Cascade of autophosphorylation in the β‐subunit of the insulin receptor

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Abstract
Insulin stimulated autophosphorylation of the β‐subunit of the insulin receptor purified from Fao hepatoma cells or purified from Chinese hamster ovary (CHO/HIRC) or Swiss 3T3 (3T3/HIRC) cells transfected with the wild‐type human insulin receptor cDNA. Autophosphorylation of the purified receptor occurred in at least two regions of the β‐subunit: the regulatory region containing Tyr‐1146, Tyr‐1150, and Tyr‐1151, and the C‐terminus containing Tyr‐1316 and Tyr‐1322. In the presence of antiphosphotyrosine antibody (α‐PY), autophosphorylation of the purified receptor was inhibited nearly 80% during insulin stimulation. Tryptic peptide mapping showed that α‐PY inhibited autophosphorylation of both tyrosyl residues in the C‐terminus and one tyrosyl residue in the regulatory region, either Tyr‐1150 or Tyr‐1151. Thus, a bis‐phosphorylated form of the regulatory region accumulated in the presence of α‐PY, which contained Tyr(P)‐1146 and either Tyr(P)‐1150 or 1151. In intact Fao, CHO/HIRC, and 3T3/HIRC cells, insulin stimulated tyrosyl phosphorylation of the β‐subunit of the insulin receptor. Tryptic peptide mapping indicated that the regulatory region of the β‐subunit was mainly (>80%) bis‐phosphorylated; however, all three tyrosyl residues of the regulatory region were phosphorylated in about 20% of the receptors. As the phosphotransferase was activated by tris‐phosphorylation but not bis‐phosphorylation of the regulatory region of the β‐subunit (White et al.: Journal of Biological Chemistry 263:2969–2980, 1988), the extent of autophosphorylation in the regulatory region may play an important regulatory role during signal transmission in the intact cell.