Transcriptional regulation of the human PRL-releasing peptide (PrRP) receptor gene by a dopamine 2 Receptor agonist: cloning and characterization of the human PrRP receptor gene and its promoter region.

Abstract

PRL-releasing peptide receptor (PrRPR) mRNA was expressed in pituitary adenomas but was not detected in patients treated with bromocriptine, a specific agonist of dopamine 2 (D2) receptor. Although medical treatment with bromocriptine is effective for patients with pituitary adenomas, little is known about the molecular mechanisms of gene regulation mediated by D2 receptors. The cloned human PrRPR gene spanned approximately 2.0 kb and contained two exons and one intron. Two functional polyadenylation signals located at 510 and 714 bp downstream from the stop codon. A primer extension analysis demonstrated two major transcriptional start sites at 139 and 140 bp upstream from the translational start site and an additional minor site at -161. The promoter region contained several putative binding sites for transcriptional factors including pituitary-specific transcription factor (Pit 1), activator protein 1 (AP-1), and specificity protein (Sp1), but no typical TATA or CAAT box. This promoter showed the strong activity in the pituitary-derived GH4C1 cells, and the region between -697 and -596 bp was responsible for the stimulation both by forskolin and overexpression of cAMP response element binding protein (CREB). These stimulations were significantly suppressed by incubation with bromocriptine in a dose- and time-dependent manner, and the mutant CREB (S133A) completely abolished the inhibitory events of bromocriptine. However, EMSA studies demonstrated that CREB did not bind to this region, to which an approximately 60-kDa protein was strongly bound, and that antibodies against CREB, c-Fos, and Sp1 did not supershift this complex. Furthermore, the amount of this unknown protein was apparently reduced by treatment with bromocriptine. A series of mutation analyses demonstrated that the specific sequence, 5'-cccacatcat-3', was required for both the binding to the 60-kDa protein and the repression by bromocriptine. Therefore, the transcriptional repression of the PrRPR gene by bromocriptine required CREB but was independent of direct binding of CREB to the gene and that the sequence -663 -- -672, 5'-cccacatcat-3', bound to the 60-kDa protein appeared to be critical for this event.