INTRACELLULAR-DISTRIBUTION OF THE ENDOGENOUS AND TRANSFECTED-BETA-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 cerbA (hTR-beta-1) protein were prepared. hTR-beta-1 was expressed in Escherichia coli and purified to apparent homogeneity. The purified hTR-beta-1 was used to produce monoclonal antibodies. Three hybridomas, secreting mAb J51, J52, and J53, were isolated. All of these mAbs recognized hTR-beta-1. 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) TR-alpha-1, TR-alpha-2, rat (r) TR-beta-1, TR-alpha-1, and TR-alpha-2. None of these three mAbs reacted with h- or rTR-alpha-1 and TR-alpha-2. J51 did not react with rTR-beta-1, but J52 and J53 cross-reacted with rTR-beta-1 with the same activity as hTR-beta-1. To localize the epitopes in the hTR-beta-1 molecule, [S-35]methionine-labeled and truncated hTR-beta-1 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 hTR-beta-1 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 TR-beta-1 in rat pituitary GH3 cells was visualized to be exclusively present in nuclei. The transfected hTR-beta-1 in monkey COS-1 and human choriocarcinoma JEG-3 cells was recognized by both J51 and J52. Interestingly, the intracellular localization of the transfected hTR-beta-1 or rTR-beta-1 in the above two cell lines depended on the level of expression. TR-beta-1 expressed at low levels was found exclusively in nuclei. However, for high level expression of TR-beta-1, cytoplasmic localization was also detected. J53, however, failed to detect nuclear fluorescence of the endogenous and transfected TR-beta-1 in fixed cells, suggesting that its antigenic site might be occluded. Localization of the endogenous and transfected TR-beta-1 in nuclei indicated that these two receptor proteins are structurally indistinguishable. Furthermore, the findings that TR-beta-1 could be localized in the cytoplasm when receptor was overexpressed suggested finite numbers of acceptor sites for TR-beta-1 in the nucleus.