Abstract

The β₁-adrenergic receptor (β₁-AR) mediates several functions of catecholamines in the heart, including the stimulation of heart rate and contractility. The expression of the rat β₁-AR gene was assessed by transiently transfecting chimeric genes containing the β₁-AR promoter, driving the luciferase reporter gene into various cell lines. β₁-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 β₁-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 β₁-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 β₁-AR gene involves widely separated sequences 3′ and 5′ to the transcriptional start site.