Goncalves E, Yamada K, Thatte H S, Backer J M, Golan D E, Kahn C R, Shoelson S E
Joslin Diabetes Center, Boston, MA 02215.
Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5762-6. doi: 10.1073/pnas.90.12.5762.
Transmembrane (TM) domains of integral membrane proteins are generally thought to be helical. However, a Gly-Pro sequence within the TM domain of the insulin receptor is predicted to act as a helix breaker. CD analyses of model TM peptides in a lipid-like environment show that substitution of Gly and Pro by Ala enhances helicity. On this basis, Gly933 and Pro934 within the TM domain of the intact human insulin receptor were mutated to Ala (G-->A, P-->A, GP-->AA) to assess effects of altered helicity on receptor functions. Mutated and wild-type receptors, expressed stably in cultured CHO cells at equivalent levels, were properly assembled, biosynthetically processed, and exhibited similar affinities for insulin. Receptor autophosphorylation and substrate kinase activity in intact cells and soluble receptor preparations were indistinguishable. In contrast, insulin-stimulated receptor internalization was accelerated 2-fold for the GP-->AA mutant, compared to a wild-type control or the G-->A and P-->A mutants. Insulin degradation, which occurs during receptor endocytosis and recycling, was similarly elevated in cells transfected with GP-->AA mutant receptors. Fluorescence photobleaching recovery measurements showed that the lateral mobility of GP-->AA mutant receptors was also increased 2- to 3-fold. These results suggest that lateral mobility directly influences rates of insulin-mediated receptor endocytosis and that rates of endocytosis and lateral mobility are retarded by a kinked TM domain in the wild-type receptor. Invariance of Gly-Pro within insulin receptor TM domain sequences suggests a physiologic advantage for submaximal rates of receptor internalization.
整合膜蛋白的跨膜（TM）结构域通常被认为是螺旋状的。然而，胰岛素受体TM结构域内的一个甘氨酸 - 脯氨酸序列预计会起到螺旋破坏者的作用。在类脂环境中对模型TM肽进行的圆二色性（CD）分析表明，用丙氨酸取代甘氨酸和脯氨酸可增强螺旋性。在此基础上，将完整人类胰岛素受体TM结构域内的甘氨酸933和脯氨酸934突变为丙氨酸（G→A、P→A、GP→AA），以评估螺旋性改变对受体功能的影响。在培养的CHO细胞中稳定表达且水平相当的突变型和野生型受体，能够正确组装、进行生物合成加工，并且对胰岛素表现出相似的亲和力。完整细胞和可溶性受体制剂中的受体自身磷酸化和底物激酶活性没有差异。相比之下，与野生型对照或G→A和P→A突变体相比，GP→AA突变体的胰岛素刺激受体内化加速了2倍。在受体胞吞作用和再循环过程中发生的胰岛素降解，在用GP→AA突变体受体转染的细胞中同样有所升高。荧光漂白恢复测量表明，GP→AA突变体受体的侧向流动性也增加了2至3倍。这些结果表明，侧向流动性直接影响胰岛素介导的受体胞吞作用速率，并且野生型受体中扭结的TM结构域会阻碍胞吞作用速率和侧向流动性。胰岛素受体TM结构域序列中甘氨酸 - 脯氨酸的不变性表明受体内化亚最大速率具有生理优势。
