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.
Yen
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Department of Medicine, Brigham and Women's Hospital, Howard Hughes Medical Institute, Boston, Massachusetts 02115.
Sunday
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Darling
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Chin
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D000818:Animals; D000918:Antibody Specificity; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D010902:Pituitary Gland / Q000737:chemistry* / Q000648:ultrastructure*; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D011988:Receptors, Thyroid Hormone / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D013237:Stereoisomerism; D014162:Transfection
D012333:RNA, Messenger; D011988:Receptors, Thyroid Hormone