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 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, 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 (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 hIR delta GPLY receptor also exhibited normal endocytic functions as hIR-WT in that: a) the internalization of surface photoaffinity labeled hIR delta GPLY was similar to that of hIR-WT, and b) the rate and extent of 125I-insulin internalization and degradation at 37 degrees 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.
Berhanu
P
P
Department of Medicine, University of Colorado Health Sciences Center, Denver 80262, USA.
Anderson
C
C
Paynter
D R
DR
Wood
W M
WM
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D004705:Endocytosis; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine; D011972:Receptor, Insulin / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives / Q000502:physiology
D017931:DNA Primers; D007328:Insulin; D019000:Phosphotyrosine; D014443:Tyrosine; D011972:Receptor, Insulin