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 demons trated in rat luteal cells by Northern blot analysis, in situ hybridiz ation as well as by reverse transcriptase polymerase chain reaction. W estern 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 i n other tissues. Further studies were performed to determine whether h uman chorionic gonadotropin (hCG) can interact with the insulin-like g rowth factor-I (IGF-I) signaling pathway to increase tyrosine phosphor ylation of IRS-I. While hCG alone was ineffective in stimulating the p hosphorylation of IRS-I, IGF-I mediated phosphorylation of IRS-I was i ncreased by prior exposure to hCG. These results were further confirme d 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 tyrosi ne phosphorylation of IRS-I seen in response to IGF-I stimulation foll owing treatment with either hCG or forskolin was not due to an increas e in IRS-I content. Furthermore. IGF-I receptor tyrosine kinase activi ty was not affected by forskolin, suggesting that the increase in IRS- I tyrosine phosphorylation was not the result of an increase in its ac tivity. Thus, we conclude that hCG/LH and IGF-I signaling pathways 'cr oss-talk' to increase the levels of IRS-I tyrosine phosphorylation. Th e observed increase in IRS-I tyrosine phosphorylation may be the resul t of an increase in the stability of the phosphorylated form of IRS-I.