Publication Date

2014-11-06

Availability

Open access

Embargo Period

2014-11-06

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Molecular and Cellular Pharmacology (Medicine)

Date of Defense

2014-07-18

First Committee Member

Vladlen Z. Slepak

Second Committee Member

Charles W. Luetje

Third Committee Member

Nirupa Chaudhari

Fourth Committee Member

R. Grace Zhai

Fifth Committee Member

Christian Faul

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.

Keywords

Gβ5-RGS7; G protein-coupled receptor; muscarinic M3 receptor; Ca2+ signaling; functional selectivity

Share

COinS