THE EFFECTS OF P-BOX SUBSTITUTIONS IN THYROID-HORMONE RECEPTOR ON DNA-BINDING SPECIFICITY

Three ''P-box'' amino acids within the DNA recognition alpha-helix of members of the steroid hormone and thyroid hormone families of nuclear receptors are known to determine the identity of two of the six base pairs within the half- sites of cognate DNA elements. We introduced P- box substitutions derived from different members of the thyroid hormon e/estrogen receptor (T3R/ER) family into the beta-isoform of human thy roid hormone receptor (hT3R beta) and tested the DNA binding and trans activation activities of these mutants using thyroid hormone response elements (TREs) with half-sites composed of different sequences and ar ranged in different orientations. Different P-box sequences derived fr om the T3R/ER family resulted in distinct DNA binding specificities de termined by the fourth base pair of the half-site. Thyroid hormone rec eptor mutants containing EGA, EAA, EGS substitutions for the wild type EGG P-box bound with wild type affinity to consensus AGGTCA half-site s, regardless of orientation. TREs composed of AGGACA half-sites bound hT3R beta s with an EGG or EAA P-box sequence, but not those with EGA or EGS P-box sequence. A reversal of this specificity was observed on a direct repeat TRE with AGGGCA half-sites. Additionally, an ESG P-bo x substitution in hT3R beta prevented the receptor from binding to a d irect repeat as a homodimer, but this mutant could bind as a heterodim er with retinoid X receptor or to the everted repeat TRE from the chic ken lysozyme promoter.