Characterization of Site-Specific Polyclonal Antibodies to c-erbA Peptides Recognizing Human Thyroid Hormone Receptors α1, α2, and β and native 3,5,3′- Triiodothyronine Receptor, and Study of Tissue Distribution of the Antigen

Abstract

The translated products of v-erbA-related cDNAs have been demonstrated to be thyroid hormone receptors, and three different forms of receptor (alpha 1, alpha 2, and beta) have been found in human tissues. We synthesized five peptides corresponding to different portions of these three receptors and raised site-specific polyclonal-antipeptide sera in rabbits. Each antibody displayed high titer and specificity for its respective antigen when tested in an enzyme-linked immunosorbent assay. Each immunoprecipitated the corresponding in vitro translated products of human c-erbA alpha 1, alpha 2, or beta. Two of the antisera were specific for beta, one for alpha 2, and one detected a sequence common to alpha 1 and alpha 2. The fifth was directed toward the DNA-binding area of the proteins and interacted with each receptor. The four antibodies against alpha 1 and beta immunoprecipitated the native thyroid hormone receptor from rat liver and caused a partial shift in the elution profile of the native receptor labeled with [125I]T3 on Sephacryl S-300 column chromatography. The antibody against alpha 2 protein did not interact with native thyroid hormone receptor from rat liver. Using the indirect immunofluorescence technique with the five antibodies, we detected immunoreactivity primarily in the nucleus of cells in several tissues. In general, there was coordinate expression of both alpha and beta receptors in each organ examined, in agreement with previous data on tissue distribution of mRNAs for human thyroid hormone receptors. These studies prove the identity of v-erbA-related gene products with native thyroid hormone receptors and the expression of both alpha and beta receptors in nuclei of human and rat tissues.

Plasma protein regulation by thyroid hormone

“…Their results revealed abundant TR 1, TR 2 and TR 1 protein in human hepatocyte. Macchia et al (1990) also Genes with a greater than twofold increase or decrease are shown. The data represent the mean of three independent experiments.…”

“…Thereafter, RNA was isolated and cDNA microarray was performed as described in the Materials and Methods. reported equal intensity of 47 kDa (TR 1) and 55 kDa (TR 1) protein bands from rat liver (Macchia et al 1990). Moreover, Flores-Morales et al (2002) used TH 1-deficient mice and cDNA microarray to determine the target genes regulated by T 3 .…”

Thyroid hormone actions on cartilage and bone: interactions with other hormones at the epiphyseal plate and effects on linear growth

“…Testosterone binding Figure 2 Longitudinal sections (original magnification 400) of decalcified rat proximal tibiae, showing immunolocalisation of T 3 R 1 (left panels) and T 3 R 1 (right panels) within distinct zones of the growth plate, using isoform-specific rabbit polyclonal antibodies against human T 3 R 1 and T 3 R 1 proteins (Macchia et al 1990, Falcone et al 1992), which we have demonstrated to recognise rat T 3 R isoforms specifically in bone cells, using western blotting , Bland et al 1997. The upper panels show reserve (RZ), proliferative (PZ) and proximal hypertrophic (HZ) zones; the lower panels show the distal hypertrophic zone (HZ) and metaphyseal primary spongiosum (MPS).…”

Hormone-induced changes in nuclear receptor stoichiometry in HL60 cells correlate with induction of monocyte or neutrophil differentiation

“…Specific receptor proteins were detected by enhanced chemiluminescent assay (ECL; Amersham) after exposure of filters to X-ray film for 1-20 min. Filters were probed with the following primary antibodies: polyclonal against human T 3 R 1, c-erb A 2 and T 3 R 1 proteins (Macchia et al 1990, Falcone et al 1992, polyclonal against rat RAR (Ali et al 1992), and monoclonal against human VDR (Pike et al 1982) (purchased from Cambridge Research Biochemicals, Zeneca, Northwich, UK). A monoclonal antibody against mouse RXR, which recognises the , and RXR isoforms (4RX 1D12, (Rochette Egly et al 1994)), was used to detect RXR protein.…”

“…There was no change in receptor protein size after induction of cell differentiation by hormones (data not shown). Estimation of the approximate molecular masses of each protein using standard markers indicated that the expressed receptors were of appropriate size (Pike et al 1982, Macchia et al 1990, Ali et al 1992, Falcone et al 1992, Rochette Egly et al 1994. The 4RX 1D12 monoclonal antibody used to detect RXR expression recognises the three RXR isoforms , and (Rochette Egly et al 1994).…”