Min L, Ascoli M
Department of Pharmacology, The University of Iowa College of Medicine, Iowa City 52242-1109, USA.
Mol Endocrinol. 2000 Nov;14(11):1797-810. doi: 10.1210/mend.14.11.0555.
The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact. Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.
研究了人类促黄体生成素受体（hLHR）的几种突变对细胞表面受体磷酸化、内化及周转的影响。使用了三种与睾丸间质细胞增生相关的功能获得性突变（L457R和D578Y）、一种与睾丸间质细胞腺瘤相关的突变（D578H）、一种与睾丸间质细胞发育不全相关的信号传导受损突变（I625K）以及两种实验室设计的信号传导受损突变（D405N和Y546F）。信号传导受损突变显示人绒毛膜促性腺激素（hCG）诱导的受体磷酸化和内化减少。这些功能丧失性突变体磷酸化位点的突变对内化几乎没有或没有额外影响。与G蛋白偶联受体激酶-2（GRK2）共转染可挽救hCG诱导的信号传导受损突变体的磷酸化和内化，但前提是磷酸化位点完整。抑制蛋白3的过表达可挽救内化速率，无论磷酸化位点是否完整。三个组成型激活突变体中只有两个显示基础磷酸化增加。尽管它们都未能对hCG作出反应而使受体磷酸化增加，但它们都比野生型hLHR（hLHR-wt）更快地内化hCG。这些组成型激活突变体磷酸化位点的突变使hCG内化的半衰期延长至接近hLHR-wt的水平。抑制蛋白3的过表达对这些突变体介导的hCG内化已经很短的半衰期几乎没有或没有影响。用hLHR的信号传导受损和磷酸化缺陷突变体获得的数据支持一种模型，即受体磷酸化和激活在hCG内化中起冗余作用。用组成型激活突变体获得的结果表明，当被hCG占据时，这些突变体呈现一种构象，绕过了内化过程中涉及的许多步骤（即激活、磷酸化和/或抑制蛋白结合）。
