Lazari M F, Bertrand J E, Nakamura K, Liu X, Krupnick J G, Benovic J L, Ascoli M
Department of Pharmacology, The University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
J Biol Chem. 1998 Jul 17;273(29):18316-24. doi: 10.1074/jbc.273.29.18316.
We have previously mapped the agonist-induced phosphorylation of the rat lutropin/choriogonadotropin receptor (rLHR) to a locus of four serines (Ser635, Ser639, Ser649, and Ser652) located in the C-terminal tail. The removal or mutation of this locus delays the time course of agonist-induced uncoupling of the rLHR from its effector system without affecting the overall magnitude of uncoupling, and it retards the endocytosis of the agonist-receptor complex. We have now prepared and analyzed four new rLHR mutants in which each of these serines were individually mutated to alanines. The data presented show that each mutation reduces agonist-promoted rLHR phosphorylation by 20-40%. Mutation of Ser635 or Ser639 delayed the time course of agonist-induced uncoupling to about the same extent as the simultaneous mutation of all four serines. Mutation of Ser635 or Ser639 also retarded agonist-induced internalization, but the magnitude of this decrease was less than that induced by the simultaneous mutation of all four serines. Mutation of Ser649 had no effect on agonist-induced uncoupling but retarded agonist-induced internalization to the same extent as the simultaneous mutation of all four serines. Mutation of Ser652 has little or no effect on either of these two parameters. Co-transfection studies with dominant-negative arrestins and dominant-negative dynamin reveal that, despite differences in their rates of internalization, rLHR-wild-type, rLHR-S639A, and rLHR-S649A are internalized by an arrestin- and dynamin-dependent pathway. These data show that the structural requirements needed for the agonist-induced uncoupling and internalization of the rLHR are distinct.
我们之前已将激动剂诱导的大鼠促黄体激素/绒毛膜促性腺激素受体（rLHR）磷酸化定位至位于C末端尾巴的四个丝氨酸位点（Ser635、Ser639、Ser649和Ser652）。该位点的去除或突变会延迟激动剂诱导的rLHR与其效应系统解偶联的时间进程，而不影响解偶联的总体幅度，并且会延缓激动剂-受体复合物的内吞作用。我们现在制备并分析了四个新的rLHR突变体，其中每个丝氨酸都分别突变为丙氨酸。所呈现的数据表明，每个突变都会使激动剂促进的rLHR磷酸化降低20%-40%。Ser635或Ser639的突变使激动剂诱导的解偶联时间进程延迟的程度与所有四个丝氨酸同时突变的情况大致相同。Ser635或Ser639的突变也会延缓激动剂诱导的内化作用，但这种降低的幅度小于所有四个丝氨酸同时突变所诱导的幅度。Ser649的突变对激动剂诱导的解偶联没有影响，但会使激动剂诱导的内化作用延缓至与所有四个丝氨酸同时突变相同的程度。Ser652的突变对这两个参数中的任何一个几乎没有影响。与显性负性抑制蛋白和显性负性发动蛋白的共转染研究表明，尽管rLHR野生型、rLHR-S639A和rLHR-S649A的内化速率存在差异，但它们都是通过抑制蛋白和发动蛋白依赖性途径内化的。这些数据表明，激动剂诱导的rLHR解偶联和内化所需的结构要求是不同的。
