Intracellular distribution of the endogenous and transfected β form of thyroid hormone nuclear receptor visualized by the use of domain-specific monoclonal antibodies

To study the regulation, tissue distribution, and subcellular localization of nuclear receptor for thyroid hormone, monoclonal antibodies (mAbs) against the human placental c-erbA. (hTRβ1) protein were prepared. hTRβ1 was expressed in Escherichia coli and purified to apparent homogeneity. The purified hTRβ1 was used to produce monoclonal antibodies. Three hybridomas, secreting mAb J51, J52, and J53, were isolated. All of these mAbs recognized hTRβ1, J51 and J52 belong to the immunoglobulin G1-_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) TRα1, TRα2, rat (r) TRβ1, TRα1, and TRα2. None of these three mAbs reacted with h- or rTRα1 and TRα2. J51 did not react with rTRβ1, but J52 and J53 cross-reacted with rTRβ1 with the same activity as hTRβ1. To localize the epitopes in the hTRβ1 molecule, [³⁵S]methionine-labeled and truncated hTRβ1 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 hTRβ1 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 hTRβ1 in monkey COS-1 and human choriocarcinoma JEG-3 cells was recognized by both J51 and J52. Interestingly, the intracellular localization of the transfected hTRβ1 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.