METABOLIC EFFECTS OF THYROID HORMONE

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Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.