ROLE OF THE TRANSMEMBRANE DOMAIN AND FLANKING AMINO-ACIDS IN INTERNALIZATION AND DOWN-REGULATION OF THE INSULIN-RECEPTOR

We have characterized the internalization and downregulation of the in sulin receptor and nine receptors with mutations in the transmembrane (TM) domain and/or flanking charged amino acids to define the role of this domain in receptor cycling, When expressed in Chinese hamster ova ry cells, all had normal tetrameric structure and normal insulin-stimu lated autophosphorylation/kinase activity, Replacement of the TM domai n with that of the platelet derived growth factor receptor, insertion of 3 amino acids, and substitution of Asp for Val(938) or of Ala for e ither Gly(933) Or Pro(934) had no effect on internalization Replacemen t of the TER domain with that of c-neu or conversion of the charged am ino acids on the cytoplasmic Bank to uncharged amino acids, on the oth er hand, resulted in a 40-60% decrease in insulin-dependent internaliz ation rate constants, By contrast, substitution of Ala for both Gly(93 3) and Pro(934) increases lateral diffusion mobility and accelerates i nternalization rate, These changes in internalization were due to decr eased or increased rates of redistribution of receptors from microvill i to the nonvillous cell surface, In all cases, receptor down-regulati on and receptor-mediated insulin degradation paralleled the changes in internalization Thus, the structure of the transmembrane domain of th e insulin receptor and flanking amino acids are major determinants of receptor internalization, insulin degradation, and receptor down-regul ation.