Alpha 2 adrenergic receptors have been shown to play an important role in homeostasis of kidney function and in the regulation of the central sympathetic system. Dysfunction of these receptors is probably involved in the development and/or maintenance of some forms of hypertension. Recent molecular analysis has shown that there are three subtypes of alpha 2 adrenergic receptors, each encoded by a separate gene in rat and human. The long term goal of this project is to understand the relative contributions of each subtype to cardiovascular function and blood pressure regulation. It has been shown that each of these receptor subtypes has a different pattern of tissue specific expression. It is our hypothesis that the diversity of alpha 2 adrenergic receptor genes allows for the tissue specific expression of genes that differ in their transcriptional regulation and vary in their response to physiological stimuli. It is the goal of this grant to understand the mechanism of tissue specific expression of various alpha 2 genes and to begin to analyze the intracellular and extracellular signals that regulate alpha 2 gene expression. The following specific aims will be undertaken: (1) to study the distribution of transcripts specific for each of the three alpha 2 genes in subregions of brain and kidney; (2) To study the cis regulatory regions of the various alpha 2 genes that may be responsible for their differential expression by comparing the ability of putative regulatory regions to direct transcription in a variety of cell lines. (3) To characterize nuclear factors that are important or tissue specific expression of a alpha 2 adrenergic receptor genes by analyzing DNA/nuclear factor interactions in DNase I footprinting and gel mobility shift assays, by assessing the effects of point mutations in nuclear factor binding sites to alter transcription and by studying the tissue distribution of key nuclear factors. (4) To study intracellular and extracellular signals that alter alpha 2 gene transcription by monitoring the ability of agents, such as cAMP or receptor agonists, to alter transcription in various cells lines and to study the cis regulatory elements and trans-acting factors that mediate these affects by methods in specific aims 2 and 3. These studies will lead to the development of intraventions to specifically affect the level of expression of each alpha 2 gene at the level of transcription. In summary, the proposed studies will provide important information concerning the expression and regulation of genes that encode the receptors that have many important biological functions.
