Lohse M J, Andexinger S, Pitcher J, Trukawinski S, Codina J, Faure J P, Caron M G, Lefkowitz R J
Laboratory of Molecular Biology, University of Munich, Max-Planck-Institute of Biochemistry, Martinsried, Germany.
J Biol Chem. 1992 Apr 25;267(12):8558-64.
Homologous desensitization of beta-adrenergic receptors, as well as adaptation of rhodopsin, are thought to be triggered by specific phosphorylation of the receptor proteins. However, phosphorylation alone seems insufficient to inhibit receptor function, and it has been proposed that the inhibition is mediated, following receptor phosphorylation, by the additional proteins beta-arrestin in the case of beta-adrenergic receptors and arrestin in the case of rhodopsin. In order to test this hypothesis with isolated proteins, beta-arrestin and arrestin were produced by transient overexpression of their cDNAs in COS7 cells and purified to apparent homogeneity. Their functional effects were assessed in reconstituted receptor/G protein systems using either beta 2-adrenergic receptors with Gs or rhodopsin with Gt. Prior to the assays, beta 2-receptors and rhodopsin were phosphorylated by their specific kinases beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, respectively. beta-Arrestin was a potent inhibitor of the function of beta ARK-phosphorylated beta 2-receptors. Half-maximal inhibition occurred at a beta-arrestin:beta 2-receptor stoichiometry of about 1:1. More than 100-fold higher concentrations of arrestin were required to inhibit beta 2-receptor function. Conversely, arrestin caused half-maximal inhibition of the function of rhodopsin kinase-phosphorylated rhodopsin when present in concentrations about equal to those of rhodopsin, whereas beta-arrestin at 100-fold higher concentrations had little inhibitory effect. The potency of beta-arrestin in inhibiting beta 2-receptor function was increased over 10-fold following phosphorylation of the receptors by beta ARK, but was not affected by receptor phosphorylation using protein kinase A. This suggests that beta-arrestin plays a role in beta ARK-mediated homologous, but not in protein kinase A-mediated heterologous desensitization of beta-adrenergic receptors. It is concluded that even though arrestin and beta-arrestin are similar proteins, they display marked specificity for their respective receptors and that phosphorylation of the receptors by the receptor-specific kinases serves to permit the inhibitory effects of the "arresting" proteins by allowing them to bind to the receptors and thereby inhibit their signaling properties. Furthermore, it is shown that this mechanism of receptor inhibition can be reproduced with isolated purified proteins.
β - 肾上腺素能受体的同源脱敏以及视紫红质的适应性变化，被认为是由受体蛋白的特异性磷酸化所触发。然而，仅磷酸化似乎不足以抑制受体功能，有人提出，在受体磷酸化之后，这种抑制作用是由额外的蛋白质介导的，对于β - 肾上腺素能受体而言是β - 抑制蛋白，对视紫红质而言是抑制蛋白。为了用分离的蛋白质来验证这一假说，通过在COS7细胞中瞬时过表达β - 抑制蛋白和抑制蛋白的cDNA来产生它们，并纯化至表观均一性。在重组的受体/G蛋白系统中，使用带有Gs的β2 - 肾上腺素能受体或带有Gt的视紫红质来评估它们的功能效应。在测定之前，β2 - 受体和视紫红质分别被它们的特异性激酶β - 肾上腺素能受体激酶（βARK）和视紫红质激酶磷酸化。β - 抑制蛋白是βARK磷酸化的β2 - 受体功能的强效抑制剂。半最大抑制发生在β - 抑制蛋白与β2 - 受体的化学计量比约为1:1时。需要浓度比其高100倍以上的抑制蛋白才能抑制β2 - 受体功能。相反，当抑制蛋白的浓度约等于视紫红质的浓度时，它能使视紫红质激酶磷酸化的视紫红质功能产生半最大抑制，而浓度高100倍的β - 抑制蛋白几乎没有抑制作用。在βARK使受体磷酸化后，β - 抑制蛋白抑制β2 - 受体功能的效力增加了10倍以上，但不受蛋白激酶A使受体磷酸化的影响。这表明β - 抑制蛋白在βARK介导的β - 肾上腺素能受体同源脱敏中起作用，但在蛋白激酶A介导的异源脱敏中不起作用。得出的结论是，尽管抑制蛋白和β - 抑制蛋白是相似的蛋白质，但它们对各自的受体表现出明显的特异性，并且受体特异性激酶对受体的磷酸化作用通过允许“抑制”蛋白与受体结合从而抑制其信号特性，来实现“抑制”蛋白的抑制作用。此外，研究表明这种受体抑制机制可以用分离纯化的蛋白质重现。
