I. High level expression of the endoplasmic reticulum-associated thyroid hormone binding protein (p55). A full-length p55 cDNA was inserted into a Harvey murine sarcoma virus-derived vector (pHTBr). pHTBr was transfected into NIH 3T3 cells using pRSVneo and pHaMOR1 as the selectable markers. Using indirect immunofluorescence, nine stable cell lines expressing p55 were identified. Among the nine, two cell lines were expressing p55 as high as that in parental cells. Furthermore, the molecular size, immunoreactivity, subcellular localization and the reactivity of the expressed p55 with an affinity analog of 3.3', 5-triiodo-L-thyronine (T3), N- bromoacetyi-T3 are indistinguishable from those of the endogenous p55. These results indicated the production of stable cell lines expressing heterologous p55. These stable cell lines could be used as a tool to further study the function of p55. II. Characterization of a cytosolic thyroid hormone binding protein (p58) using monoclonal antibodies. A cellular hormone binding protein (p58) was purified to homogeneity. Two hybridomas, J11 and J12, secreting monoclonal antibodies to p58 were isolated. Using these antibodies, p58 was found to be not post- translationally modified by glycosylation, sulfation or phosphorylation. It has a cellular degradation rate, t1/2 = 2.1 hour. Immunocytochemical studies indicate that p58 is located in the non-membranous cytoplasm (cytosol). Its role in the intracellular transport of T3 is being investigated. III. Antipeptide antibodies to the Tq nuclear receptor. Recent studies have shown that c-erbA protein is the T3 nuclear receptor. To study its tissue distribution and subcellular localization, antipeptide antibodies to the human placenta c-erbA (hc-erbA-B) protein were prepared. Two antibodies, NP-98 and CP-91, specifically reacted with the hc-erbA-B protein synthesized in vitro. These antibodies also specifically immunoabsorb the T3 binding activity of the T3 nuclear receptors isolated from placenta tissue and cultured cells. These antibodies are a valuable tool in studying the thyroid hormone action.
