AN IRREGULARITY IN THE TRANSMEMBRANE DOMAIN HELIX CORRELATES WITH THERATE OF INSULIN-RECEPTOR INTERNALIZATION

Internalization of insulin and its receptor via receptor-mediated endo cytosis is an important step in insulin-induced signal transduction. T o investigate the structural determinants underlying the enhanced inte rnalization rate observed for the insulin receptor transmembrane mutan t Gly(933)-Pro(934) --> Ala-Ala (GP --> AA), we have designed and chem ically synthesized two peptides, IR(TM)-GP and IR(TM)-AA, correspondin g respectively to the N-terminal portion of the wild-type and the muta nt insulin receptor TM segment containing these sites. Conformational studies by circular dichroism (CD) spectroscopy on these two peptides in their monomeric states revealed that peptide IR(TM)-GP forms an irr egular helix in the membrane-mimetic environments of sodium dodecyl su lfate (SDS) micelles with a possible ''kink'' in the helix imposed by its Gly-Pro sequence, while peptide IR(TM)-AA assumes largely classica l alpha-helical structure under corresponding conditions. The helical pattern of peptide IR(TM)-AA was maintained at elevated temperatures, while the shape of the CD curve for peptide IR(TM)-GP was found to alt er as a function of temperature. At higher concentrations, both peptid es formed high molecular weight aggregates in SDS micelles, as demonst rated by SDS-PAGE gels, but peptide IR(TM)-AA was shown to aggregate m ore readily and more extensively than peptide IR(TM)-GP. Fluorescent d ye-leakage experiments indicated that peptide IR(TM)-GP produces an en hanced disruption of the membrane bilayer in phosphatidylglycerol vesi cles vs that induced by IR(TM)-AA. The overall results are consistent with the notion that a Gly-Pro kink in the TM helix of the wild-type i nsulin receptor may submaximize the rate of its internalization by ret arding its lateral movement through the plasma membrane and, according ly, indicate an active role for the TM segment in receptor-mediated en docytosis.