Thyroid hormone-responsive tissues contain chromatin-localized receptor proteins that appear to be involved in mediating many of the diverse actions of these hormones. In order to understand the details of how thyroid hormone works, it will be critical to purify these proteins. This has been a difficult task, because the proteins are of low abundancy. Nevertheless, to date work in the laboratory has resulted in solubilization of the receptors from chromatin and in a 500-fold purification of the receptor in a relatively stable form (ca. 1% pure) that exhibits reversible hormone binding properties and other characteristics of receptors in crude preparations. It is hoped to adapt the procedures already developed, including affinity chromatography, so that much larger quantities of purified receptors can be obtained. This will involve a modification of the extraction procedure, a precipitation step, heparin-agarose chromatography, high pressure liquid chromatography and polyacrylamide gel electrophoresis. The major goal will be to obtain enough receptors (ca. 100 pmoles) to perform microsequencing. From the sequence, it is hoped to synthesize receptor peptides for raising antibodies and synthesizing oligonucleotide probes for use in screening for cloned cDNA to receptor mRNA. The cDNA will be used to clone the chromosomal receptor gene and related genes. The cloned genes will then be used to synthesize larger quantities of receptors in bacteria, yeasts and/or mammalian cells. The information from receptor gene structure and the use of receptor genes should provide more information about receptor structure, biosynthesis and function. The receptors, both purified and in crude extracts and synthesized with the use of recombinant DNA technology will also be used to study receptor properties relevant to thyroid hormone action, such as DNA and other binding effects, capabilities to stimulate cell-free transcription of thyroid hormone-responsive genes and potential enzyme activity. Also receptors will be made available for other detailed structure studies such as x-ray crystallography. It is hoped that the progress resulting from this work will lead to a better understanding of the mechanisms of thyroid hormone action. This information may also be relevant to a better understanding of gene expression in general.
