Thyroid Hormone Receptors Form Distinct Nuclear Protein-Dependent and Independent Complexes with a Thyroid Hormone Response Element (original) (raw)

Abstract

We have examined the binding of nuclear proteins and recombinant thyroid hormone receptors (TRs) to the palindromic thyroid hormone responsive element AGGTCATGACCT (TREp) using a gel electrophoretic mobility shift assay. Four specific protein- DNA complexes were detected after incubation of nuclear extracts (NE) from T3-responsive pituitary (GH3) cells with a TREp-containing DNA fragment. This was compared with the TREp binding of reticulocyte lysate-synthesized TRs. TRα1 and TRβ2 each formed a single major TR:TREp complex which comigrated with the least retarded complex formed by GH3 NE, while TRβ1 formed multiple complexes suggesting that it can bind to TREp as an oligomer. Interestingly, coincubation of 35S-TRα1, GHM3 NE, and unlabeled TREp resulted in not only the 35S-TR:TREp complex, but in two additional more greatly retarded complexes containing 35S-TRα1 and comigrating with those formed by GH3 extract alone. Incubation of each of the TRs with NE from COS-7 cells, which do not possess sufficient endogenous TRs to mediate T3-responses, resulted in formation of a new, more greatly shifted complex. A similar, heat labile activity which altered mobility of the TR:TRE complex was also present in NE from T3-unresponsive JEG-3 cells. At high concentration of NE, all of the TR bound to TREp was more greatly retarded than in the absence of NE. Truncation of TRα1 at amino acid 210 prevented additional complex formation in the presence of NE without affecting DNA binding, suggesting that the carboxyl-terminus of the TRs is essential for interaction with nuclear proteins. These results demonstrate that the gel mobility shift assay can be used to distinguish two distinct properties of the TRs, i.e. TRE-binding, and interactions with proteins present in NE from various cell types. Nuclear proteins retard the electrophoretic mobility of the TR:TRE complex, either by directly binding to the complex or by modifying the TR. These proteins could play an important functional role in T3 action.

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Author notes

This work was supported by the University of Pennsylvania Department of Medicine and by the McCabe Fund.