Gurevich V V, Richardson R M, Kim C M, Hosey M M, Benovic J L
Department of Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611.
J Biol Chem. 1993 Aug 15;268(23):16879-82.
Arrestins play an important role in regulating the activity of the G protein-coupled receptors rhodopsin and the beta 2-adrenergic receptor. Recently, we described the expression and functional characterization of visual arrestin using an in vitro translation system. Here we report the expression of beta-arrestin and development of a direct binding assay to study the interaction of arrestins with a muscarinic cholinergic receptor. In vitro translated beta-arrestin was found to specifically bind to purified reconstituted human m2 muscarinic cholinergic receptor (hm2 mAChR) in an agonist- and phosphorylation-dependent manner. Visual arrestin also bound to the hm2 mAChR, albeit to a lesser extent and with lower affinity. In an attempt to dissect the major domains responsible for determining the receptor binding specificity of arrestin and beta-arrestin, we generated several chimeric arrestins. One contained the first 340 residues of beta-arrestin followed by residues 346-404 of arrestin (BRV4), another consisted of the first 207 residues of beta-arrestin and residues 214-404 of visual arrestin (BV3), and a third had residues 1-43 of beta-arrestin replaced by residues 1-47 of arrestin (VIN1). All of these arrestins were able to specifically bind to the activated and phosphorylated form of both the hm2 mAChR and rhodopsin, with a clear preference for the muscarinic receptor. The Kd values for beta-arrestin, BRV4, BV3, VIN1, and visual arrestin binding to the hm2 mAChR were 0.48 +/- 0.06, 0.51 +/- 0.19, 1.38 +/- 0.26, 1.13 +/- 0.26, and 7.2 +/- 1.2 nM, respectively. These data demonstrate that: 1) beta-arrestin binds to the hm2 mAChR in an activation- and phosphorylation-dependent fashion, 2) visual arrestin has 15-fold lower affinity for the hm2 mAChR as compared to beta-arrestin, and 3) the N-terminal half of beta-arrestin plays a key role in determining receptor binding specificity. The use of in vitro translated arrestins to directly assess receptor binding may serve as a viable approach for elucidating the specificity and molecular mechanisms involved in receptor-arrestin interaction.
抑制蛋白在调节G蛋白偶联受体视紫红质和β2-肾上腺素能受体的活性中发挥着重要作用。最近，我们利用体外翻译系统描述了视觉抑制蛋白的表达及功能特性。在此，我们报告β-抑制蛋白的表达以及一种直接结合测定法的开发，以研究抑制蛋白与毒蕈碱型胆碱能受体的相互作用。发现体外翻译的β-抑制蛋白以激动剂和磷酸化依赖性方式特异性结合纯化的重组人m2毒蕈碱型胆碱能受体（hm2 mAChR）。视觉抑制蛋白也与hm2 mAChR结合，尽管程度较低且亲和力较低。为了剖析负责确定抑制蛋白和β-抑制蛋白受体结合特异性的主要结构域，我们构建了几种嵌合抑制蛋白。一种包含β-抑制蛋白的前340个残基，接着是抑制蛋白的346 - 404位残基（BRV4），另一种由β-抑制蛋白的前207个残基和视觉抑制蛋白的214 - 404位残基组成（BV3），第三种是将β-抑制蛋白的1 - 43位残基替换为抑制蛋白的1 - 47位残基（VIN1）。所有这些抑制蛋白都能够特异性结合hm2 mAChR和视紫红质的活化及磷酸化形式，且明显更倾向于毒蕈碱受体。β-抑制蛋白、BRV4、BV3、VIN1和视觉抑制蛋白与hm2 mAChR结合的Kd值分别为0.48±0.06、0.51±0.19、1.38±0.26、1.13±0.26和7.2±1.2 nM。这些数据表明：1）β-抑制蛋白以激活和磷酸化依赖性方式结合hm2 mAChR；2）与β-抑制蛋白相比，视觉抑制蛋白对hm2 mAChR的亲和力低15倍；3）β-抑制蛋白的N端一半在确定受体结合特异性中起关键作用。利用体外翻译的抑制蛋白直接评估受体结合可能是阐明受体 - 抑制蛋白相互作用所涉及的特异性和分子机制的一种可行方法。
