The members of the nuclear hormone receptor superfamily regulate biological processes that range from complex metabolic responses in adults to pattern formation in early development. This family of related transcription factors includes the receptors for numerous important biological regulatory compounds (eg. steroids, thyroid hormone and retinoids), and a even larger group of related proteins, called the orphan receptors, that do not have known ligands. This proposal is directed toward understanding the function of a two closely related human and murine orphan receptors initially isolated and characterized in this laboratory. Based on the ability of these proteins to transactivate expression from a subset of retinoic acid response elements (RAREs) in the absence of retinoids or other exogenously added compounds, we call them CAR (Constitutive Activator of Retinoid response). CAR is expressed primarily in the liver in both humans and mice, and is similar to the retinoic acid receptors (RA) in that it binds to the elements it transactivates as a heterodimer with the 9-cis-retinoic acid receptors (RXR). In contrast to the rather broad specificity of the RARs, however, CAR targets only RAREs that consist of direct repeats of the receptor binding consensus hexamer separated 5 base pairs (DR-5). Moreover, since binding of such elements by CAR is quite dependent on the sequence of the 5 intervening base pairs, CAR/RXR heterodimers bind only a subset of DR-5 sites bound by RA/RXR heterodimers. We have recently used the yeast two- hybrid system to isolate clones encoding several interesting new proteins that interact with CAR. These include a new kinase, a novel component of the basal transcriptional apparatus, and a new receptor superfamily member that can apparently heterodimerize with RA and other conventional receptors.
The specific aims of this proposal are to: i. Continue to characterize the structure, expression and function of CAR proteins. 2. Define the amino acid sequences responsible for the differential binding specificity of CAR and RA. 3. Characterize the function of the CAR interacting proteins. 4. Test the hypothesis that decreasing CAR expression in vivo using antisense CAR sequences will decrease expression of putative CAR target genes such as the beta2 isoform of RA. These studies will provide new understanding of the complex response to retinoids and new insights in the mechanisms of action of the nuclear hormone receptors.
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