A Glucocorticoid/Retinoic Acid Receptor Chimera That Displays Cytoplasmic/Nuclear Translocation in Response to Retinoic Acid

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.The glucocorticoid receptor (GR) 1 is a member of the translocating class of steroid receptors. Unliganded GR is found in the cytoplasm and moves rapidly into the nucleus in response to hormone stimulation. Other members of the nuclear receptor superfamily are either complex in their intracellular distributions (e.g. mineralocorticoid receptor (1), progesterone receptor (2, 3), androgen receptor (4), and dioxin receptor (5)) or are found primarily in the nucleus (e.g. estrogen receptor (6), thyroid hormone receptor (see Refs. 7 and 8), and retinoic acid receptor (RAR; see Refs. 7 and 8)). The recent characterization of functional GFP fusions with the steroid/nuclear receptors allows study of real time movement for these molecules within living cells (9). The unliganded GFP-GR is found completely in the cytoplasm, whereas the hormone-activated receptor is located almost exclusively in the nucleus shortly after hormone activation (9 -11). Because nuclear translocation by this intrinsically fluorescent receptor is quite dramatic and easily monitored in real time in vivo, it can provide a straightforward methodology to detect physiological concentrations of receptor ligand in the cellular environment.The availability of chimeric proteins with the nuclear/cytoplasmic translocation properties of GR, and the ligand responsiveness of the nuclear receptors, would provide a powerful translocation assay for nuclear receptor ligands and potentially for novel analogues, as well. Many chimeric receptors have been described (e.g. see Ref. 12), but these fusions have been characterized exclusively in terms of their gene activation potential. We describ...

Molecular Identification and Characterization of a Novel Nuclear Protein Whose Expression Is Up-regulated in Insulin-resistant Animals

“…It was reported that subcellular localization of CARP is altered by a change of circumstance of the cell, such as serum depletion in vitro (20). Since the function of protein is sometimes regulated by its subcellular localization (25,26), detailed analysis of DARP localization may provide important clues to clarify the physiological function of DARP.…”

Activity and subcellular compartmentalization of peroxisome proliferator-activated receptor α are altered by the centrosome-associated protein CAP350

“…In some cases, receptors have been shown to accumulate in distinct nuclear foci in the presence of ligand (van Steensel et al, 1995). Intracellular trafficking and subcellular or subnuclear compartmentalization may be a general phenomenon of type II nuclear hormone receptors and could be differentially influenced by ligand interaction, association with cofactors and/or through heterodimerization with RXR (Baumann et al, 2001b;Mackem et al, 2001;Akiyama et al, 2002;Barsony and Prufer, 2002;.…”

An estrogen receptor chimera senses ligands by nuclear translocation

“…pCI-nGFP-C656G containing a polylinker replacing sites downstream of the coding region of helix 1 of the LBD of rGR [17] was digested sequentially with StuI and EcoRI at the polylinker. The ER␣ expression vector HEGO was digested with BlpI and the overhang filled in prior to digestion with EcoRI.…”

“…Like other nuclear receptors, the estrogen receptor shuttles between the cytoplasm and the nucleus but its constitutive location is predominantly nuclear [12][13][14][15][16], so it does not undergo translocation to the nucleus in response to ligand. Our earlier studies showed that the translocation properties of GR can be conferred to the ligand binding domain of another nuclear receptor, the retinoic acid receptor [17]. We therefore initiated efforts to generalize the concept that the intrinsic translocation properties of GR can be transferred to non-translocating receptors.…”