Vol. 58, Issue 4, 821-827, October 2000

Transcriptional Down-Regulation of the Human 2C-Adrenergic Receptor by cAMP

Stéphane Schaak, Cécile Cayla, Anastasios Lymperopoulos, Christodoulos Flordellis, Daniel Cussac, Colette Denis, and Hervé Paris

Institut National de la Santé et de la Recherche Médicale Unit 388, Institut L. Bugnard, CHU Rangueil, Toulouse, France (S.S., C.C., D.C., C.D., H.P.); and Department of Pharmacology, School of Medicine, University of Patras, Patras, Greece (A.L., C.F.)

The heterologous regulation of the 2C-adrenergic receptor (2C-AR) was investigated in the HepG2 cell line. Binding of [³H]MK912 (2-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 2C-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 2-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 2C4-mRNA. As assessed by experiments in the presence of actinomycin D, the stability of 2C4-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 2C4 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- and trans-acting elements involved will be the object of future investigation, but the present study provides evidence for transcriptional regulation of human 2C-AR by cAMP.