The Amino Acid Sequence GPLY Is Not Necessary for Normal Endocytosis of the Human Insulin Receptor B Isoform

Abstract

The human insulin receptor (hIR) cytoplasmic juxtamembrane domain contains two tyrosine (Y) residues which exist in GPLY and NPEY motifs that have been implicated in endocytic function. We have previously shown that the NPEY motif is not necessary for endocytosis of the B isoform (exon 11+) of hIR. To examine the role of the GPLY sequence in transmembrane insulin signaling and endocytic functions of hIR-B, we constructed a mutant receptor, hIRΔGPLY, that lacks the GPLY sequence (residues 962-965), and stably expressed it in CHO cells. When compared to wild type hIR-B (hIR-WT) similarly expressed in CHO cells, the hIRΔGPLY mutant exhibited higher insulin binding affinity (EC50 of 1.0 vs 3.5 nM) and normal insulin-stimulated receptor tyrosine autophosphorylation and kinase activity towards the endogenous 185 kDa insulin receptor substrate. The hIRΔGPLY receptor also exhibited normal endocytic functions as hIR-WT in that: a) the internalization of surface photoaffinity labeled hIRΔGPLY was similar to that of hIR-WT, and b) the rate and extent of 125I-insulin internalization and degradation at 37°C were also unimpaired. Therefore, these results demonstrate that the GPLY sequence is not necessary for transmembrane insulin signaling and endocytic functions of the hIR-B isoform.