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

Kameyama, K.; Haga, K.; Haga, T.; Moro, O.; Sadée, W.

European Journal of Biochemistry 226(2): 267-276

1994


ISSN/ISBN: 0014-2956
PMID: 8001544
Accession: 008100415

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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 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-o and G-i2, 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 K-i 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 beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of (3H)quinuclidinyl benzilate or (35S)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.