Regulation of beta2-adrenergic receptor (beta2AR) levels by glucocorticoids is a physiologically important mechanism for altering beta2AR responsiveness. Glucocorticoids increase beta2AR density by increasing the rate of beta2AR 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 beta2AR gene is necessary for glucocorticoid-dependent stimulation of receptor gene expression. Using a nested set of deletion fragments of the rat beta2AR 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 beta2AR gene mediates glucocorticoid stimulation of beta2AR gene transcription.

Corticotropin releasing hormone receptor (CRHR) and the VT 2 arginine vasotocin receptor (VT2R) are vital links in the hypothalamic-pituitary-adrenal axis that enable a biological response to stressful stimuli in avian species. CRHR and VT2R are both G-protein coupled receptors (GPCRs), and have been shown by us to form a heterodimer via fluorescent resonance energy transfer (FRET) analysis in the presence of their respective ligands, corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT). The dimerization interface of the heterodimer is unknown, but computational analyses predict transmembrane domains (TMs) as likely sites of the interaction. We constructed chimerical VT2Rs, tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the fourth transmembrane region (TM4) of VT2R with TM4 of the β 2 -adrenergic receptor (β 2 AR). The VT2R/β 2 AR chimeras were expressed in HeLa cells and proper trafficking is confirmed by observing cell membrane localization using confocal microscopy. VT2R/β 2 AR-YFP chimera functionality was confirmed with a Fura-2 acetoxymethyl ester (Fura-2AM) assay. FRET analysis was then performed on VT2 / β 2 AR-chimera/CRHR pairs, and the calculated distance was observed to be >10 nm apart, indicating that heterodimerization was partly disrupted by mutating TM4 of the VT2R. Therefore, TM4 may form one region of the possible dimerization interfaces between the VT2R and CRHR.

As early postnatal development of the male rat proceeds, there is a decline in transcription of the beta2-adrenergic receptor gene in liver which is associated with a decline in beta2-adrenergic receptor mediated glucose mobilization. In this study, primary cultures of rat hepatocytes transiently transfected with fusion genes containing various segments of beta2-adrenergic receptor gene 5'-flanking DNA fused to a promoterless luciferase reporter gene were used to identify genetic elements that might control beta2-adrenergic receptor gene expression during the first 10 days of postnatal life. We found that 261 bp of beta2-adrenergic receptor gene 5'-flanking region (-372 to -95, start of translation is +1) was sufficient to direct high luciferase expression in fetal day 18 hepatocytes and therefore included the beta2-adrenergic receptor gene promoter. Luciferase activities in fetal day 18 hepatocytes transfected with pbeta2AR(-372/-95), pbeta2AR(-1,335/-95) and pbeta2AR(-3,349/-95) were fourfold greater than that in either postnatal day 5 or postnatal day 10 hepatocytes transfected with the same fusion genes. By use of gel mobility shift assays, we observed increased protein binding to a 50 bp segment (-372 to -323) of the beta2-adrenergic receptor gene 5'-flanking region with nuclear extracts prepared from postnatal day 5 and postnatal day 10 hepatocytes compared to fetal day 18 hepatocytes. These findings suggest the presence of a regulatory element in the 5'-flanking region of the beta2-adrenergic receptor gene that appears to be involved in suppression of transcription of the beta2-adrenergic receptor gene in liver during early postnatal development.

Corticotropin releasing hormone receptor (CRHR) and the VT 2 arginine vasotocin receptor (VT2R) are vital links in the hypothalamic-pituitary-adrenal axis that enable a biological response to stressful stimuli in avian species. CRHR and VT2R are both G-protein coupled receptors (GPCRs), and have been shown by us to form a heterodimer via fluorescent resonance energy transfer (FRET) analysis in the presence of their respective ligands, corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT). The dimerization interface of the heterodimer is unknown, but computational analyses predict transmembrane domains (TMs) as likely sites of the interaction. We constructed chimerical VT2Rs, tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the fourth transmembrane region (TM4) of VT2R with TM4 of the β 2 -adrenergic receptor (β 2 AR). The VT2R/β 2 AR chimeras were expressed in HeLa cells and proper trafficking is confirmed by observing cell membrane localization using confocal microscopy. VT2R/β 2 AR-YFP chimera functionality was confirmed with a Fura-2 acetoxymethyl ester (Fura-2AM) assay. FRET analysis was then performed on VT2 / β 2 AR-chimera/CRHR pairs, and the calculated distance was observed to be >10 nm apart, indicating that heterodimerization was partly disrupted by mutating TM4 of the VT2R. Therefore, TM4 may form one region of the possible dimerization interfaces between the VT2R and CRHR.

Background: β 2 -Adrenergic receptors (β 2 AR) play important regulatory roles in a variety of cells and organ systems and are important therapeutic targets in the treatment of airway and cardiovascular disease. Prolonged use of β-agonists results in tolerance secondary to receptor down-regulation resulting in reduced therapeutic efficiency. The purpose of this work is to evaluate the signaling capabilities of the β 2 AR expressed by a recombinant adeno-associated viral (AAV) vector that also included an enhanced green fluorescent protein (EGFP) gene (AAV-β 2 AR/EGFP).