TRANSCRIPTIONAL EFFECTS IN GH(3) CELLS OF G(S)ALPHA MUTANTS ASSOCIATED WITH HUMAN PITUITARY-TUMORS - STIMULATION OF ADENOSINE-3',5'-MONOPHOSPHATE RESPONSE ELEMENT-BINDING PROTEIN-MEDIATED TRANSCRIPTION AND OF PROLACTIN AND GROWTH-HORMONE PROMOTER ACTIVITY VIA PROTEIN-KINASE-A

Somatic mutations of the alpha-subunit of G(s) (G(s) alpha) have been detected previously at high frequency in human PRL- and/or GH-producin g pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransf ection assays to analyze their genomic effects in GH(3) rat pituitary cells. We first show that guanosine triphosphatase (GTPase)deficient G (s) alpha subunits (mutated at amino acid 201 or 227) stimulate transc ription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constru cts, we further show that this stimulatory effects of G(s) alpha on th e CRE is probably mediated by the transacting factor CRE-binding prote in. Then, in experiments using a reporter gene driven by the human pro moters for either the PRL (position -250 to 18) or GH (position -500 t o 13) genes, we show that these mutant G(s) alpha subunits stimulate e xpression driven by either the PRL or GH promoter. Finally, we show th at a dominant inhibitory mutant of cAMP-dependent kinase (protein kina se A) completely blocks the ability of these G(s) alpha mutants to sti mulate the activity of either the PRL or GH promoter, implying that GT Pase-deficient G(s) alpha subunits stimulation of the activities of th ese promoters is mediated entirely via the cAMP/protein protein kinase A pathway. Taken together, these results imply that activation of thi s pathway by the GTPase-deficient mutants found in human pituitary tum ors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the sec retory phenotype and endocrine disorders associated with these tumors.