Dataset Information

Helix 8 and the i3 loop of the muscarinic M3 receptor are crucial sites for its regulation by the G?5-RGS7 complex.

ABSTRACT: The muscarinic M3 receptor (M3R) is a Gq-coupled receptor and is known to interact with many intracellular regulatory proteins. One of these molecules is G?5-RGS7, the permanently associated heterodimer of G protein ?-subunit G?5 and RGS7, a regulator of G protein signaling. G?5-RGS7 can attenuate M3R-stimulated release of Ca(2+) from intracellular stores or enhance the influx of Ca(2+) across the plasma membrane. Here we show that deletion of amino acids 304-345 from the central portion of the i3 loop renders M3R insensitive to regulation by G?5-RGS7. In addition to the i3 loop, interaction of M3R with G?5-RGS7 requires helix 8. According to circular dichroism spectroscopy, the peptide corresponding to amino acids 548-567 in the C-terminus of M3R assumes an ?-helical conformation. Substitution of Thr553 and Leu558 with Pro residues disrupts this ?-helix and abolished binding to G?5-RGS7. Introduction of the double Pro substitution into full-length M3R (M3R(TP/LP)) prevents trafficking of the receptor to the cell surface. Using atropine or other antagonists as pharmacologic chaperones, we were able to increase the level of surface expression of the TP/LP mutant to levels comparable to that of wild-type M3R. However, M3R-stimulated calcium signaling is still severely compromised. These results show that the interaction of M3R with G?5-RGS7 requires helix 8 and the central portion of the i3 loop.

SUBMITTER: Karpinsky-Semper D

PROVIDER: S-EPMC4318586 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Helix 8 and the i3 loop of the muscarinic M3 receptor are crucial sites for its regulation by the Gβ5-RGS7 complex.

Karpinsky-Semper Darla D Tayou Junior J Levay Konstantin K Schuchardt Brett J BJ Bhat Vikas V Volmar Claude-Henry CH Farooq Amjad A Slepak Vladlen Z VZ

Biochemistry 20150120 4

The muscarinic M3 receptor (M3R) is a Gq-coupled receptor and is known to interact with many intracellular regulatory proteins. One of these molecules is Gβ5-RGS7, the permanently associated heterodimer of G protein β-subunit Gβ5 and RGS7, a regulator of G protein signaling. Gβ5-RGS7 can attenuate M3R-stimulated release of Ca(2+) from intracellular stores or enhance the influx of Ca(2+) across the plasma membrane. Here we show that deletion of amino acids 304-345 from the central portion of the ...[more]

PMID: 25551629

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Project description:The complex of the regulator of G protein signaling (RGS), Gbeta(5)-RGS7, can inhibit signal transduction via the M3 muscarinic acetylcholine receptor (M3R). RGS7 consists of three distinct structural entities: the DEP domain and its extension DHEX, the Ggamma-like (GGL) domain, which is permanently bound to Gbeta subunit Gbeta(5), and the RGS domain responsible for the interaction with Galpha subunits. Inhibition of the M3R by Gbeta(5)-RGS7 is independent of the RGS domain but requires binding of the DEP domain to the third intracellular loop of the receptor. Recent studies identified the dynamic intramolecular interaction between the Gbeta(5) and DEP domains, which suggested that the Gbeta(5)-RGS7 dimer could alternate between the "open" and "closed" conformations. Here, we identified point mutations that weaken DEP-Gbeta(5) binding, presumably stabilizing the open state, and tested their effects on the interaction of Gbeta(5)-RGS7 with the M3R. We found that these mutations facilitated binding of Gbeta(5)-RGS7 to the recombinant third intracellular loop of the M3R but did not enhance its ability to inhibit M3R-mediated Ca(2+) mobilization. This led us to the idea that the M3R can effectively induce the Gbeta(5)-RGS7 dimer to open; such a mechanism would require a region of the receptor distinct from the third loop. Indeed, we found that the C-terminus of M3R interacts with Gbeta(5)-RGS7. Truncation of the C-terminus rendered the M3R insensitive to inhibition by wild-type Gbeta(5)-RGS7; however, the open mutant of Gbeta(5)-RGS7 was able to inhibit signaling by the truncated M3R. The GST fusion of the M3R C-tail could not bind to wild-type Gbeta(5)-RGS7 but could associate with its open mutant as well as with the separated recombinant DEP domain or Gbeta(5). Taken together, our data are consistent with the following model: interaction of the M3R with Gbeta(5)-RGS7 causes the DEP domain and Gbeta(5) to dissociate from each other and bind to the C-tail, and the DEP domain also binds to the third loop, thereby inhibiting M3R-mediated signaling.

Dataset Information

Helix 8 and the i3 loop of the muscarinic M3 receptor are crucial sites for its regulation by the G?5-RGS7 complex.

Publications

Helix 8 and the i3 loop of the muscarinic M3 receptor are crucial sites for its regulation by the Gβ5-RGS7 complex.

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