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Characterization of Site-Specific Polyclonal Antibodies to c-erbA Peptides Recognizing Human Thyroid Hormone Receptors 1, 2, and β and native 3,5,3'- Triiodothyronine Receptor, and Study of Tissue Distribution of the Antigen

Thyroid Study Unit/Immunodermatology Laboratory, Department of Medicine, University of Chicago Chicago, Illinois 60637

Address all correspondence and requests for reprints to: Leslie J. DeGroot, M.D., Thyroid Study Unit, Box 138, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.

Abstract

The translated products of v-erbA-related cDNAs have been demonstrated to be thyroid hormone receptors, and three different forms of receptor (l, 2, and β) have been found in human tissues. We synthesized five peptides corresponding to different portions of these three receptors and raised site-specific polyclonal antipeptide sera in rabbits. Each antibody displayed high titer and specificity for its respective antigen when tested in an enzyme-linked immunosorbent assay. Each immunoprecipitated the corresponding in vitro translated products of human c-erbA l, 2, or β. Two of the antisera were specific for β, one for 2, and one detected a sequence common to l and 2. The fifth was directed toward the DNA-binding area of the proteins and interacted with each receptor. The four antibodies against l and β immunoprecipitated the native thyroid hormone receptor from rat liver and caused a partial shift in the elution profile of the native receptor labeled with [¹²⁶I]T₃ on Sephacryl S-300 column chromatography. The antibody against 2 protein did not interact with native thyroid hormone receptor from rat liver. Using the indirect immunofluorescence technique with the five antibodies, we detected immunoreactivity primarily in the nucleus of cells in several tissues. In general, there was coordinate expression of both and β receptors in each organ examined, in agreement with previous data on tissue distribution of mRNAs for human thyroid hormone receptors. These studies prove the identity of v-erbA-related gene products with native thyroid hormone receptors and the expression of both and β receptors in nuclei of human and rat tissues. (Endocrinology 126: 3232–3239,1990)

Received December 8, 1989.

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