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 exami ned for their contribution to insulin binding, tyrosine kinase activit y, and receptor biosynthesis. Asn397 and Asn418 were replaced by Gln u sing 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 fi ndings compared with cells that transiently expressed the wild-type hu man insulin receptor. 0-397 and Q-418 mutant insulin receptors had ins ulin-binding characteristics similar to the wild-type human insulin re ceptor, whereas no insulin-binding activity could be detected above th e control level in cells transfected with Q-D. Flow cytometry with ant ibodies against the human insulin receptor indicated the presence of Q -397, Q-418, and wild-type human insulin receptors in the surface of C OS cells and failed to demonstrate a Q-D receptor. insulin-induced aut ophosphorylation was similar in Q-397, 0-418, and wild-type human insu lin receptors as was their ability to phosphorylate an artificial subs trate, poly Glu-Tyr (4:1). Our inability to detect Q-D receptors was n ot caused by a lack of Q-D mRNA. COS cells transfected with Q-D cDNA g enerated as much Q-D mRNA as the amount of wild-type human insulin rec eptor mRNA present in cells transfected with wild-type receptor cDNA. Finally, pulse-chase experiments with [S-35]Met were able to detect 19 0,000-M(r) proreceptors and the alpha-subunits for Q-397, Q-418, and w ild-type human insulin receptors. In these experiments, Q-D-transfecte d cells produced a small amount of 170,000-M(r) protein (presumably th e Q-D proreceptor) that disappeared after 2 h and was not processed in to alpha- and beta-subunits. The marked discrepancy in the synthesis a nd/or processing of the single and double mutants suggests a functiona l 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.