Insulin stimulates the phosphorylation of its own receptor. In the work reported here, the kinase activity responsible for the insulin-stimulated phosphorylation of the insulin receptor was localized. In a first approach, partially purified insulin receptors derived from normal rat hepatocytes were immunoprecipitated with antibodies specific for the insulin receptor; thereafter, the immunoprecipitates were incubated with [gamma-(32)P]-ATP in the absence or presence of insulin (1 muM). NaDodSO(4)/polyacrylamide gel electrophoretic analysis of the immunoprecipitates under reducing conditions revealed autophosphorylation of the beta subunit (M(r) 95,000) of the insulin receptor; the alpha subunit (M(r) 130,000) was not phosphorylated. Further, insulin specifically increased 3- to 4-fold the labeling of its own receptor beta subunit, indicating that anti-receptor antibodies precipitate a functional and insulin-stimulable protein kinase that appears to be independent of cyclic AMP and calcium. To localize more precisely the insulin receptor-related kinase activity, we searched for an ATP-binding site on solubilized insulin receptors. By using covalent labeling with oxidized [alpha-(32)P]ATP, a labeled polypeptide with precisely the same electrophoretic mobility as that of the beta subunit of the insulin receptor (M(r) 95,000) was specifically immunoprecipitated with anti-receptor antibodies. Further, its appearance was prevented when the immunoprecipitation was preceded by incubation with unlabeled insulin. In conclusion, we have shown that an insulin-stimulated phosphorylation site and an ATP-binding site coexist on the beta subunit of the insulin receptor. The simultaneous presence of these two sites on the same receptor subunit indicates that the insulin receptor acts as its own protein kinase.

The structure of rat intestinal cell receptors for Escherichia coli heat-stable enterotoxin (ST) was investigated by affinity cross-linking to 125I-ST and analysis by denaturing gel electrophoresis. Cross-linking of labeled toxin to intestinal membranes and analysis by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed five specifically labeled proteins with molecular masses of 160, 136, 78, 71, and 56 (kilodaltons) kDa. Exhaustive reduction of these samples resulted in a similar pattern of labeling. Affinitylabeled proteins were further analyzed by nonreducing SDS-PAGE, reduction of the resulting separated proteins, and further separation by SDS-PAGE in the presence of ,-mercaptoethanol. Thus, the 160-kDa band on nonreducing gels consisted of two different receptors: a 160-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 78-kDa subunit. Similarly, the 136-kDa band on nonreducing gels consisted of a 136-kDa polypeptide not further reducible and one composed of at least two subunits, one of which was the 71-kDa subunit. The 78-, 71-, and 56-kDa subunits were not further reducible.

In intact rat hepatocytes insulin stimulates the phosphorylation of the beta-subunit of its receptor exclusively on serine residues, which are also phosphorylated in the absence of insulin. In contrast, in partially purified insulin receptors derived from these same cells and in highly purified insulin receptors obtained by immunoprecipitation with anti-receptor antibodies, the receptor beta-subunit is phosphorylated solely on tyrosine residues. For both cell-free systems, insulin's stimulatory action on receptor phosphorylation leads to an increase in phosphotyrosine. When partially purified receptors were used to phosphorylate two exogenous substrates, casein and histone, insulin was found to stimulate the phosphorylation of both tyrosine and serine. However, the basal and insulin-stimulated kinase activity of immunoprecipitated receptors was only tyrosine-specific. From these observations we propose that the insulin-receptor complex consists of two different insulin-stimulatable kinase activities: (1) a tyrosine-specific kinase, which is a constituent of the insulin-receptor structure and whose activation is likely to be the first post-binding event in insulin action; and (2) a serine-specific kinase, which is closely associated with the receptor in the cell membrane.

Guanylate cyclase is regulated by adenine nucleotides in membranes of intestinal mucosal cells. Basal guanylate cyclase was activated about twofold by adenine nucleotides. Activation was specific for adenine, as compared with the pyrimidine nucleotides UTP and CTP. In addition, enzyme activation was obtained in the presence of saturating concentrations of GTP, the substrate for guanylate cyclase. The most potent adenine nucleotide was the nonhydrolyzable analog of ATP, adenosine 5'-O-(3-thiotriphosphate). Adenine nucleotide activation was specific for the particulate form of guanylate cyclase, as compared with the soluble form. Also, adenine nucleotides potentiated the activation of guanylate cyclase by the heat-stable enterotoxin produced by Escherichia coli. Indeed, enzyme activation by adenine nucleotides and toxin was greater than the sum of individual activations by these agents. Adenine nucleotides regulate guanylate cyclase by increasing the maximum velocity of the enzyme without altering its affinity for substrate or its cooperativity. In addition to stimulating guanylate cyclase, adenine nucleotides decreased the specific binding of the heat-stable enterotoxin to receptors in intestinal membranes. The coordinated regulation of the toxin-receptor interaction and guanylate cyclase activity by a process utilizing nonhydrolyzable analogs of a purine nucleotide is similar to the mechanisms involved in the hormone regulation of adenylate cyclase by guanine nucleotide-binding proteins. These data suggest that an adenine nucleotide-dependent protein may couple the toxin-receptor interaction to the regulation of particulate guanylate cyclase in intestinal membranes.

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