Revised Manuscript Received March 12, 1993 abstract: Autophosphorylation sites of the human insulin receptor were identified bymicrosequence analysis of 19 discrete tryptic [32P] phosphopeptides, purified from the autophosphorylated cytoplasmic kinase domain (CKD). Seventeen phosphopeptides were generated by cleavage at Arg and/or Lys, but two phosphopeptides were generated reproducibly by anomalous cleavages. Two new sites were identified in the juxtamembrane region of the intact insulin receptor 8-subunit (the amino terminus of the CKD), including phosphotyrosines 965 and 972. Three sites in the central region, including phosphotyrosines 1158, 1162, and 1163, were identified from six phosphopeptides; tyrosine at 1158 was the least phosphorylated. Monophosphopeptides contained phosphotyrosine at either residue 1158 or 1163, but not at 1162. Bisphosphorylation included phosphotyrosines only at 1162 and 1163. The two autophosphorylation sites near the car boxy terminus were found in seven phosphopeptides, including phosphotyrosines at 1328 and 1334. When mapped byreverse-phase high-performance liquid chromatography, these phosphopeptides eluted in the order central domain, first; carboxy-terminal region, second; and juxtamembrane (aminoterminal) domain, last.

The insulin receptor is a hormonally regulated protein (ty-rosine) kinase, first reported by Kasuga et al.(1983a, b). The intracellular kinase activity of this receptor’s transmembrane 8-subunit is stimulated by insulin binding to the extracellular-subunit. In addition, autophosphoryaltion can be stimulated by polycations (Morrison et al., 1989; Biener & Zick, 1990; Fujita-Yamaguchi et al., 1989; Sacks & McDonald, 1988; Kohanski, 1989) and certain polypeptides derived from the high-affinity substrate RCAM-lysozyme (Kohanski & Schen-ker, 1991). For the native insulin receptor, similar tryptic