Chinese hamster ovary (CHO) cells expressing human insulin receptor (hIR) of the wild-type (CHO R) or hIR mutated at tyrosines 1162 and 1163 (CHO Y2) were compared for agonist-induced receptor phosphorylation of serine/threonine residues and receptor desensitization. Relative to CHO R cells, CHO Y2 cells exhibited a marked decrease in their response to insulin and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) for hIR phosphorylation on serine residues. Moreover, the tyr1162,1163 mutant hIR could not be normally phosphorylated by purified protein kinase C (PKC) in vitro. Finally, in contrast to CHO R cells, CHO Y2 cells were refractory to PMA-induced IR desensitization for subsequent activation by insulin of exogenous tyrosine kinase and glycogen synthesis. These results strongly suggest that the replacement of tyrosines 1162 and 1163 by phenylalanine residues changes the IR beta-subunit conformation and thus impedes phosphorylation of the IR at crucial serine residues and prevents PMA-induced desensitization. This supports the hypothesis that IR serine phosphorylation and desensitization are related.