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 resid...

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Bibliographic Details
Published in:Biochemistry (Easton) 1994-04, Vol.33 (14), p.4343-4351
Main Authors: Bernier, Michel, Liotta, Anthony S, Kole, Hemanta K, Shock, David D, Roth, Jesse
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Biological and medical sciences
Blotting, Western
Cell Membrane Permeability - drug effects
Cell receptors
Cell structures and functions
CHO Cells
Cricetinae
Digitonin
Fundamental and applied biological sciences. Psychology
Hormone receptors. Growth factor receptors. Cytokine receptors. Prostaglandin receptors
Humans
Insulin Receptor Substrate Proteins
Molecular and cellular biology
Molecular Sequence Data
Phosphoproteins - metabolism
Phosphorylation
Receptor, Insulin - genetics
Receptor, Insulin - metabolism
Recombinant Proteins - metabolism
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00180a031