cAMP regulates transcription of the alpha 2A adrenergic receptor gene in HT-29 cells

Abstract

Since cAMP has a number of important effects regulating activity of adrenergic receptor pathways, we wondered if expression of the alpha 2A adrenergic receptor gene is regulated by this second messenger. We have examined the effects of a cAMP analog (Bt2cAMP) on the expression of alpha 2A adrenergic receptors in HT-29 cells. Bt2cAMP induced a 5.3 +/- 0.8-fold increase in alpha 2A receptor mRNA abundance as did forskolin and vasoactive intestinal polypeptide which both increase cAMP accumulation in these cells. Bt2cAMP increased alpha 2A receptor number up to 2.4 +/- 0.3-fold. The rate of alpha 2A receptor gene transcription increased 7.8 +/- 3.2-fold in cells treated with Bt2cAMP for 2 h; after 24 h, the transcription rate was 3.7 +/- 1.7-fold higher than in controls. The increased rate of transcription occurred in the presence of the protein synthesis inhibitor cycloheximide. The half life of the alpha 2A receptor mRNA in cells incubated with Bt2cAMP for 2 h increased by 1.5-fold but returned to the original value after exposure to Bt2cAMP for 24 h. The increased expression of alpha 2A receptors was associated with an increased efficacy of inhibition of cAMP accumulation mediated by the alpha 2 adrenergic agonist UK14304. Using a chloramphenicol acetyltransferase (CAT) reporter plasmid containing 5'-flanking sequences of the alpha 2A receptor gene, we found that co-transfection of JEG-3 cells with expression vectors containing cAMP-dependent protein kinase regulatory subunit cDNA with mutations at both cAMP binding sites inhibited basal and Bt2cAMP-stimulated expression of CAT activity. These results demonstrate that an alpha 2A adrenergic receptor gene is regulated by the second messenger cAMP via cAMP-dependent protein kinase, mainly by controlling the rate of transcription, which leads to an increased expression of these receptors.