Abstract
Members of the nuclear receptor superfamily play key roles in a host of physiologic and pathologic processes from embryogenesis to cancer. Some members, including the retinoic acid receptor (RAR), are activated by ligand binding but are unaffected in their subcellular distribution, which is predominantly nuclear. In contrast, several members of the steroid receptor family, including the glucocorticoid receptor, are cytoplasmic and only translocate to the nucleus after ligand binding. We have constructed chimeras between RAR and glucocorticoid receptor that selectively respond to RAR agonists but display cytoplasmic localization in the absence of ligand. These chimeric receptors manifest both nuclear translocation and gene activation functions in response to physiological concentrations of RAR ligands. The ability to achieve regulated subcellular trafficking with a heterologous ligand binding domain has implications both for current models of receptor translocation and for structural-functional conservation of ligand binding domains broadly across the receptor superfamily. When coupled to the green fluorescent protein, chimeric receptors offer a powerful new tool to 1) study mechanisms of steroid receptor translocation, 2) detect dynamic and graded distributions of ligands in complex microenvironments such as embryos, and 3) screen for novel ligands of "orphan" receptors in vivo.
Highlights
From the ‡Laboratory of Pathology and the ¶Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892
We describe here a glucocorticoid receptor (GR)-RAR chimeric protein2 that resides primarily in the cytoplasm of untreated cells but manifests complete nuclear translocation when cells are induced with the normal RAR ligand, all-trans-retinoic acid
With increased ligand binding affinity fused to GFP; RAR, retinoic acid receptor; GR-RAR, chimera containing GFP fused to GR* with the LBD replaced by that of RAR; ATRA, all-trans-retinoic acid; Dex, dexamethasone; HSP, heat shock protein; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; H1, helix 1; MCS, multiple cloning site; EGFP, enhanced GFP; BisTris, 2-[bis(2-hydroxyethyl)amino]-2(hydroxymethyl)propane-1,3-diol
Summary
From the ‡Laboratory of Pathology and the ¶Laboratory of Receptor Biology and Gene Expression, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892. We have constructed chimeras between RAR and glucocorticoid receptor that selectively respond to RAR agonists but display cytoplasmic localization in the absence of ligand These chimeric receptors manifest both nuclear translocation and gene activation functions in response to physiological concentrations of RAR ligands. We describe here a GR-RAR chimeric protein that resides primarily in the cytoplasm of untreated cells but manifests complete nuclear translocation when cells are induced with the normal RAR ligand, all-trans-retinoic acid. This chimera forms the basis for a new, in vivo translocation assay for nuclear receptor ligands and has important implications for mechanisms of receptor subcellular trafficking
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