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-producing pituitary tumors. To test whether these mutants are responsible for the increased production of these hormones, we used transient cotransfection 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 transcription from a reporter construct bearing the consensus cAMP response element (CRE; TGACGTCA). Using GAL4-CRE-binding protein fusion constructs, we further show that this stimulatory effects of G(s) alpha on the CRE is probably mediated by the transacting factor CRE-binding protein. Then, in experiments using a reporter gene driven by the human promoters for either the PRL (position -250 to 18) or GH (position -500 to 13) genes, we show that these mutant G(s) alpha subunits stimulate expression driven by either the PRL or GH promoter. Finally, we show that a dominant inhibitory mutant of cAMP-dependent kinase (protein kinase A) completely blocks the ability of these G(s) alpha mutants to stimulate the activity of either the PRL or GH promoter, implying that GTPase-deficient G(s) alpha subunits stimulation of the activities of these promoters is mediated entirely via the cAMP/protein protein kinase A pathway. Taken together, these results imply that activation of this pathway by the GTPase-deficient mutants found in human pituitary tumors stimulates the expression of PRL and GH genes. The transcriptional effects exerted via this pathway may thus provide a basis for the secretory phenotype and endocrine disorders associated with these tumors.