Dynamic regulation of intact and C-terminal truncated insulin receptor phosphorylation in permeabilized cells. 
Using digitonin-permeabilized Chinese hamster ovary (CHO) cells that were transfected with intact human insulin receptors (CHO/HIRc 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/delta CT 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/delta CT 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/HIRc 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.(ABSTRACT TRUNCATED AT 250 WORDS)