Activation of a GTP‐Binding Protein and a GTP‐Binding‐Protein‐Coupled Receptor Kinase (β‐Adrenergic‐Receptor Kinase‐1) by a Muscarinic Receptor m2 Mutant Lacking Phosphorylation Sites

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Abstract
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 β‐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 β‐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), Go and Gi2, displayed guanine‐nucleotide‐sensitive high‐affinity agonist binding, as assessed by displacement of [3H] quinuclidinylbenzilate binding with carbamoylcholine, and both stimulated guanosine 5′‐3‐O ‐[35S] thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H] quinuclidinyl‐benzilate binding were indistinguishable for the mutant and wild‐type m2 receptors. Moreover, the phosphorylation of the wild‐type m2 receptor by β‐adrenergic‐receptor kinase‐1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]qunuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of β‐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 β‐adrenergic‐receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.