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 c-erbA (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, [35S]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.(ABSTRACT TRUNCATED AT 400 WORDS)
Lin
K H
KH
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Willingham
M C
MC
Liang
C M
CM
Cheng
S Y
SY
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003429:Cross Reactions; D000939:Epitopes; D013006:Growth Hormone / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism*; D007536:Isomerism; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011988:Receptors, Thyroid Hormone; D013006:Growth Hormone