Expression of constitutively active Gs alpha-subunits in GH3 pituitary cells stimulates prolactin promoter activity

Although G protein alpha subunits are known to regulate such cellular functions as growth and enzymatic activity, the ability of these proteins to regulate target gene expression has not yet been directly investigated. Transient expression in GH3 pituitary cells of a target rat prolactin promoter-chloramphenicol acetyltransferase construct, (-1957)PRL-CAT, was increased by coexpressed constitutively active alpha s mutant Q227L-alpha s but not by wild-type alpha s. Thus activated alpha s but not basal state alpha s can stimulate prolactin promoter activity. Q227L-alpha s also stimulated expression of construct (-187)PRL-CAT, showing that only the proximal prolactin promoter region is required for a response to activated alpha s. The promoter specificity of the transcriptional influence of activated alpha s was demonstrated by the inability of either Q227L-alpha s or wild-type alpha s to stimulate expression of control target constructs containing either the rat growth hormone promoter or the thymidine kinase promoter. Previous studies have shown that the most proximal prolactin promoter binding site for the pituitary-specific transcription factor pit-1, site 1P, can act as an independent response element for either thyrotropin-releasing hormone or Ca2+. Two copies of site 1P conferred upon a heterologous metallothionein promoter a response to Q227L-alpha s. This implies that site 1P can also serve as an independent response element for alpha s and suggests that pit-1 may be a mediator of the cellular regulation by alpha s of the prolactin promoter.