Differential expression of the adenylyl cyclase-stimulatory guanosine triphosphate-binding protein Gsα in the human myometrium during pregnancy and labor involves transcriptional regulation by cyclic adenosine 3,5′-monophosphate and binding of phosphorylated nuclear proteins to multiple GC boxes within the promoter

G(s)alpha is the G protein subunit that stimulates adenylyl cyclase activity in the myometrium during pregnancy, raising intracellular levels of the smooth muscle relaxant cAMP. The promoter region of the gene encoding G,a is GC rich and contains multiple putative binding sites for the specificity protein (Sp) transcription factor family. In electrophoretic mobility shift assays, four of these Sp sites were bound by recombinant Sp1 protein. Binding was dependent on phosphorylation of Sp1 by protein kinase A. Phosphorylated Sp1-4 proteins were observed in extracts of cultured human myometrial cells, but in electrophoretic mobility shift assays G,a promoter sequence binding by Sp1 was not apparent. Instead, these assays showed phosphorylation-dependent G(s)alpha promoter binding by lower molecular weight myometrial proteins that could not be supershifted by antibodies specific to Sp1-4 proteins. To investigate the regulation of G,a expression, the GC-rich promoter region was used to direct transcription of a firefly luciferase reporter gene in transient transfection assays of primary human myometrial cell cultures, COS-7 and HEK 293 cells. Reporter gene expression was found to follow a biphasic response to forskolin and 8-bromo-cAMP, with an initial, concentration-dependent increase in luciferase activity, followed by a prolonged decrease. In myometrial cells, this pattern was also seen in response to treatment with human chorionic gonadotropin.