DUAL ROLE OF A DILEUCINE MOTIF IN INSULIN-RECEPTOR ENDOCYTOSIS

Two leucines (Leu(986) and Leu(987)) have recently been shown to take part in the control of human insulin receptor (HIR) internalization (R enfrew-Haft, C,, Klausner, R, D,, and Taylor, S, I, (1994) J, Biol, Ch em, 269, 26286-26294), The aim of the present study was to further inv estigate the exact mechanism of this control process, Constitutive and insulin-induced HIR internalizations were studied biochemically and m orphologically in NIH 3T3 cells overexpressing either a double alanine (amino acid residues 986-987) mutant NIR (HIR AA1) or HIR truncated a t either amino acid residue 981 (HIR Delta 981) or 1000 (HIR Delta 100 0). Data collected indicate that: (a) the three mutant HIR show a redu ced association with microvilli as compared with HIR wild-type; (b) th e two receptors containing the dileucine motif (HIR WT and HIR Delta 1 000) show the highest propensity to associate with clathrin-coated pit s, independently of kinase activation; (c) the two receptors lacking t he dileucine motif but containing two tyrosine-based motifs, previousl y described as participating in clathrin-coated pit segregation, assoc iate with these surface domains with a lower affinity than the two oth ers, (d) in the presence of the kinase domain, an unmasking of the tyr osine-based motifs mediated by kinase activation is required. These re sults indicate that the dileucine motif is not sufficient by itself, b ut participates in anchoring HIR on microvilli and that another sequen ce, located downstream from position 1000 is crucial for this event, T his dileucine motif also plays a role in HIR segregation in clathrin-c oated pits, This latter function is additive with that of the tyrosine -based motifs but the role of the dileucine motif predominates, Eventu ally, the clathrin-coated pit anchoring function of the dileucine moti f is independent of receptor kinase activation in contrast to the tyro sine-based motifs.