Richardson R M, Kim C, Benovic J L, Hosey M M
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
J Biol Chem. 1993 Jun 25;268(18):13650-6.
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.
对人m2（hm2）毒蕈碱型胆碱能受体（mAChR）进行了研究，以进一步深入了解β-肾上腺素能受体激酶（βARK）的同工型对这些受体的潜在调节作用。hm2 mAChR以及βARK1和βARK2同工型分别在感染重组杆状病毒的昆虫Sf9细胞中表达并纯化。使用与完整细胞中相似的受体和激酶浓度，在体外将表达的hm2受体作为βARK1和βARK2的底物进行测试。hm2 mAChR被βARK1或βARK2以激动剂依赖的方式磷酸化，达到每摩尔受体4 - 5摩尔磷酸盐。这些反应高度依赖激动剂；拮抗剂阿托品和βARK抑制剂肝素均能阻止βARK介导的磷酸化。两种同工型催化的磷酸化速率相似，在不到5分钟内达到最大磷酸化的一半。在所采用的条件下，异三聚体G蛋白G(o)或转导蛋白的βγ亚基使两种激酶催化的磷酸化化学计量比（而非速率）增加2 - 3倍。磷酸肽图谱实验表明，两种βARK同工型磷酸化的位点相似。为了测试βARK同工型介导的磷酸化的功能效应，将受体与纯化的G(o)重组，并测试其刺激鸟苷5'-3-O-(硫代)三磷酸（GTPγS）结合的能力。导致最大受体磷酸化的条件使受体刺激GTPγS与G(o)结合的能力降低了30 - 50%。结果表明，hm2 mAChR在体外是βARK1和βARK2的优良底物，并且在G蛋白的βγ亚基存在下，这些酶会发生广泛的磷酸化。βARK介导的m2 mAChR磷酸化会导致受体/G蛋白偶联的扰动。
