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.
Gene expression, receptor binding and growth-promoting activity of insulin-like growth factor I (IGF I) was studied in cultured astrocytes from developing rat brain. Northern blot analysis of poly(A)+ RNAs from astrocytes revealed an IGF I mRNA of 1.9 kb. Competitive binding and receptor labelling techniques revealed two types of IGF receptor in astroglial cells. Type I IGF receptors consist of a-subunits (Mr 130 000) which bind IGF I with significantly higher affinity than IGF 11, and fl-subunits (Mr 94 000) which show IGF Isensitive tyrosine kinase activity. Type II IGF receptors are monomers (Mr 250 000) which bind IGF H with three times higher affinity than IGF I. Both types of IGF receptor recognize insulin weakly. DNA synthesis measured by cellular thymidine incorporation was stimulated 2-fold by IGF I and IGF H. IGF I was more potent than IGF H, and both were significantly more potent than insulin. Our findings suggest that IGF I is synthesized in fetal rat astrocytes and acts as a growth promoter for the same cells by activation of the type I IGF receptor tyrosine kinase. We propose that IGF I acts through autocrine or paracrine mechanisms to stimulate astroglial cell growth during normal brain development.
Using a solution-hybridization assay and specific oligonucleotidic probes, we have studied IGF-I and insulin receptor mRNAs in the rat central nervous system during development. The expression of mRNAs was maximal at embryonic day 15 and 20 for IGF-I receptors, and at embryonic day 20 and the day of birth for insulin receptors. After birth, the expression of both receptor transcripts decreased and reached minimal levels in the adult. At the time at which these transcripts were maximally expressed (embryonic day 20), the regional analysis indicated that IGF-I receptor transcripts were widely distributed in the brain. In contrast, insulin receptor transcripts were restricted to certain areas in which they were coexpressed with the IGF-I receptor transcripts. We next analyzed which cells at embryonic day 20 expressed those receptor transcripts. Late embryonic neurons, astrocytes, and neonatal progenitors of oligodendrocytes synthesized both IGF-I and insulin receptor mRNAs after a short time in culture. However, astrocytes expressed preferentially IGF-I receptor transcripts, while young progenitors for oligodendrocytes expressed high levels of insulin receptor transcripts. As a whole, our data indicate that during rat CNS development expression of IGF-I and insulin receptors appears to be stage- and cell-specific. The differences observed between the expression of both receptors might point to a specific, but coordinative role of IGF-I and insulin and their receptors during that time.
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