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.

Capacitation and the acrosome reaction of mammalian spermatozoa are essential for fertilization. In vitro results are presented that demonstrate that catecholamines stimulate activation (a whiplash flagellar movement characteristic of capacitated hamster spermatozoa) and the acrosome reaction. Protein-free ultrafiltrates of bovine adrenal cortex and medulla preparations stimulated motility, activation, and acrosome reactions of hamster spermatozoa in the presence of bovine serum albumin. The medulla preparation was more effective than the cortex preparation in the stimulation of activation and acrosome reactions. Epinephrine (0.5-50 paM) and norepinephrine (50.0 pM) in the presence of bovine serum albumin and a partially purified protein-free cortex preparation also stimulated activation and the acrosome reactions. Both activation and acrosome reactions in the presence of epinephrine were inhibited by the adrenergic antagonists phentolamine and propranolol, suggesting the involvement of a-and a-adrenergic receptors in the stimulation of capacitation and the acrosome reaction. In addition, phenylephrine, an a-adrenergic agonist, was as potent as epinephrine in the stimulation of acrosome reactions, but activation was reduced. Isoproterenol, a P-adrenergic agonist, was as potent as epinephrine in the stimulation of activation, but acrosome reactions were reduced. High percentages of both activation and acrosome reactions were observed only in the presence of epinephrine, norepinephrine, or phenylephrine and isoproterenol together.Sperm capacitation (subtle cellular changes occurring within the female reproductive tract or in vitro) and the resulting acrosome reaction (a fusion and vesiculation of the outer acrosomal membrane and its overlying sperm head plasma membrane) are essential for mammalian fertilization (1, 2). The molecular events of capacitation and of the acrosome reaction, including its initiation, are only partially understood (3). Capacitation and the acrosome reaction can be induced in hamster spermatozoa in vitro by detoxified follicular fluid (4,5) or by blood sera (6). A nondialyzable serum or follicular fluid factor (labile at 900) was required for the acrosome reaction to occur and a dialyzable heat-stable factor derived from serum or follicular fluid was necessary for sperm motility, activation (the whiplash flagellar movement characteristic of capacitated hamster spermatozoa), and the occurrence of the optimum number of acrosome reactions (4-6). It has now been shown that certain serum albumins can replace the nondialyzable factor (6-9) and that the bovine follicular fluid factor is serum albumin (9).Recently, it has been reported that the dialyzable factor can be replaced by a "motility factor" of less than 1000 molecular weight obtained from extracts of hamster adrenal gland (10) or hamster, guinea pig, or human spermatozoa (11,12). The motility factor derived from spermatozoa also seems to maintain hamster sperm viability upon dilution in vitro (12). In the present paper the bovin...

In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15-18%, and the distance between receptors was 5-6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent protein-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction.

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