Ramkumar V, Kwatra M, Benovic J L, Stiles G L, Stilesa G L
Department of Medicine, Duke University Medical Center, Durham, NC 27710.
Biochim Biophys Acta. 1993 Oct 7;1179(1):89-97. doi: 10.1016/0167-4889(93)90075-z.
Treatment of smooth-muscle cells with R-phenylisopropyladenosine (R-PIA) leads to a loss of A1 adenosine receptor (A1AR)-mediated inhibition of adenylate cyclase, a decrease in receptor number and an increase in receptor phosphorylation. In this study, the role of the beta-adrenergic receptor kinase (beta ARK) in the phosphorylation and inactivation of the A1AR was examined. A1ARs were purified from bovine brain and reconstituted into phospholipid vesicles, with or without a 10-fold excess of Gi/Go (a 50:50 mixture). The reconstituted receptor preparations were phosphorylated with beta ARK in the absence (control) or presence (treated) of R-PIA. R-PIA stimulated A1AR phosphorylation by 2-3-fold over control. Phosphorylation of the A1AR was blocked by XAC, and A1AR antagonist, underscoring its agonist dependence. The stoichiometry of phosphorylation obtained was approx. 1.3 mol of phosphate per mol of A1AR. Phosphorylation of the A1AR by beta ARK was enhanced by an additional 42% when G beta gamma (30 nM) was included in the phosphorylation mixture. In order to test the role of phosphorylation on receptor function, the purified A1AR was reconstituted with a mixture of Gi/Go, phosphorylated with beta ARK and used to determine high-affinity [125I]APNEA (A1AR agonist) binding. Agonist binding was reduced by about 50% in the treated preparations compared to control. In contrast, antagonist ([3H]XAC) binding was increased by about 50%. These data are consistent with an uncoupling of the A1AR from G proteins following receptor phosphorylation. In control preparations, R-PIA stimulated GTPase activity from 0.08 to 0.164 pmol Pi released/pmol Gi/Go per min. Phosphorylation of receptor by beta ARK reduced R-PIA-stimulated GTPase activity by 35%. In addition, phosphorylation of the A1AR by beta ARK decreased R-PIA-stimulated GTP gamma S binding by 62%. These data provide evidence that A1AR phosphorylation by beta ARK results in a diminished receptor-G-protein interaction.
用R-苯异丙基腺苷（R-PIA）处理平滑肌细胞会导致A1腺苷受体（A1AR）介导的腺苷酸环化酶抑制作用丧失、受体数量减少以及受体磷酸化增加。在本研究中，检测了β-肾上腺素能受体激酶（βARK）在A1AR磷酸化和失活中的作用。从牛脑中纯化A1AR，并将其重构成磷脂囊泡，添加或不添加10倍过量的Gi/Go（50:50混合物）。在不存在（对照）或存在（处理）R-PIA的情况下，用βARK对重构的受体制剂进行磷酸化。与对照相比，R-PIA刺激A1AR磷酸化增加了2 - 3倍。A1AR拮抗剂XAC可阻断A1AR的磷酸化，突出了其对激动剂的依赖性。获得的磷酸化化学计量约为每摩尔A1AR 1.3摩尔磷酸盐。当磷酸化混合物中包含Gβγ（30 nM）时，βARK对A1AR的磷酸化作用增强了42%。为了测试磷酸化对受体功能的作用，将纯化的A1AR与Gi/Go混合物重构，用βARK进行磷酸化，并用于测定高亲和力[125I]APNEA（A1AR激动剂）结合。与对照相比，处理后的制剂中激动剂结合减少了约50%。相反，拮抗剂（[3H]XAC）结合增加了约50%。这些数据与受体磷酸化后A1AR与G蛋白解偶联一致。在对照制剂中，R-PIA刺激GTP酶活性从每分钟释放0.08皮摩尔无机磷/皮摩尔Gi/Go增加到0.164皮摩尔。βARK对受体的磷酸化使R-PIA刺激的GTP酶活性降低了35%。此外，βARK对A1AR的磷酸化使R-PIA刺激的GTPγS结合减少了62%。这些数据提供了证据，表明βARK对A1AR的磷酸化导致受体 - G蛋白相互作用减弱。
