ACTIVATION OF A GTP-BINDING PROTEIN AND A GTP-BINDING-PROTEIN-COUPLEDRECEPTOR 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 syste m. The mutant was not phosphorylated by beta-adrenergic-receptor kinas e, as expected from the previous assignment of phosphorylation sites t o the central part of the third intracellular loop. However, the m2 re ceptor mutant was capable of stimulating beta-adrenergic-receptor-kina se-1-mediated phosphorylation of a glutathione S-transferase fusion pr otein 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 bin ding, as assessed by displacement of [H-3]quinuclidinyl-benzilate bind ing with carbamoylcholine, and both stimulated guanosine 5'-3-O-[S-35] thiotriphosphate ([S-35]GTP[S]) binding in the presence of carbamoylch oline and GDP. The K-i values of carbamoylcholine effects on [H-3]quin uclidinyl-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 in teraction with G proteins as assessed by the binding of [H-3]qunuclidi nyl 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-adrene rgic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G prot eins and its phosphorylation by beta-adrenergic-receptor kinase does n ot uncouple the receptor and G proteins in reconstituted lipid vesicle s.