The long-term objective of these studies is the understanding of the molecular mechanisms through which aldosterone stimulates Na transport and thereby regulates blood pressure. This project focuses on the initial nuclear events initiated by the hormone-liganded mineralocorticoid receptor (MR), in cells of its major target, the cortical collecting duct (CCD). The foundation of these studies is our recent observation that aldosterone induces MR clustering in the nucleus, whereas MR antagonists dissociate such clusters. Our studies also indicate that MR clusters are associated with the nuclear matrix but not with pallindromic hormone response elements. Our central hypothesis is that clustering of MRs is a critical step in the mechanism of action of aldosterone, and that foci of high MR density are also enriched in proteins involved in chromatin remodeling and in basal transcription factors. Consequently, the main goal of this project is to identify proteins interacting with the MR, that are responsible for its clustering and/or play a critical role in the major biological action of aldosterone.
In aim 1, we will identify the nuclear subcompartment where the agonist-liganded MR clusters reside, and will determine which residues of the MR are involved in receptor clustering. These studies will employ side-directed mutagenesis of MR-green fluorescent protein chimeras, immunocytochemistry and confocal microscopy.
Under Aim 2, we will identify and characterize proteins expressed in the CCD that directly interact with the agonist-liganded MR, using transcription- and signal transduction-based yeast two-hybrid screens. We will identify regions of interaction of MR with MR-interacting proteins (MRIPs), generate antibodies against MRIPs, and construct expression vectors capable of disrupting MR/MRIP interactions in vivo.
In Aim 3, we will determine the role of MRIPs in MR clustering and in the biological responses initiated by aldosterone. We will determine the effects of manipulating the expression of MRIPs and disruption of their interaction with the MR, on aldosterone-induced receptor clustering, transcriptional regulation of an endogenous aldosterone target gene and on changes in Na transport in CCD cells. Identification of the molecular events involved in MR action should not only yield new insights into the mechanism of transcriptional regulation by aldosterone, but could also lead to the identification of genetic defects resulting in derangement of Na homeostasis, leading to hypertension (as in Liddle syndrome) or salt wasting (as in pseudohypoaldosteronism).
|Naray-Fejes-Toth, Aniko; Boyd, Cary; Fejes-Toth, Geza (2008) Regulation of epithelial sodium transport by promyelocytic leukemia zinc finger protein. Am J Physiol Renal Physiol 295:F18-26|
|Martel, Jessica A; Michael, Donna; Fejes-Toth, Geza et al. (2007) Melanophilin, a novel aldosterone-induced gene in mouse cortical collecting duct cells. Am J Physiol Renal Physiol 293:F904-13|
|Naray-Fejes-Toth, Aniko; Fejes-Toth, Geza (2007) Novel mouse strain with Cre recombinase in 11beta-hydroxysteroid dehydrogenase-2-expressing cells. Am J Physiol Renal Physiol 292:F486-94|
|Cordas, Emily; Naray-Fejes-Toth, Aniko; Fejes-Toth, Geza (2007) Subcellular location of serum- and glucocorticoid-induced kinase-1 in renal and mammary epithelial cells. Am J Physiol Cell Physiol 292:C1971-81|
|Boyd, Cary; Naray-Fejes-Toth, Aniko (2005) Gene regulation of ENaC subunits by serum- and glucocorticoid-inducible kinase-1. Am J Physiol Renal Physiol 288:F505-12|
|Helms, My N; Fejes-Toth, Geza; Naray-Fejes-Toth, Aniko (2003) Hormone-regulated transepithelial Na+ transport in mammalian CCD cells requires SGK1 expression. Am J Physiol Renal Physiol 284:F480-7|
|Pearce, David; Naray-Fejes-Toth, Aniko; Fejes-Toth, Geza (2002) Determinants of subnuclear organization of mineralocorticoid receptor characterized through analysis of wild type and mutant receptors. J Biol Chem 277:1451-6|
|Fejes-Toth, G; Naray-Fejes-Toth, A (2001) Immunohistochemical localization of colonic H-K-ATPase to the apical membrane of connecting tubule cells. Am J Physiol Renal Physiol 281:F318-25|