ISOFORM-SPECIFIC THYROID-HORMONE RECEPTOR ANTIBODIES DETECT MULTIPLE THYROID-HORMONE RECEPTORS IN RAT AND HUMAN PITUITARIES

There are three known isoforms of the thyroid hormone receptor (TR) in the rat: TR-alpha-1, TR-beta-1, and TR-beta-2. The TR-alpha-1 and TR-beta-1 mRNAs are found in many tissues, whereas TR-beta-2 mRNA is detected only in the pituitary gland. Thus far, TR-alpha-1 and TR-beta-1 mRNAs have been found in humans and are highly homologous to their counterparts in rats; however, TR-beta-2 mRNA has not yet been demonstrated in humans. To examine the expression of these TRs at the protein level, we have raised isoform-specific polyclonal antibodies in female New Zealand White rabbits against the rat TRs and c-erbA-alpha-2, a carboxy-terminal variant of TR-alpha-1 that does not bind thyroid hormone. The rabbits were immunized with synthetic peptides that contained the following amino acid sequences: TR-alpha-common-(10-31), c-erbA-alpha-2-(428-442), TR-beta-1-(73-93), and TR-beta-2-(86-101, 113-133). All immune sera could bind specifically to their respective immunizing peptides on enzyme-linked immunosorbent assay as well as immunoprecipitate specifically in vitro translated rat and human TRs. Anti-TR-beta-1 and anti-TR-alpha-common antibodies could immunoprecipitate TR-beta-1 or TR-alpha-1, respectively, in transfected COS-7 cells. We also immunostained normal adult rat and human pituitary glands. Each isoform-specific antibody could immunostain almost all of the anterior pituitary cells, suggesting that TR-alpha-1, TR-beta-1, TR-beta-2, and c-erbA-alpha-2 are most likely expressed in all anterior pituitary cell types in rats and humans. The staining of rat pituitary glands by the anti-TR-beta-2 antibodies demonstrates for the first time that TR-beta-2 is expressed as a protein in pituitary cells. Furthermore, the staining of human pituitary glands by the anti-TR-beta-2 antibodies suggests that there is a human homolog of the rat pituitary-specific TR-beta-2 that shares similar epitopes with the rat TR-beta-2. In summary, we have prepared isoform-specific antibodies against TRs that can recognize in vitro translated, transiently transfected, and in situ rat and human pituitary TRs. These antibodies will be useful in examining tissue- and cell type-specific expression of rat and human TRs at the protein level.