Jewell-Motz E A, Liggett S B
Department of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0564, USA.
J Biol Chem. 1996 Jul 26;271(30):18082-7. doi: 10.1074/jbc.271.30.18082.
The alpha2-adrenergic receptor (alpha2AR) subtype alpha2C10 undergoes rapid agonist-promoted desensitization which is due to phosphorylation of the receptor. One kinase that has been shown to phosphorylate alpha2C10 in an agonist-dependent manner is the betaAR kinase (betaARK), a member of the family of G protein-coupled receptor kinases (GRKs). In contrast, the alpha2C4 subtype has not been observed to undergo agonist-promoted desensitization or phosphorylation by betaARK. However, the substrate specificities of the GRKs for phosphorylating alpha2AR subtypes are not known. We considered that differential capacities of various GRKs to phosphorylate alpha2C10 and alpha2C4 might be a key factor in dictating in a given cell the presence or extent of agonist-promoted desensitization of these receptors. COS-7 cells were co-transfected with alpha2C10 or alpha2C4 without or with the following GRKs: betaARK, betaARK2, GRK5, or GRK6. Intact cell phosphorylation studies were carried out by labeling cells with 32Pi, exposing some to agonist, and purifying the alpha2AR by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. BetaARK and betaARK2 were both found to phosphorylate alpha2C10 to equal extents (>2-fold over that of the endogenous kinases). On the other hand, GRK5 and GRK6 did not phosphorylate alpha2C10. In contrast to the findings with alpha2C10, alpha2C4 was not phosphorylated by any of these kinases. Functional studies carried out in transfected HEK293 cells expressing alpha2C10 or alpha2C4 and selected GRKs were consistent with these phosphorylation results. With the marked expression of these receptors, no agonist-promoted desensitization was observed in the absence of GRK co-expression. However, desensitization was imparted to alpha2C10 by co-expression of betaARK but not GRK6, while alpha2C4 failed to desensitize with co-expression of betaARK. These results indicate that short term agonist-promoted desensitization of alpha2ARs by phosphorylation is dependent on both the receptor subtype and the expressed GRK isoform.
α2 - 肾上腺素能受体（α2AR）亚型α2C10会经历快速的激动剂促进的脱敏作用，这是由于受体的磷酸化所致。已证明一种能以激动剂依赖方式使α2C10磷酸化的激酶是β - 肾上腺素能受体激酶（βARK），它是G蛋白偶联受体激酶（GRK）家族的一员。相比之下，尚未观察到α2C4亚型会经历激动剂促进的脱敏作用或被βARK磷酸化。然而，GRK对α2AR亚型进行磷酸化的底物特异性尚不清楚。我们认为，各种GRK对α2C10和α2C4进行磷酸化的不同能力可能是决定在给定细胞中这些受体激动剂促进的脱敏作用的存在或程度的关键因素。将COS - 7细胞与α2C10或α2C4共转染，转染时有的不加GRK，有的则加入以下GRK：βARK、βARK2、GRK5或GRK6。完整细胞磷酸化研究通过用32Pi标记细胞、使部分细胞暴露于激动剂以及通过免疫沉淀和SDS - 聚丙烯酰胺凝胶电泳纯化α2AR来进行。发现βARK和βARK2都能使α2C10磷酸化，且程度相同（比内源性激酶高2倍以上）。另一方面，GRK5和GRK6不能使α2C10磷酸化。与α2C10的结果相反，α2C4未被这些激酶中的任何一种磷酸化。在表达α2C10或α2C4以及选定GRK的转染HEK293细胞中进行的功能研究与这些磷酸化结果一致。在这些受体大量表达的情况下，在没有GRK共表达时未观察到激动剂促进的脱敏作用。然而，通过βARK共表达可使α2C10产生脱敏作用，而GRK6共表达则不能，同时α2C4与βARK共表达时不会产生脱敏作用。这些结果表明，通过磷酸化实现的α2ARs短期激动剂促进的脱敏作用既取决于受体亚型，也取决于所表达的GRK同工型。
