In the normal human heart, cardiac rate and contractility are stimulated primarily through signal transduction pathways which involve the beta-1- adrenergic receptor (beta-1-AR). The sympathetic neurotransmitter norepinephrine binds to the beta-1-AR which by activating the GTP-binding protein, GS, modulates cardiac Na+, Ca++ channels and the sinoatrial pacemaker current by both direct and cAMP-dependent mechanisms. In the failing human heart, a reduction in the density of the beta-1-AR, in part, severely limits cardiac function, possibly as a protective mechanism. The mechanisms which modulate beta-1-adrenergic receptor expression are clearly of great physiological significance considering the central role the beta-1-AR occupies in the functioning of the normal and the failing heart. We have recently cloned and sequenced the entire coding region of the human beta-1-adrenergic receptor gene and over 3 kb of the 5' promoter region. Whereas amino acid sequences of the human beta-1-AR and the beta-2-AR are 75% identical, no sequence homology is found when the two promoter regions are compared. This suggests that the transcription of each beta-AR subtype is regulated by different mechanisms. The beta-1-AR promoter contains consensus sites for insulin (ISRE), glucocorticoid (GRE), estrogen/thyroid (ERE/TRE), cAMP (CRE) and nerve growth factor (NGFI) dependent regulation. Also present are consensus sites for the cardiac myocyte specific E-box, CArG box and M-CAT transcriptional elements and a binding site for MEF-2, a cardiac specific transcription factor all of which suggest a cardiac myocyte-specific transcriptional regulation of the beta-1-AR. Transcriptional regulation conferred by these elements and by as yet unidentified regulatory sequences will be examined by assessing the ability of mutated and deleted (5',3' and internal) beta-1-AR promoter regions to drive transcription of the luciferase reporter gene. In particular, NGF-dependent and cardiac myocyte-specific transcription will be studied using appropriately mutated promoter sequences to transect neuronal cardiac myocyte and control cells. Using nested deletions at the 5' end of the promoter, we have demonstrated the existence of both positive and negative regulatory regions which modulate luciferase expression in rat C6 glioma cells. Internal deletions within these 5' regions have been constructed to more precisely localized transcriptionally important elements and will be assayed for transcriptional activity in all appropriate cell lines. A consensus CCAAT box at -366 bp and CCAAT and TATA boxes at -1400 bp suggest the existence of two transcriptional start sites. Northern analysis of heart mRNA, RNAse protection and primer extension analyses all suggest the existence of two alternate promoters, one of which may require splicing within the 5' noncoding region. PCR amplification of cDNAs synthesized from the 5' termini of poly(A+ ) mRNAs will be used to conclusively identify alternate promoters. The tissue specific use of these alternate promoters will also be determined using a combination of Northern and RNAse protection analyses of poly(A+ ) mRNA isolated from various human tissues and promoter dependent luciferase expression in appropriate cell lines.
NIH ExPorter
NIH ExPorter