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

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 i3 loop renders M3R insensitive to regulation by Gβ5-RGS7. In addition to the i3 loop, interaction of M3R with Gβ5-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 α-helical conformation. Substitution of Thr553 and Leu558 with Pro residues disrupts this α-helix and abolished binding to Gβ5-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 Gβ5-RGS7 requires helix 8 and the central portion of the i3 loop.