Helix 8 and the i3 loop of the Muscarinic M3 Receptor are Crucial Sites for its Regulation by the Gβ5-RGS7 Complex.

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 Ca2+ release from intracellular stores or enhance Ca2+ influx across the plasma membrane. Here we show that deletion of amino acids 304-345 in 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 M3R C-terminus assumes an alpha helical conformation. Substitution of the Thr553 and Leu558 with Pro residues disrupts this helix and abolished binding to Gβ5-RGS7. Introduction of the double Pro substitution into the full-length M3R (M3RTP/LP) prevents trafficking of the receptor to the cell surface. Using atropine or other muscarinic antagonists as a pharmacologic chaperones we were able to increase the surface expression of TP/LP mutant to levels comparable to WT 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.