Using digitonin-permeabilized Chinese hamster ovary (CHO) cells that were transfected with intact human insulin receptors (CHO/HlRc cells), we examined insulin receptor phosphorylation and dephosphorylation using pulse-chase techniques. Insulin activated receptor autophosphorylation on tyrosyl residues to a level severalfold over basal, reaching maximal levels after 2, 5, and 10 min of stimulation at 34, 18, and 6-degrees-C, respectively. Phosphopeptide analysis revealed that the triply phosphorylated form of the 1146-kinase domain of the insulin receptor was the major species, which is characteristic of the fully active tyrosine kinase function. The dephosphorylation reaction was time- and temperature-dependent with t1/2 values of 0.67 and 2 min at 18 and 6-degrees-C, respectively. Vanadate completely inhibited dephosphorylation. Under similar permeabilization conditions when compared with CHO/HIRc cells, CHO/DELTACT cells (CHO cells overexpressing a mutated form of the receptor with a 43 amino acid deletion at the C-terminus) stimulated with insulin exhibited larger increases in receptor autophosphorylation levels and in tyrosine kinase activity toward a synthetic peptide substrate; the rate of CHO/DELTACT receptor dephosphorylation was not reduced. There was near-complete absence of insulin receptor substrate 1 (IRS-1) in the cell ghosts after permeabilization. We therefore examined the pattern of tyrosine phosphorylation and dephosphorylation of residual cellular proteins in permeabilized CHO/HlRc cells by Western blot analysis. In addition to the 95-kDa receptor beta-subunit, we detected the phosphorylation of two glycoproteins which included the commonly found 120-kDa protein and a novel 195-kDa protein whose dephosphorylation rate is slower than that of receptor beta-subunit. We conclude that there is an intimate association between insulin receptor and the protein(s) involved in the dephosphorylation cascade which is maintained in these permeabilized cells, even in the absence of IRS-1. This cell system allows new insights into insulin action (independent of IRS-1) by studying the effect of biological substances on the dynamic regulation of receptor phosphorylation and dephosphorylation as well as tyrosine kinase activity of the receptor beta-subunit.
