Glycosylation of Asn397 or Asn418 is required for normal insulin receptor biosynthesis and processing.
Two N-linked sites of glycosylation in the insulin receptor were examined for their contribution to insulin binding, tyrosine kinase activity, and receptor biosynthesis. Asn397 and Asn418 were replaced by Gln using site-directed mutagenesis either as single mutations, i.e., Q-397 and Q-418, or as a double mutation in which both sites were removed (Q-D). The mutations were transiently expressed in COS cells and the findings compared with cells that transiently expressed the wild-type human insulin receptor. Q-397 and Q-418 mutant insulin receptors had insulin-binding characteristics similar to the wild-type human insulin receptor, whereas no insulin-binding activity could be detected above the control level in cells transfected with Q-D. Flow cytometry with antibodies against the human insulin receptor indicated the presence of Q-397, Q-418, and wild-type human insulin receptors in the surface of COS cells and failed to demonstrate a Q-D receptor. Insulin-induced autophosphorylation was similar in Q-397, Q-418, and wild-type human insulin receptors as was their ability to phosphorylate an artificial substrate, poly Glu-Tyr (4:1). Our inability to detect Q-D receptors was not caused by a lack of Q-D mRNA. COS cells transfected with Q-D cDNA generated as much Q-D mRNA as the amount of wild-type human insulin receptor mRNA present in cells transfected with wild-type receptor cDNA. Finally, pulse-chase experiments with [35S]Met were able to detect 190,000-M(r) proreceptors and the alpha-subunits for Q-397, Q-418, and wild-type human insulin receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
University of Rochester Medical Center, Department of Medicine, New York.
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W
Bastian
J
J
Zhu
B
B
Way
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D
Lockwood
J
J
Livingston
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D001483:Base Sequence; D002460:Cell Line; D005434:Flow Cytometry; D006031:Glycosylation; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D011972:Receptor, Insulin / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014162:Transfection
D007328:Insulin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D001216:Asparagine; D008715:Methionine; D011972:Receptor, Insulin