Receptor-mediated internalization of insulin requires a 12-amino acid sequence in the juxtamembrane region of the insulin receptor β-subunit

The juxtamembrane region of the insulin receptor (IR) β-subunit contains an unphosphorylated tyrosyl residue (Tyr⁹⁶⁰) that is essential for insulin-stimulated tyrosyl phosphorylation of some endogenous substrates and certain biological responses (White, M.F., Livingston, J.N., Backer, J.M., Lauris, V., Dull, T.J., Ullrich, A., and Kahn, C.R. (1988) Cell 54, 641-649). Tyrosyl residues in the juxtamembrane region of some plasma membrane receptors have been shown to be required for their internalization. In addition, a juxtamembrane tyrosine in the context of the sequence NPXT is required for the coated pit-mediated internalization of the low density lipoprotein receptor. To examine the role of the juxtamembrane region of the insulin receptor during receptor-mediated endocytosis, we have studied the internalization of insulin by Chinese hamster ovary (CHO) cells expressing two mutant receptors: IR(F960), in which Tyr⁹⁶⁰ has been substituted with phenylalanine, and IR(Δ960), in which 12 amino acids (Ala⁹⁵⁴-Asp⁹⁶⁵), including the putative consensus sequence NPXT, were deleted. Although the in vivo autophosphorylation of IR(F960) and IR(Δ960) was similar to wild type, neither mutant could phosphorylate the endogenous substrate pp185. CHO/IR(F960) cells internalized insulin normally whereas the intracellular accumulation of insulin by CHO/IR(Δ960) cells was 20-30% of wild-type. However, insulin internalization in the CHO-IR(Δ960) cells was consistently more rapid than that occurring in CHO cells expressing kinase-deficient receptors (CHO/IR(A1018)). The degradation of insulin was equally impaired in CHO/IR(Δ960) and CHO/IR(A1018) cells. These data show that the juxtamembrane region of the insulin receptor contains residues essential for insulin-stimulated internalization and suggest that the sequence NPXT may play a general role in directing the internalization of cell surface receptors.