Amino acid sequences Gly-Pro-Leu-Tyr and Asn-Pro-Glu-Tyr in the submembranous domain of the insulin receptor are required for normal endocytosis.

We have recently shown that the immediately submembranous domain of the human insulin receptor (hIR) is required for rapid ligand-dependent internalization (Thies, R. S., Webster, N. J., and McClain, D. A. (1990) J. Biol. Chem. 265, 10132-10137). This region contains one copy of an NPXY sequence that is required for endocytosis of the low density lipoprotein receptor. In order to dissect and analyze the specific sequences involved in endocytosis of the insulin receptor, we have mutated the NPXY sequence from NPEY (residues 957-960) to APEA (NPEY/APEA). In addition, we have mutated a similar sequence in the same region, changing GPLY (residues 950-953) to APLA (GPLY/APLA). The cDNAs encoding the normal hIR and these mutant receptors were transfected into Rat 1 fibroblasts. The expressed receptors bound insulin with high affinity and retained insulin-stimulated tyrosine kinase activity. Despite the ability of these mutant receptors to bind insulin and undergo autophosphorylation, the GPLY/APLA receptor internalized insulin at only 32% of the rate of normal hIR at low receptor occupancy. On the other hand, the NPEY/APEA receptor internalized insulin at 87% of the normal rate. These results were confirmed by measuring internalization of photoaffinity-labeled insulin receptors. Another receptor with both the NPEY/APEA and GPLY/APLA mutations internalized to a lesser degree than the GPLY/APLA receptor and at a rate equivalent to that seen for a receptor with the entire submembranous domain deleted. A receptor with the complete normal submembranous domain but with the tyrosine kinase and C-terminal region of the hIR deleted exhibited only a basal internalization rate. We conclude that the information contained in the GPLY and, to a lesser extent, the NPEY sequences are necessary but not sufficient for signaling internalization of the insulin receptor.