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Although the GH3 line of somatolactotropic rat pituitary cells has proven useful for many regulation studies, the absence of functional D2 receptors on these cells long prevented their use in studies of dopaminergic action. However, it is now possible to employ GH3 cells expressing recombinant D2 receptors for such investigations. We have investigated both the level at which expression of functional D2 receptors in GH3 cells is blocked, and the cellular pathways employed by the major pituitary D2 receptor isoform, D2A, to inhibit prolactin (PRL) gene transcription. In run-off transcription assays with nuclei from either parental GH3 cells or a GH3 cell line stably expressing a D2A expression vector, Pit-1 gene transcription was detectable in either cell line, but only the latter cell line yielded detectable D2 receptor transcription, implying that the block in D2 receptor expression by GH3 cells is transcriptional. Further investigations employed GH3 cells transiently co-transfected with a D2A expression vector plus a rat PRL promoter construct (-1957)PRL-CAT. Pertussis toxin blocked repression by quinpirole, a D2 agonist, of PRL-CAT activity, demonstrating that this action is mediated by a pertussis toxin-sensitive G protein. The observations that neither of two agents expected to raise intracellular Ca2+, Bay K8644 or thyrotropin-releasing hormone, prevented quinpirole repression of PRL-CAT activity, and that the repressive effects on this construct of quinpirole and the Ca2+ channel antagonist were independent, suggested that regulation of intracellular Ca2+ levels does not play a major role in D2A-mediated repression of the PRL promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

The transcription factor Pit-1 has been shown to be important for both the developmental and homeostatic regulation of expression of the PRL and GH genes in pituitary cells. However, little is known about possible covalent modifications in Pit-1 that might mediate its transactivational properties. Previous studies showing that Pit-1 is a phosphorylation substrate for either protein kinase A or C, or their cellular inducers, led us to investigate whether phosphorylation of Pit-1 is required for its function in either basal or induced cellular activity of either the PRL or GH promoters. The transactivational properties of wild type Pit-1 were compared with those of Pit-1(A3), mutated in the three known phosphorylation sites. At saturating levels of Pit-1 expression vectors, activation of transient basal expression in HeLa cells of constructs (-1957)PRL-CAT or (-244)GH-CAT by RSV-Pit-1(A3) was, respectively, about 50% and 65% as strong as by RSV-Pit-1. Hence, phosphorylation at the sites mutated in Pit-1(A3) is not critically required for basal transactivation of either promoter but may modulate this activity. RSV-Pit-1 and RSV-Pit-1(A3) were equally effective in mediating estrogen receptor stimulation of (-1957)PRL-CAT expression in HeLa cells, thus revealing no phosphorylation requirement for the prerequisite for Pit-1 in estrogen receptor action on the PRL estrogen response element.(ABSTRACT TRUNCATED AT 250 WORDS)