stimulated Ca2+ signaling, and the newly discovered positive effect on Ca2+ influx plays an important role in hormone and/or neurotransmitter secretion stimulated by the M3R pathway.