Vol. 54, Issue 6, 1016-1023, December 1998

Identification of a Glucocorticoid Response Element in the Rat ₂-Adrenergic Receptor Gene

Lawrence E. Cornett, F. Charles Hiller, Sandie E. Jacobi, Wenhui Cao, and Dennis W. McGraw

Division of Critical and Pulmonary Care Medicine, Department of Medicine (L.E.C., F.C.H., S.E.J., D.W.M.), and Department of Physiology and Biophysics (L.E.C., W.C.), University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205

Regulation of ₂-adrenergic receptor (₂AR) levels by glucocorticoids is a physiologically important mechanism for altering ₂AR responsiveness. Glucocorticoids increase ₂AR density by increasing the rate of ₂AR gene transcription, but the cis-elements involved have not been well characterized. We now show that one of six potential glucocorticoid response elements (GREs) in the 5'-flanking region of the rat ₂AR gene is necessary for glucocorticoid-dependent stimulation of receptor gene expression. Using a nested set of deletion fragments of the rat ₂AR gene 5'-flanking region fused to a luciferase reporter gene, glucocorticoid-dependent induction of reporter gene expression in HepG2 cells was localized to a region between positions 643 and 152, relative to the transcription initiation site. In electrophoretic mobility shift assays, a double-stranded oligonucleotide incorporating a near-consensus GRE from this region (positions 379 to 365) formed complexes with the human recombinant glucocorticoid receptor, as well as with nuclear protein from dexamethasone-treated HepG2 cells. Mutation of a single base within this GRE sequence greatly diminished interaction of the mutated oligonucleotide with the human recombinant glucocorticoid receptor. The functional activity of the GRE was characterized using a luciferase reporter construct driven by a minimal thymidine kinase promoter. In HepG2 cells transfected with constructs containing the GRE, dexamethasone increased reporter gene expression approximately 3-fold, whereas a dexamethasone effect was not observed with constructs lacking the GRE. Taken together, these findings show that a GRE located at positions 379 to 365 in the 5'-flanking region of the rat ₂AR gene mediates glucocorticoid stimulation of ₂AR gene transcription.