ANALYSIS OF THE JUXTAMEMBRANE DILEUCINE MOTIF IN THE INSULIN-RECEPTOR

Dileucine-containing motifs are involved in trans-Golgi sorting, lysos omal targeting, and internalization. Previously, we have shown that th e dileucine motif (EKITLL, residues 982-987) in the juxtamembrane regi on of the insulin receptor is involved in receptor internalization. Su bstitution of alanine residues for Leu(986) and Leu(987) led to a 3- t o 5-fold decrease in the ability of the receptors to mediate insulin u ptake. In the current study, we show that mutation of the same motif t o Met(986)Ser(987), the sequence found in the homologous position in t he type I insulin-like growth factor receptor, did not affect insulin uptake. Therefore, we inquired whether the sequence EKITMS as an isola ted motif could mediate the targeting of a reporter molecule to endoso mes and then lysosomes, as was shown previously with the EKITLL motif of the normal receptor. Chimeric molecules containing Tac antigen fuse d to different hexapeptide sequences showed distinct patterns of subce llular localization by immunofluorescence microscopy. Tac-EKITLL and T ac-EKITAA were found predominantly in lysosomes and the plasma membran e, respectively. In contrast, Tac-EKITMS was found at the plasma membr ane, in the trans-Golgi network, and in endosomes, but only small amou nts were found in lysosomes. Thus, the dileucine motif (EKITLL) plays an important role in directing endocytosis of the intact insulin recep tor and in mediating efficient endocytosis and lysosomal targeting as an isolated motif. Substitution of AA for LL inhibits endocytosis and lysosomal targeting in both systems. In contrast, substitution of MS f or LL permits rapid endocytosis in the intact receptor, but mediates m odest endocytosis and very little targeting to lysosomes as an isolate d motif. Our observations support the idea that sorting signals are re cognized at multiple steps in the cell, and that specific amino acid s ubstitutions may differentially affect each of these sorting steps.