CONSTITUTIVELY ACTIVE G(S) ALPHA-SUBUNITS STIMULATE PIT-1 PROMOTER ACTIVITY VIA A PROTEIN-KINASE A-MEDIATED PATHWAY ACTING THROUGH DEOXYRIBONUCLEIC-ACID BINDING-SITES BOTH FOR PIT-1 AND FOR ADENOSINE-3',5'-MONOPHOSPHATE RESPONSE ELEMENT-BINDING PROTEIN

Constitutively active mutations of the G protein alpha(S) subunit are detected at a high frequency in human pituitary adenomas that secrete GH or PRL. It seems possible that over-expression of the pituitary cel l-specific transcription factor Pit-1/GHF-1 (Pit-1) gene in response t o active alpha(S) subunits contributes to the formation of these adeno mas. We have examined whether expression in pituitary cells of one of these constitutively active alpha(S) subunits, Q227L-alpha(S), stimula tes expression directed by the Pit-1 promoter. Transient expression of Q227L-alpha(S) yielded a strong stimulation of a target Pit-1 promote r-chloramphenicol acetyl transferase (CAT) construct, (-200)Pit-1-CAT. Expression of wild-type alpha(S) or an inactive alpha(S) mutant yield ed, respectively, reduced or no stimulation of CAT activity. A dominan t inhibitor of protein kinase A (PKA), R(AB), blocked almost completel y either forskolin (FSK) or Q227L-alpha(S) stimulation of (-200)Pit-1- CAT expression, implying that PKA is required for the action of Q227L- alpha(S) on the Pit-1 promoter. The Pit-1 promoter contains a binding site for Pit-1 and two CREB binding sites. Mutation of the Pit-1 bindi ng site reduced but did not eliminate either FSK or Q227L-alpha(S) sti mulation of Pit-1 promoter activity, implying a partial but incomplete requirement for this element for a PKA-mediated response to Q227L-alp ha(S). The CREB dominant inhibitor S133A-CREB yielded a partial reduct ion in either FSK or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT exp ression, implying that one or both of the Pit-1 promoter adenosine 3'5 '-monophosphate response element binding protein (CREB) binding sites is/are also required for a complete PKA-mediated response to Q227L-alp ha(S). The observation that S13SA-CREB completely blocked the response to FSK or Q227L-alpha(S) of a Pit-1 promoter containing a mutated sit e PitB1 implies that the binding sites for Pit-1 and CREB account for all of the response elements for FSK or alpha(S) in the Pit-1 promoter .