Figure 4.

The selective activation of ERK1/2 in granulosa cells provokes the trans-phosphorylation of the EGFR in test cells in the absence of hCG stimulation. A, Primary cultures of granulosa cells were infected with Ad-hLHR and Ad-βgal alone or Ad-hLHR and Ad-CAMEK as indicated. Phosphorylated ERK1/2, total ERK2, and total MEK1/2 were measured using Western blots at different times after infection. The blot shown is representative of four experiments with similar results. B, Test cells expressing the EGFR-GFP were cocultured with primary granulosa cells expressing the hLHR and βgal (white symbols) or the hLHR and CAMEK (black symbols) at time zero. The phosphorylated and total EGFR-GFP was measured at different times after the cocultures were initiated. Note that the three time points shown in this panel correspond to the same time points shown in A and C for the individual cultures of granulosa cells. Each point is the mean ± sem of three independent experiments. Asterisks denote significant differences between the cells expressing CAMEK or βgal at a given time point (t test, P < 0.05). C, Primary cultures of granulosa cells were infected with Ad-hLHR and Ad-bgal (white symbols) or Ad-hLHR and Ad-CAMEK (black symbols) as indicated. Epiregulin and GAPDH mRNA were quantitated using RT followed by real-time PCR as described in Materials and Methods. Each point is the mean ± sem of five to six independent experiments. Asterisks denote significant differences between the cells expressing CAMEK or βgal at a given time point (t test, P < 0.05).