title: Helix 8 and the i3 loop of the muscarinic M3 receptor are crucial sites for its regulation by the Gβ5-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 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.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami Miller School of Medicine , 1600 NW 10th Avenue, RMSB6024A, Miami, Florida 33136, United States.
authors.forename: Darla
authors.initials: D
authors.lastname: Karpinsky-Semper
authors.forename: Junior
authors.initials: J
authors.lastname: Tayou
authors.forename: Konstantin
authors.initials: K
authors.lastname: Levay
authors.forename: Brett J
authors.initials: BJ
authors.lastname: Schuchardt
authors.forename: Vikas
authors.initials: V
authors.lastname: Bhat
authors.forename: Claude-Henry
authors.initials: CH
authors.lastname: Volmar
authors.forename: Amjad
authors.initials: A
authors.lastname: Farooq
authors.forename: Vladlen Z
authors.initials: VZ
authors.lastname: Slepak
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D018678:Cholinergic Agents / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D044387:GTP-Binding Protein beta Subunits / Q000737:chemistry* / Q000502:physiology*; D008969:Molecular Sequence Data; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000737:chemistry* / Q000502:physiology*
chemical_list: D018678:Cholinergic Agents; D044387:GTP-Binding Protein beta Subunits; D043587:Receptor, Muscarinic M3