Dominant negative and DNA-binding properties of mutant thyroid hormone receptors that are defective in homodimerization but not heterodimerization.
Thyroid hormone receptors (TRs) bind to thyroid hormone response elements (TREs) as monomers, homodimers, and heterodimers. Mutations that cause resistance to thyroid hormone (RTH) have proven useful for identifying important functional domains in the receptor. Previous studies have shown that RTH mutants must retain the ability to form heterodimers with RXR to exert dominant negative inhibition of wild-type receptor function. In this report, we examined in detail the dimerization properties, function, and dominant negative activity of RTH mutations at R316H and R338W--two mutations that have a propensity to cause the pituitary form of RTH. These mutants show selective loss of homodimerization, with preservation of heterodimerization with RXR alpha. The selective loss of homodimerization was independent of the orientation of the half sites in the TRE. The R316H mutant was transcriptionally inactive in transient expression assays, consistent with its markedly reduced T3 binding. In contrast, R338W was activated at nanomolar concentrations of T3, precluding quantitative analyses of its dominant negative properties. In cotransfection assays with wild-type TR beta, the R316H mutant functioned in a dominant negative manner to block positively (TRE-pal; DR4) and negatively (TSH alpha) regulated reporter genes, although its inhibitory potential was reduced compared with other RTH mutants. Introduction of the R316H mutation into a receptor containing a potent RTH mutant (G345R) reduced its dominant negative activity to the level of the R316H mutant alone. These results suggest that mutations that alter homodimerization have reduced dominant negative activity for some target genes, a feature that may account, in part, for phenotypic variability in RTH.
Kitajima
K
K
Division of Endocrinology, Metabolism, and Molecular Medicine, Northwestern University Medical School, Chicago, Illinois 60611, USA.
Nagaya
T
T
Jameson
J L
JL
D001483:Base Sequence; D002460:Cell Line; D002822:Choriocarcinoma; D004247:DNA / Q000378:metabolism*; D015292:Glycoprotein Hormones, alpha Subunit / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011988:Receptors, Thyroid Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D014162:Transfection; D014284:Triiodothyronine / Q000378:metabolism / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015292:Glycoprotein Hormones, alpha Subunit; D046911:Macromolecular Substances; D011988:Receptors, Thyroid Hormone; D011993:Recombinant Fusion Proteins; D014284:Triiodothyronine; D004247:DNA; D008156:Luciferases