Figure 6.

Activation of cAMP Signaling Is Involved in EGFR Transactivation by LH

A, Fsk stimulation of EGFR phosphorylation in POFs. POFs were preincubated for 30 min in the absence or presence of DMSO, AG1478 (0.5 μm), or GM6001 (20 μm) followed by 30 min culture in the presence of rLH (5 IU) or Fsk (50 μm). EGFR was immunoprecipitated from POF extracts, and Western blot analyses were performed. Fsk was as effective as rLH in promoting EGFR phosphorylation. AG1478 and GM6001 each inhibited Fsk-induced EGFR phosphorylation. B, LH-induced CREB phosphorylation. POFs were preincubated for 30 min in the absence or presence of DMSO, AG1478 (0.5 μm), GM6001 (20 μm), or TAPI-1 (30 μm) followed by culture in the presence of rLH (5 IU) for another 30 min. Western blot analyses were performed to detect phosphorylated and nonphosphorylated CREB protein. The inhibitors AG1478, GM6001, and TAPI-1 had no effect on LH-stimulated CREB phosphorylation. C, LH-induced EGFR phosphorylation involves PKA. POFs were preincubated for 30 min with DMSO or H89 (20 μm) and then stimulated by rLH (5 IU) for 30 min. EGFR was immunoprecipitated from POF extracts and Western blot analyses performed. The ratio of phosphorylated EGFR to total EGFR was normalized to control. Results are shown as the mean ± sem of three separate experiments and representative blots. *, P < 0.05.