Autoregulation of the human D1A dopamine receptor gene by cAMP.

Abstract

Stimulation of the D1A dopamine receptor increases intracellular cAMP concentration and down-regulates the receptor protein. We evaluated the possibility that this second messenger system could affect the expression of the D1A gene as a positive autoregulatory mechanism. Treatment of the D1A-expressing cells SK-N-MC with 100 microM dopamine resulted in an initial increase in steady-state levels of the D1A mRNA beginning at 30 min, followed by decline below the baseline and then recovery by 24 hr. Forskolin/IBMX (100 microM each) treatment also resulted in a decline followed by recovery. To determine if these changes in D1A message levels are due to transcriptional control, transient expression assays were done using reporter gene constructs of the human D1A gene 5'-flanking region. Forskolin/IBMX treatment for 19 hr resulted in a four- to seven-fold increase in trans-activation of the human D1A gene 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 D1A gene between these two cAMP-responsive regions contained two additional DNA-protein interaction sites, one of which bound to nuclear factors considerably stronger following forskolin/IBMX treatment. Several consensus sequences for classical transcription factors known to mediate the cAMP response, such as CREB, AP2, and AP1, are found in the human D1A gene. However, the location of all but one AP2 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 response. Thus, dopamine treatment results in delayed cAMP-mediated trans-activation of the D1A gene via an indirect mechanism.