A truncated human insulin receptor missing the COOH-terminal 365 amino acid residues does not undergo insulin-mediated receptor migration or aggregation.

Abstract

A previous study of tyrosine kinase-defective insulin receptors demonstrated that receptor autophosphorylation or tyrosine kinase activity was required for concentrating insulin receptors in coated pits, but not for their migration or aggregation on the cell surface. Furthermore, receptor migration and aggregation on the cell surface were not sufficient to cause internalization of the occupied receptors in coated pits. In the present study, biochemical and ultrastructural techniques were used to compare insulin receptor mobility and internalization in Rat 1 fibroblasts expressing wild-type human insulin receptors (HIRc) with those in cells expressing receptors truncated at residues 978 (HIR delta 978) or 1301 of the carboxyl-terminus (HIR delta CT). There were no significant differences in the mobility or internalization of insulin receptors on HIR delta CT cells compared to those of insulin receptors on HIRc cells. Ultrastructural analysis revealed that truncated insulin receptors on HIR delta 978 cells failed to migrate from their initial location on the microvilli, move to the plasma membrane, and aggregate in coated pits. Receptor-mediated insulin internalization in HIR delta 978 cells was markedly decreased due entirely to a decrease in ATP-dependent, coated pit-mediated internalization. ATP-independent endocytosis in non-coated pinocytotic invaginations was not affected by receptor truncations. These results provide evidence of the roles that regions of the beta-subunit play in the processing of occupied insulin receptors. 1) The carboxyl-terminus of the insulin receptor is not involved in the events leading to receptor internalization, i.e. migration, aggregation, and concentration in coated pits. 2) Internalization of insulin receptors by the ATP-independent noncoated invagination pathway is not regulated by residues in the insulin receptor beta-subunit distal to 978. 3) Sequences in the beta-subunit between 978-1300, but not the autophosphorylation and kinase domains, are involved in insulin-induced receptor migration and aggregation.