G protein-coupled receptors comprise a large family of receptors which interact with diverse chemical structures to activate GTP-binding regulatory proteins. The objective of the proposed studies is to determine the mechanism by which the expression of these receptors is regulated, using the beta-adrenergic receptor (beta-AR) as the model. The beta-AR appears to be a dominant receptor for mediating the functions of catecholamines in the heart, which include regulation of heart rate, force and rhythmic contractions. The overall density of cardiac beta-AR in vivo and in cultured ventricular myocytes (VM) in vitro is increased by thyroid hormone (T3). T3 increased the number, mRNA level, and gene transcription of beta1-AR in VM. The transcriptional effects of T3 are mediated by nuclear T3 receptors (T3R) which bind to T3 response elements (TRE) in T3 responsive genes. The major goal of this proposal is to characterize beta- AR expression in VM. The primary hypothesis underlying this proposal is that T3 increases cardiac beta-AR expression by a transcriptional mechanism orchestrated by heterodimer formation between nuclear T3R and auxiliary nuclear proteins at the TRE in the promoter region of the beta1-AR gene.
The aim of this proposal is to identify the TREs in the beta1-AR gene and to characterize the proteins that bind to the TRE and stimulate the transcription of the beta1-AR gene. Neonatal rat VM will be used to study the regulation of cardiac beta-AR by T-3.
The first aim i s to identify the DNA sequences (TREs) in the beta1-AR gene that confer transcriptional regulation by T3. Genomic fragments from the 5' flanking region of the beta1-AR gene will be ligated to chloramphenicol acetyl transferase (CAT) gene to generate chimeric beta1-AR CAT vectors. These vectors will be transiently transfected into VM, followed by measuring the effect of T3 on CAT activity. In the second aim, we will examine DNA-protein interactions in the promoter regulatory region of the beta1-AR gene. The interaction between DNA sequences in the beta1-AR promoter and rat heart nuclear proteins will be characterized by means of DNAse I footprint analysis to define those domains that bind nuclear proteins. Gel mobility shift assays with purified T3R and nuclear proteins will be used to evaluate their specificity in binding to the beta1-AR TRE. In the third aim, we will identify the proteins that bind to the beta1-AR TRE and heterodimerize with the T3R to enhance hormonal transactivation of beta1-AR gene. These experiments will involve the use of gel mobility assays an other related techniques to characterize the T3R binding partner at the beta1-AR TRE. The functional significance of these proteins will be determined by transient transfection assays to confirm their role in the transcriptional regulation by T3. Other experiments will determine the role of retinoic acid and its nuclear receptor in regulating beta1-AR gene expression. Characterization of the beta1-AR TRE and the proteins that bind to it, is an important step in elucidating the mechanism by which T3 directly stimulates transcription. These studies will be relevant to regulation of many G protein-linked receptor genes whose expression is controlled transcriptionally by hormones.
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