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.