OPTIMIZING TRANSMEMBRANE DOMAIN HELICITY ACCELERATES INSULIN-RECEPTOR INTERNALIZATION AND LATERAL MOBILITY

被引:29
作者
GONCALVES, E
YAMADA, K
THATTE, HS
BACKER, JM
GOLAN, DE
KAHN, CR
SHOELSON, SE
机构
[1] JOSLIN DIABET CTR,1 JOSLIN PL,BOSTON,MA 02215
[2] BRIGHAM & WOMENS HOSP,DEPT MED,BOSTON,MA 02115
[3] HARVARD UNIV,SCH MED,DEPT MED,BOSTON,MA 02115
[4] BRIGHAM & WOMENS HOSP,DIV HEMATOL ONCOL,BOSTON,MA 02115
[5] HARVARD UNIV,SCH MED,DEPT MED,BOSTON,MA 02115
[6] HARVARD UNIV,SCH MED,DEPT BIOL CHEM,BOSTON,MA 02115
[7] HARVARD UNIV,SCH MED,DEPT MOLEC PHARMACOL,BOSTON,MA 02115
关键词
D O I
10.1073/pnas.90.12.5762
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:5762 / 5766
页数:5
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