The long range goal of this research is to explore the molecular basis by which the nuclear thyroid hormone receptor mediates changes in specific gene expression. Recent reports have suggested that the thyroid hormone receptor is the normal cellular counterpart of the v-erbA oncogene. We will test this hypothesis by isolating full-length cDNA for rat liver which are homologous to the v-erbA oncogene. The ability of antibodies raised against the purified hepatic c-erbA polypeptide, produced in E. coli, to recognize the nuclear thyroid hormone binding activity in rat liver will be tested. In addition, the hepatic c-erbA cDNA in an appropriate eucaryotic expression vector will be introduced into hepatoma cells lacking the thyroid hormone receptor. The expression of nuclear thyroid hormone binding activity and regulation of thyroid hormone responsive genes following transfection will be measured. Finally, the ability of the expressed c-erbA polypeptide to bind to specific DNA sequences of a thyroid hormone responsive gene (the S14 gene) and it transcript will be assessed. Preliminary evidence suggest that there are at least two erbA- homologous genes which are expressed in the rat. We propose that alternate erbA genes could encode variant forms of the thyroid hormone receptor which differ in their target gene specificities. We further propose that the specificity of such alternate forms of receptor will be determined by the 70 amino acid domain involved in binding to DNA. To test this hypothesis, alternate forms of erbA-related cDNA clones will be selected from appropriate rat cDNA libraries (eg., brain). The sequence of these variant forms will be compared to that found in adult liver, particularly within the DNA-binding domain. The expression of variant c-erbA genes in different tissues and developmental stages of the rat will be tested. The ability of variant forms of c- erbA polypeptide to bind to and stimulate expression of hepatic thyroid hormone responsive genes will be determined. Finally, using the techniques of in vitro mutagenesis, sequences within the DNA-binding domains of variant forms of c-erbA will be systematically changed to more closely resemble the hepatic form. The ability of these altered forms of c-erbA to bind to DNA sequences on the S14 gene and to stimulate the expression of this gene will be monitored. These studies should provide insight into the amino acid residues which play a critical role in determining the specificity of the interaction with DNA target sites.
