Determinants of subnuclear organization of mineralocorticoid receptor characterized through analysis of wild type and mutant receptors.
The mineralocorticoid receptor (MR) is a hormone-dependent regulator of gene transcription that in the absence of ligand resides both in the cytoplasm and the nucleus. Agonists but not antagonists increase the number of MRs residing in the nucleus and cause aggregation of MRs into distinct clusters. To identify the functional determinants of MR nuclear organization, we examined the localization pattern of wild type MR and a series of mutants in the presence and absence of ligands using fluorescent protein chimeras in living cells. Our data show that although MR DNA binding is not necessary to mediate nuclear localization, it is absolutely required for wild type cluster formation as is an intact N-terminal or C-terminal activation function. In contrast, destabilization of a dimerization motif within the DNA-binding domain has no effect on subnuclear receptor architecture. These data suggest that normal MR cluster formation is dependent on both DNA binding and intact transcriptional activation functions but not on DNA-dependent receptor dimerization. Because dimer mutants bind with high affinity to hormone response element DNA multimers but not to single palindromic DNA sites, we suggest that clusters represent MR aggregates bound to DNA response element multimers in the vicinity of regulated genes.
Pearce
David
D
Division of Nephrology, Department of Medicine and Cellular & Molecular Pharmacology, University of California, San Francisco, California 94143, USA. pearce@medicine.ucsf.edu
Náray-Fejes-Tóth
Anikó
A
Fejes-Tóth
Géza
G
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000450:Aldosterone / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011940:Receptor Aggregation / Q000502:physiology*; D018161:Receptors, Mineralocorticoid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D018161:Receptors, Mineralocorticoid; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000450:Aldosterone