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. In these experiments, Q-D-transfected cells produced a small amount of 170,000-M_r protein (presumably the Q-D proreceptor) that disappeared after 2 h and was not processed into α- and β-subunits. The marked discrepancy in the synthesis and/or processing of the single and double mutants suggests a functional redundancy that requires the presence of carbohydrates in a certain region of the receptor, but the precise location of the carbohydrates within the region can vary.