Identification of mechanisms underlying angiotensinII receptor (AIIR)- mediated regulation of gene expression is central to our understanding of how angiotensin II (AII) exerts long-lasting effects on cells with physiological and pathological consequences to the organism. While the structure of AIIR and the short-term membrane or cytoplasmic effects initiated by the ligand-occupied receptor have been extensively investigated, few studies have directly addressed mechanisms by which AIIR controls gene transcription. In this proposal the principal objective is to determine molecular mechanisms by which AIIR activates expression of the gene which encodes tyrosine hydroxylase (THE), the rate limiting enzyme in catecholamine biosynthesis. Catecholaminergic cells are established effectors of cardiovascular and other homeostatic functions of AII. We have shown that the long-term induction of catecholamine release from the adrenal medullary cells by AIIR is associated with the transcriptional activation of the TH gene. We have characterized second messenger systems and the roles of protein kinases mediating this activation. Pathways involved in AIIR activation were different from those utilized during nicotinic receptor induction of the TH gene. We have identified THE gene promoter region that mediates AII activation and additional regions that modulate this activation. The promoter region mediating AII stimulation was different from the region involved in synaptic activation of the TH gene. The specific promoter sequences involved in the regulation by AIIR remain to be identified. An in vitro DNA-protein binding assay detected several different protein complexes forming with the regulatory region of the TH promoter. Stimulation of AIIR induced formation of a specific nuclear protein complex with the regulatory region of the TH gene promoter. This effect was unique to AIIR, and was not reproduced by other treatments which activate THE gene expression (i.e. stimulation of nicotinic receptors or protein kinase C). The nature of nuclear proteins forming AIIR-induced complex and other complexes binding to THE promoter regulatory regions are unknown, and will be elucidated in the proposed studies. Their role in the regulation of THE gene expression by AIIR will be determined. Specifically the aims are: (i) to identify individual regulatory elements in THE gene - promoter that participate in transcriptional regulation by AIIR; (ii) to further characterize nuclear proteins which interact with the TH promoter AIIR regulatory elements; and (iii) to purify and clone selected nuclear protein(s) that mediate transcriptional activation by AIIR. To achieve these goals we have developed an in vitro model which allows the investigation of regulatory elements of the TH gene and trans-acting factors that participate in the regulatory cascades initiated from native AIIR activation. The recombinant genes consisting of THE promoter and luciferase reporter gene are transiently expressed in primary cultures of nontransformed adrenal medullary cells and their regulation by AIIR is examined. DNA-affinity protein selection will be employed in order to identify, purify and clone nuclear proteins that mediate the transcriptional effects of AIIR. Future studies will determine mechanisms by which the AIIR controls the function of specific transcriptional factors. Elucidation of these mechanisms will contribute towards a better understanding of how the long-term homeostatic and pathologic effects of AII are produced.
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