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Phosphorylation and desensitization of human m2 muscarinic cholinergic receptors by two isoforms of the beta-adrenergic receptor kinase

1993; Elsevier BV; Volume: 268; Issue: 18 Linguagem: Inglês

10.1016/s0021-9258(19)38697-1

1083-351X

Ricardo M. Richardson, C. Kim, Jeffrey Benovic, Marie Thérèse Hosey,

Neurobiology and Insect Physiology Research

Studies of the human m2 (hm2) muscarinic cholinergic receptors (mAChR) have been performed to provide further insights into the potential regulation of these receptors by isoforms of the beta-adrenergic receptor kinase (beta ARK). The hm2 mAChR and the isoforms beta ARK1 and beta ARK2 were individually expressed in, and purified from, insect Sf9 cells infected with recombinant baculoviruses. The expressed hm2 receptors were tested as substrates for beta ARK1 and beta ARK2 in vitro using concentrations of receptors and kinases similar to those found in intact cells. The hm2 mAChR were phosphorylated in an agonist-dependent manner to 4-5 mol of phosphate/mol of receptor by beta ARK1 or beta ARK2. The reactions were highly dependent on agonist; the antagonist atropine, and heparin, a beta ARK inhibitor, both prevented the beta ARK-mediated phosphorylation. The rates of phosphorylation catalyzed by both isoforms were similar, with half-maximal phosphorylation occurring in less than 5 min. Under the conditions employed the stoichiometries, but not the rates, of phosphorylation catalyzed by both kinases were increased 2-3-fold by either the heterotrimeric G-protein G(o) or the beta gamma subunits of transducin. Phosphopeptide mapping experiments indicated that similar sites were phosphorylated by the two beta ARK isoforms. In order to test for functional effects of the phosphorylation mediated by the beta ARK isoforms, the receptors were reconstituted with purified G(o) and were tested for their ability to stimulate guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding. The conditions leading to maximal receptor phosphorylation resulted in a 30-50% reduction in the ability of the receptors to stimulate GTP gamma S binding to G(o). The results demonstrate that the hm2 mAChR are excellent substrates in vitro for both beta ARK1 and beta ARK2 and that extensive phosphorylation by these enzymes occurs in the presence of the beta gamma subunits of G proteins. The beta ARK-mediated phosphorylation of the m2 mAChR causes a perturbation of receptor/G-protein coupling.