IRS-I expression on the luteinized rat ovary: IGF-I and cyclic AMP effects on IRS-I tyrosine phosphorylation

The expression of insulin receptor substrate-I (IRS-I) mRNA was demonstrated in rat luteal cells by Northern blot analysis, in situ hybridization as well as by reverse transcriptase polymerase chain reaction. Western blot with a polyclonal anti IRS-I antibody showed the presence of a 183 kDa protein which corresponds to the size of IRS-I reported in other tissues. Further studies were performed to determine whether human chorionic gonadotropin (hCG) can interact with the insulin-like growth factor-I (IGF-I) signaling pathway to increase tyrosine phosphorylation of IRS-I. While hCG alone was ineffective in stimulating the phosphorylation of IRS-I, IGF-I mediated phosphorylation of IRS-I was increased by prior exposure to hCG. These results were further confirmed by the immunoprecipitation of IRS-I from the lysate of hCG- and IGF-I-treated luteal cell cultures followed by Western blotting with anti-phosphotyrosine antibody. Similarly, pretreatment with forskolin also increased IGF-I stimulated IRS-I phosphorylation. The increased tyrosine phosphorylation of IRS-I seen in response to IGF-I stimulation following treatment with either hCG or forskolin was not due to an increase in IRS-I content. Furthermore, IGF-I receptor tyrosine kinase activity was not affected by forskolin, suggesting that the increase in IRS-I tyrosine phosphorylation was not the result of an increase in its activity. Thus, we conclude that hCG/LH and IGF-I signaling pathways 'cross-talk' to increase the levels of IRS-I tyrosine phosphorylation. The observed increase in IRS-I tyrosine phosphorylation may be the result of an increase in the stability of the phosphorylated form of IRS-I.