An essential role of domain D in the hormone-binding activity of human beta 1 thyroid hormone nuclear receptor.
By analogy with steroid receptors, human placental thyroid hormone nuclear receptor (hTR beta 1) could be divided into four functional domains: A/B (Met1-Leu101), C (Cys102-Ala170), D (Thr171-Lys237), and E (Arg238-Asp456). The E domain was thought to bind thyroid hormone. To evaluate whether domain E alone is sufficient to bind T3 or requires the presence of other domains for functional T3-binding activity, a series of deletion mutants was constructed. The mutants were expressed in Escherichia coli, and the expressed proteins were purified. Analysis of the T3-binding affinity and analog specificity of the purified truncated hTR beta 1 indicated that domain E alone did not have T3-binding activity. Extension of the amino-terminal sequence of domain E to include part of domain D yielded a mutant (Lys201-Asp456) with a Ka for T3 of 0.5 +/- 0.2 x 10(9) M-1. Further extension to include the entire domain D (Met169-Asp456) yielded a mutant with T3-binding activity with a Ka of 0.8 +/- 0.1 x 10(9) M-1. Further extension of the amino-terminal sequence to include domain C increased the affinity for T3 by nearly 2-fold (Ka = 1.5 +/- 0.4 x 10(9) M-1). The Ka for the wild-type hTR beta 1 is 1.5 +/- 0.2 x 10(9) M-1. Furthermore, mutant (Met169-Asp456) binds to 3',5',3-triiodo-L-thyropropionic acid, D-T3, L-T4, and L-T3 with 307%, 37%, 7%, and 0.1%, respectively, of the activity of L-T3. This order of analog affinity is similar to that of the wild-type hTR beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
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Lin
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Parkison
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McPhie
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Cheng
D001483:Base Sequence; D001667:Binding, Competitive; D015152:Blotting, Northern; D015153:Blotting, Western; D002872:Chromosome Deletion; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D014162:Transfection; D014284:Triiodothyronine / Q000378:metabolism
D012333:RNA, Messenger; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine