Promoter analysis of the rat beta1-adrenergic receptor gene identifies sequences involved in basal expression

The beta1-adrenergic receptor (beta1-AR) mediates several functions of catecholamines in the heart, including the stimulation of heart rate and contractility. The expression of the rat beta1-AR gene was assessed by transiently transfecting chimeric genes containing the beta1-AR promoter, driving the luciferase reporter gene into various cell lines. beta1-AR/luciferase vectors containing 3 kb of the 5'-flanking region and extending to -126 relative to the start site of translation were expressed at high levels in ventricular myocytes, SK-N-MC cells, and HepG2 cells. The addition of 26 nucleotides from -125 to -100 to the -3311 beta1-AR/luciferase chimeric gene reduced expression in myocytes and SK-N-MC cells while eliminating expression in HepG2 cells. This element is located 125 base-pairs 3' to the transcriptional start site. The mutation of four nucleotides between -121 and -118 diminished the inhibitory effect of this element. The inhibitory activity of the -125 to -100 sequence was completely dependent on promoter context and positioning. In addition to this 3' element, sequences between -3311 and -2740 in the 5'-flanking region of the beta1-AR gene were required for the full transcriptional suppression. Using DNase I footprinting and gel mobility assays, it was determined that within the 26-bp region, rat heart nuclear proteins bound to two sites between nucleotides -123 and -112 and -106 and -100. Therefore, appropriate basal expression of the beta1-AR gene involves widely separated sequences 3' and 5' to the transcriptional start site.