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Intracellular Distribution of the Endogenous and Transfected β Form of Thyroid Hormone Nuclear Receptor Visualized by the Use of Domain-Specific Monoclonal Antibodies

Laboratory of Molecular Biology, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, National Institutes of Health (K.-H.L., M.C. W., S.-y.C); and the Division of Blood and Blood Products, Food and Drug Administration (C.-M.L) Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Sheue-yann Cheng, Building 37, Room 4B09, National Cancer Institute, Bethesda, Maryland 20892.

Abstract

To study the regulation, tissue distribution, and subcellular localization of nuclear receptor for thyroid hormone, monoclonal antibodies (mAbs) against the human placental cerbA (hTRβl) protein were prepared. hTRβ was expressed in Escherichia coli and purified to apparent homogeneity. The purified hTRβ was used to produce monoclonal antibodies. Three hybridomas, secreting mAb J51, J52, and J53, were isolated. All of these mAbs recognized hTRβ. J51 and J52 belong to the immunoglobulin Gl_-k subclass; J53 is an IgM. To evaluate cross-reactivity with other classes of c-erbAs, the three mAbs were used to immunoprecipitate the in vitro translation products of human (h) TRl, TR2, rat (r) TRβ1, TRl, and TR2. None of these three mAbs reacted with h- or rTRl and TR2. J51 did not react with rTRβ1, but J52 and J53 cross-reacted with rTRβ1 with the same activity as hTRβ. To localize the epitopes in the hTRβ molecule, [³⁵S]methionine-labeled and truncated hTRβ containing the hormone-binding domain E (Lys²³⁵-Asp⁴⁵⁶; Lys²⁰¹-Pro⁴¹⁴), domain D (Met¹⁶⁹-Asp⁴⁵⁶), or the DNA-binding domain C (Glu¹⁰⁰-Asp⁴⁵⁶) were expressed in E. coli and purified. Immunoprecipitation of the above truncated hTRβ with mAbs indicated that the epitopes for J51 and J52 were located in two different sites in the A/B domain. The epitope for J53 was located in the E domain. Using immunocytochemistry and mAb J52, the endogenous TRβ1 in rat pituitary GH₃ cells was visualized to be exclusively present in nuclei. The transfected hTRβ in monkey COS-1 and human choriocarcinoma JEG-3 cells was recognized by both J51 and J52. Interestingly, the intracellular localization of the transfected hTRβ or rTRβ1 in the above two cell lines depended on the level of expression. TRβ1 expressed at low levels was found exclusively in nuclei. However, for high level expression of TRβ1, cytoplasmic localization was also detected. J53, however, failed to detect nuclear fluorescence of the endogenous and transfected TRβ1 in fixed cells, suggesting that its antigenic site might be occluded. Localization of the endogenous and transfected TRβ1 in nuclei indicated that these two receptor proteins are structurally indistinguishable. Furthermore, the findings that TRβ1 could be localized in the cytoplasm when receptor was overexpressed suggested finite numbers of acceptor sites for TRβ1 in the nucleus. (Endocrinology 128: 2601–2609, 1991)

Received December 11, 1990.

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