Abstract

The heterologous regulation of the α2C-adrenergic receptor (α2C-AR) was investigated in the HepG2 cell line. Binding of [³H]MK912 (α2-antagonist) to membranes from cells submitted to various treatments showed that exposure to insulin, phorbol 12-myristate 13-acetate, or dexamethasone did not affect receptor density. On the other hand, treatment with forskolin resulted in a large reduction of α2C-AR number. The effect of forskolin was mimicked by 8-br-cAMP and was abolished by the protein kinase A inhibitor, H89. The action of cAMP was slow (t _1/2 = 23 h), dose-dependent, and additive to the receptor down-regulation elicited by the α2-agonist, UK14304. Furthermore, the diminution of receptor was not caused by an increased rate of its degradation but resulted from a decrease in the steady state amounts of α2C4-mRNA. As assessed by experiments in the presence of actinomycin D, the stability of α2C4-mRNA was not affected by 8-br-cAMP or forskolin. By contrast, the activity of a luciferase construct containing the entire promoter region of the α2C4 gene (1.9 kilobase pairs) was inhibited, indicating that the primary mechanism of action of the two compounds is at the transcriptional level. Deletions in the 5′-end of this construct showed that the elements responsible for cAMP responsiveness lie within a 242-base-pair fragment of the gene promoter (nucleotides −236/+6 relative to transcription start). Band-shift experiments indicated that nuclear factors bind to this region in a cAMP-dependent manner. The determination of the actual cis- andtrans-acting elements involved will be the object of future investigation, but the present study provides evidence for transcriptional regulation of human α2C-AR by cAMP.