- This proposal focuses o the molecular mechanisms through which aldosterone stimulates Na transport in the collecting duct. Mineralocorticoid specificity of tissues is thought to be conferred, in part, by the presence of the enzyme 11-beta hydroxysteroid dehydrogenase (11-b-HSD2) which inactivates glucocorticoids and blocks illicit occupancy and activation of MR. Two isoforms (at least) of this enzyme exist, bidirectional enzyme 11B-HSD1 present in liver, and a unidirectional isoform 11B-HSD2, which has been cloned by the principal investigator and others and i specifically localized to mineralocorticoid-responsive tissues. Previous work has established that the enzyme (11-b-HSD2) is located in endoplasmic reticulu and in addition to catalyzing hydride transfer, is a high-affinity binder of glucocorticoids. Its intracellular location and binding activity are both important for reducing cytoplasmic glucocorticoids.
The first aim of the proposal is to identify residues responsible for glucocorticoid binding and ER localization of the enzyme. Targeted mutations will be introduced based on 3D computer models and the steroid-binding profile of such mutants determined. Residues important for ER localization will be mapped using GFP fusion proteins. Expression of 11-b-HSD2 is a hallmark of ALDO target cells. Therefore, identification of cis- and trans-factors that direct its cell-specific expression, which is the goal of Aim 2, should help to understan the molecular mechanism involved in generating the mineralocorticoid target cell phenotype. Cis-elements will be identified by 5' deletion analysis and in vivo footprinting, their function verified in transgenic mice. Transcription factors binding to cis-elements will be identified by gel mobility shift and supershift analysis. Novel transcription factors will be cloned using the yeas one-hybrid system.
Aim 3 is to identify ALDO-regulated early response genes using a PCR-based subtraction hybridization technique. Full-length cDNAs of ALDO-regulated genes (either induced or repressed) will be isolated from cultured CCD cells and their effect on Na transport verified by expression in CCD cell lines and oocytes.
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