THE AMINO-ACID-SEQUENCE GPLY IS NOT NECESSARY FOR NORMAL ENDOCYTOSIS OF THE HUMAN INSULIN-RECEPTOR-B ISOFORM

The human insulin receptor (hIR) cytoplasmic juxtamembrane domain cont ains two tyrosine (Y) residues which exist in GPLY and NPEY motifs tha t 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 transm embrane insulin signaling and endocytic functions of hIR-B, we constru cted a mutant receptor, hIR Delta GPLY, 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 hIR Delta GPLY mutant exhibited higher insulin binding affinity (EC(50) o f 1.0 vs 3.5 nM) and normal insulin-stimulated receptor tyrosine autop hosphorylation and kinase activity towards the endogenous 185 kDa insu lin receptor substrate. The hIR Delta GPLY receptor also exhibited nor mal endocytic functions as hIR-WT in that: a) the internalization of s urface photoaffinity labeled hIR Delta GPLY was similar to that of hIR -WT, and b) the rate and extent of I-125-insulin internalization and d egradation at 37 degrees C were also unimpaired. Therefore, these resu lts demonstrate that the GPLY sequence is not necessary for transmembr ane insulin signaling and endocytic functions of the hIR-B isoform. (C ) 1995 Academic Press, Inc.