2 STEPS OF INSULIN-RECEPTOR INTERNALIZATION DEPEND ON DIFFERENT DOMAINS OF THE BETA-SUBUNIT

The internalization of signaling receptors such as the insulin recepto r is a complex, multi-step process. The aim of the present work was to determine the various steps in internalization of the insulin recepto r and to establish which receptor domains are implicated in each of th ese by the use of receptors possessing in vitro mutations. We find tha t kinase activation and autophosphorylation of all three regulatory ty rosines 1146, 1150, and 1151, but not tyrosines 1316 and 1322 in the C OOH-terminal domain, are required for the ligand-specific stage of the internalization process; i.e., the surface redistribution of the rece ptor from microvilli where initial binding occurs to the nonvillous do main of the cell. Early intracellular steps in insulin signal transduc tion involving the activation of phosphatidylinositol 3'-kinase are no t required for this redistribution. The second step of internalization consists in the anchoring of the receptors in clathrin-coated pits. I n contrast to the first ligand specific step, this step is common to m any receptors including those for transport proteins and occurs in the absence of kinase activation and receptor autophosphorylation, but re quires a juxtamembrane cytoplasmic segment of the beta-subunit of the receptor including a NPXY sequence. Thus, there are two independent me chanisms controlling insulin receptor internalization which depend on different domains of the beta-subunit.