ACTIVATION OF A GTP-BINDING PROTEIN AND A GTP-BINDING-PROTEIN-COUPLED RECEPTOR KINASE (BETA-ADRENERGIC-RECEPTOR KINASE-1) BY A MUSCARINIC RECEPTOR M2 MUTANT LACKING PHOSPHORYLATION SITES

A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(0) and G(12), displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [H-3]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[S-35]thiotriphosphate ([S-35]GTP[S]) binding in the presence of carbamoylcholine and GDP. The K-i values of carbamoylcholine effects on [H-3]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [H-3]qunuclidinyl benzilate or [S-35]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.