AUTOREGULATION OF THE HUMAN D-1A DOPAMINE-RECEPTOR GENE BY CAMP

Stimulation of the D-1A dopamine receptor increases intracellular cAMP concentration and down-regulates the receptor protein, We evaluated t he possibility that this second messenger system could affect the expr ession of the D-1A gene as a positive autoregulatory mechanism, Treatm ent of the D-1A-expressing cells SK-NMC with 100 mu M dopamine resulte d in an initial increase in steady-state levels of the D-1A mRNA begin ning at 30 min, followed by decline below the baseline and then recove ry by 24 hr, Forskolin/IBMX (100 mu M each) treatment also resulted in a decline followed by recovery, To determine if these changes in D-1A message levels are due to transcriptional control, transient expressi on assays were done using reporter gene constructs of the human D-1A g ene 5'-flanking region, Forskolin/IBMX treatment for 19 hr resulted in a four- to sevenfold increase in trans-activation of the human D-1A g ene promoter, Two cAMP-responsive regions in exon 1 of this gene with nuclear protein binding sites within both regions were identified, The segment of the D-1A gene between these two cAMP-responsive regions co ntained two additional DNA-protein interaction sites, one of which bou nd to nuclear factors considerably stronger following forskolin/IBMX t reatment, Several consensus sequences for classical transcription fact ors known to mediate the cAMP response, such as CREB, AP2, and AP1, ar e found in the human D-1A gene, However, the location of all but one A P2 site in other parts of this gene and lack of AP2 expression in SK-N -MC cells suggest that these factors are unlikely to transduce this re sponse, Thus, dopamine treatment results in delayed cAMP-mediated tran s-activation of the D-1A gene via an indirect mechanism.