Constitutively active Gq-alpha stimulates prolactin promoter activity via a pathway involving Raf activity.
We have investigated the ability of a constitutively active Gq-alpha mutant, Q209L-alpha q, to regulate target gene expression. Transient expression in GH3 pituitary cells of a rat proximal prolactin promoter-chloramphenicol acetyltransferase construct (-187)PRL-CAT, was stimulated by co-expression of Q209L alpha q, but not by wild-type alpha q. Q209L-alpha q stimulated expression of constructs driven by promoters for either rat prolactin or growth hormone, but not of a control construct driven by the thymidine kinase promoter. Thus, transcriptional effects of alpha q are specific both for the activated state of this G-alpha subunit and the promoter examined. Since both the prolactin and growth hormone promoters are activated by the pituitary cell-specific transcription factor Pit-1, we examined whether a Pit-1 binding site could direct a response to Q209L-alpha q. Two copies of prolactin promoter Pit-1 binding site 1P conferred upon a heterologous metallothionein promoter a response to Q209L-alpha q, implying an involvement of this site in the transcriptional action of Q209L-alpha q on the prolactin promoter. The phorbol ester activator of protein kinase C, 12-O-tetradecanoylphorbol-13-acetate, stimulated (-187)PRL-CAT activity, but opposed the action of Q209L-alpha q on activity of this PRL-CAT construct. Q209L-alpha q stimulation of (-187)PRL-CAT activity was inhibited by co-expression of a dominant negative Raf mutant, Raf-C4, but not by a point mutant of Raf-C4 with reduced inhibitory properties. These results imply that activated alpha q subunits can stimulate prolactin promoter activity via a pathway that involves a Pit-1 DNA binding site(s), is opposed by protein kinase C, and is mediated by a pathway in which Raf-1 kinase plays a role.
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000502:physiology*; D010902:Pituitary Gland / Q000378:metabolism; D010957:Plasmids; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D019908:Proto-Oncogene Proteins c-raf; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D018025:Receptors, Thyrotropin-Releasing Hormone / Q000502:physiology; D011993:Recombinant Fusion Proteins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014162:Transfection
D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D018025:Receptors, Thyrotropin-Releasing Hormone; D011993:Recombinant Fusion Proteins; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D015500:Chloramphenicol O-Acetyltransferase; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate