Insulin receptor kinase activation releases a constraint maintaining the receptor on microvilli

To examine whether the surface redistribution of the insulin receptor from microvilli, where it sits in its unoccupied form, to the nonvillous domain, where it is internalized through clathrin-coated pits, is an active movement or a passive redistribution linked to the release of a restraint maintaining it on microvilli, we have generated a mutated insulin receptor with a truncation of exons 17-22 and tracked it biochemically and morphologically. Biochemical analysis indicates that this mutated receptor is constitutively internalized and recycled even in the absence of ligand. Quantitative electron microscope autoradiography analysis reveals that it does not preferentially associate with microvilli in its unoccupied form but is normally segregated in clathrin-coated pits through the preserved signal sequence(s) of exon 16. We conclude that (a) insulin receptor internalization initiated through receptor kinase activation and autophosphorylation, which free the receptor from constraints maintaining it on microvilli; (b) the signal sequences contained in exon 16 are entirely sufficient to promote clathrin-coated pit-mediated internalization of insulin receptors; (c) these sequences are not uncovered by kinase activation; and (d) the "code" maintaining the unoccupied receptors on microvilli is contained within exons 17-21 of the receptor.