Evidence for a novel cardiac-enriched retinoid X receptor partner.
Recent studies indicate that retinoid-mediated pathways play a pivotal role in cardiac morphogenesis and function. To identify proteins that serve as interacting partners of the retinoid X receptor alpha (RXRalpha) in heart, DNA-protein binding studies were performed with an RXR-responsive element (NRRE-1) derived from the medium chain acyl-CoA dehydrogenase gene promoter and nuclear protein extracts prepared from adult rat heart. NRRE-1 is a pleiotropic RXR-responsive element comprised of three potential recognition sites for class II members of the nuclear receptor superfamily. Gel mobility shift assays performed with an NRRE-1 probe in the absence or presence of bacterially overproduced RXRalpha and nuclear protein extracts prepared from adult rat heart, liver, or brain identified a cardiac-specific, RXR-dependent DNA-protein interaction. The NRRE-1-RXR.cardiac-enriched RXR-interacting protein (CERIP) complex exhibited a distinct mobility compared with NRRE-1-RXR.peroxisome proliferator-activated receptor, NRRE-1-RXR.retinoic acid receptor, or NRRE-1-RXR.thyroid receptor complexes. Mutational analysis demonstrated that two of the three potential binding half-sites of NRRE-1 (an everted repeat separated by an 8-base pair spacer) are required for the NRRE-1-RXR. CERIP interaction. Gel mobility shift assays demonstrated that CERIP interacted with RXRalpha and RXRgamma but not with RXRbeta, indicating a receptor subtypespecific binding preference and suggesting an RXR AB region-dependent interaction. The RXR.CERIP complex did not form on NRRE-1 when a mutant GST-RXRalpha fusion protein lacking the NH(2)-terminal AB region (but containing the receptor dimerization domain) of RXRalpha was added in place of the full-length RXRalpha, confirming a role for the AB region in the RXR. CERIP interaction. DNA-protein cross-linking studies demonstrated that CERIP is a DNA-binding protein of approximately 110 kDa. These results provide evidence for the existence of a cardiac-enriched DNA-binding protein that interacts with RXRalpha via the AB region and suggest a mechanism whereby cardiac retinoid signaling is controlled in an RXR subtype-specific manner.
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Center for Cardiovascular Research, Washington University School of Medicine, St. Louis, Missouri 62110, USA.
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Kelly
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D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000235:genetics; D000818:Animals; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D051381:Rats; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D047488:Retinoid X Receptors; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase