FUNCTIONAL-ANALYSIS OF A TRANSACTIVATION DOMAIN IN THE THYROID-HORMONE BETA-RECEPTOR

Hormone-dependent transcriptional activation (AF-2) by the thyroid hor mone beta receptor (TR beta) localizes to its carboxyl-terminal domain . A putative transactivation sequence within this domain was analyzed by mutating individual residues to alanine. Mutant receptor carboxyl t erminal domains were tested coupled to the heterologous DNA binding do main of Ga14. A single mutant receptor (E460A) showed normal hormone b inding and activation, whereas several others (P453A, F455A, L456A, F4 59A) exhibited impaired transactivation which correlated with their re duced ligand binding. Two mutations (L454A, E457A) were able to dissoc iate these properties, generating transcriptionally defective mutant p roteins with preserved hormone binding. A further conservative substit ution (E457D) was also non functional, and these three mutations were equally deleterious when tested in the context of full-length TR beta with a natural thyroid hormone response element containing promoter. T his loss of activity was not due to altered DNA binding or expression of mutant receptors in cultured cells. They also retained the ability to recruit VP16-tagged retinoid X receptor in vivo as well as bind the basal transcription factors TFIIB and TBP in vitro. Our observations indicate that conserved hydrophobic (Leu(454)) and charged (GlU(457)) residues mediate AF-2 activity of TR beta, possibly via a co-activator that has yet to be identified.