Addition of a GnRH agonist (GnRH-A) to alphaT3-1 cells stimulates different MAPK cascades: ERK, Jun N-terminal kinase (JNK), and p38. Activation of JNK, ERK, and p38 shows a unique fold activation ratio of 25:12:2, which might encode signal specificity. ERK is translocated to the nucleus within 20 min with a peak at 120 min of GnRH-A stimulation. We used the human alpha-subunit promoter linked to chloramphenicol acetyl transferase (alphaCAT) to examine the role of ERK, JNK, and c-Src, which is implicated in MAPK activation, in basal and GnRH-stimulated alphaCAT. Addition of GnRH-A resulted in a 3-fold increase in alphaCAT, whereas the Ca(2+) ionophore ionomycin and the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA) had no effect. Addition of GnRH-A and TPA, but not GnRH-A and ionomycin, produced a synergistic response, whereas removal of Ca(2+), but not down-regulation of TPA-sensitive PKCs, abolished GnRH-A-stimulated alphaCAT. Thus, regulation of alpha-promoter activity by GnRH is Ca(2+) dependent and is further augmented by PKC. Cotransfection of alphaCAT and constitutively active or dominant negative plasmids of ERK and JNK cascade members, or the use of the ERK inhibitor PD98059, revealed that ERK, but not JNK, is involved in basal and GnRH-A-stimulated alphaCAT. Because c-Src participates in MAPK activation by GnRH, we also studied its role. Cotransfection of alphaCAT and the dominant negative form of c-Src or incubation with the c-Src inhibitor PP1 reduced GnRH-A-stimulated alphaCAT. The 5'-deletion analysis revealed that the -846/-420 region participated in basal alpha-transcription. In addition, the -346/-156 region containing the pituitary glycoprotein hormone basal element, alpha-basal elements, glycoprotein-specific element, and upstream response element is involved in basal and GnRH-A-stimulated alphaCAT. ERK contribution to GnRH maps to -346/-280 containing the pituitary glycoprotein hormone basal element and alpha-basal elements 1/2. Surprisingly, although c-Src is involved in GnRH-A-stimulated ERK, its involvement is mapped to another region (-280/-180) containing the glycoprotein-specific element. Thus, ERK and c-Src but not JNK are involved in basal and GnRH-A-stimulated-alphaCAT, whereas c-Src contribution is independent of ERK activation.