When insulin binds to the insulin receptor, this stimulates endocytosis of the hormone-receptor complex. Intact tyrosine kinase activity is required for the receptor to undergo ligand-stimulated endocytosis. In this project, we are undertaking to identify other sequence motifs in the intracellular domain of the insulin receptor that are required for endocytosis. We have obtained evidence that the dileucine motif (EKITLL, residues 982-987) found in the juxtamembrane region of the insulin receptor is involved in receptor internalization. Substitution of alanine for the two leucines in this juxtamembrane sequence resulted in a 3- to 5-fold decrease of insulin uptake. In contrast, mutation of the same motif to EKITMS (the sequence found in the homologous position in the type I insulin-like growth factor receptor) does not affect insulin uptake. Therefore, we inquired whether the sequence EKITMS could mimic the ability of the dileucine-containing sequence (EKITLL) to direct targeting to lysosomes. To address this question, we expressed chimeric receptors containing the human Tac antigen fused to three different hexapeptide sequences. The three chimeric proteins showed distinct patterns of subcellular localization. Tac-EKITLL and Tac-EKITAA were found predominantly in lysosomes and the plasma membrane, respectively. In contrast, Tac-EKITMS was found in plasma membrane, endosomes, and trans-Golgi network with only small amounts in lysosomes. Furthermore, although Tac-EKITLL mediated rapid endocytosis of 125I-labeled anti-Tac antibody, endocytosis of 125I-labeled anti-Tac antibody proceeded very slowly in cells expressing Tac-EKITMS or Tac-EKITAA. Thus substitution of AA for LL inhibited endocytosis in both systems. However, the MS substitution did not inhibit endocytosis in the intact insulin receptor, but retarded the rate of endocytosis of the Tac-chimra. Nevertheless, because the two mutations (MS vs. AA) differentially affected the subcellular localization of the Tac-chimeras, this strongly suggests that dileucine motifs function at multiple steps in intracellular targeting, and that specific mutations may differentially.
