Structural basis of receptor/G protein coupling selectivity studied with muscarinic receptors as model systems

Different muscarinic acetylcholine receptor subtypes were used as model systems to study the structural basis of receptor/G protein coupling selectivity. Extensive mutagenesis studies have previously led to the identification of single amino acids on the m3 muscarinic receptor protein (located in the second intracellular loop (i2) and at the N- and C-terminus of the third intracellular loop (i3)) that dictate selective recognition of Gq/11 proteins by this receptor subtype. Based on these results, we proposed a model of the intracellular m3 receptor surface in which the functionally critical residues project into the interior of the transmembrane receptor core. To identify specific regions on the G protein(s) that are contacted by these different, functionally critical receptor sites, we recently employed a novel experimental strategy involving the coexpression of hybrid m2/m3 muscarinic receptors with hybrid G alpha-subunits. Using this approach, we could demonstrate that the C-terminus of G protein alpha i/o-subunits is recognized by a short sequence element in the m2 muscarinic receptor ("VTIL") that is located at the junction between the sixth transmembrane domain (TM VI) and the i3 loop. We could show that this interaction is critically involved in determining coupling selectivity and triggering G protein activation. By using a similar strategy (coexpression of mutant muscarinic receptors with hybrid G alpha-subunits), other major receptor/G protein contact sites are currently being identified. These studies, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.