OPTIMIZING TRANSMEMBRANE DOMAIN HELICITY ACCELERATES INSULIN-RECEPTORINTERNALIZATION AND LATERAL MOBILITY

Transmembrane (TM) domains of integral membrane proteins are generally thought to be helical. However, a Gly-Pro sequence within the TM doma in 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 su bstitution of Gly and Pro by Ala enhances helicity. On this basis, Gly 933 and Pro934 within the TM domain of the intact human insulin recept or were mutated to Ala (G --> A, P --> A, GP --> AA) to assess effects of altered helicity on receptor functions. Mutated and wild-type rece ptors, expressed stably in cultured CHO cells at equivalent levels, we re properly assembled, biosynthetically processed, and exhibited simil ar affinities for insulin. Receptor autophosphorylation and substrate kinase activity in intact cells and soluble receptor preparations were indistinguishable. In contrast, insulin-stimulated receptor internali zation was accelerated 2-fold for the GP --> AA mutant, compared to a wild-type control or the G --> A and P --> A mutants. Insulin degradat ion, which occurs during receptor endocytosis and recycling, was simil arly elevated in cells transfected with GP --> AA mutant receptors. Fl uorescence photobleaching recovery measurements showed that the latera l mobility of GP --> AA mutant receptors was also increased 2- to 3-fo ld. These results suggest that lateral mobility directly influences ra tes of insulin-mediated receptor endocytosis and that rates of endocyt osis and lateral mobility are retarded by a kinked TM domain in the wi ld-type receptor. Invariance of Gly-Pro within insulin receptor TM dom ain sequences suggests a physiologic advantage for submaximal rates of receptor internalization.