FUNCTIONAL-CHARACTERIZATION OF A MUTANT THYROID-HORMONE RECEPTOR IN XENOPUS-LAEVIS

Thyroid hormone plays a causative role during frog metamorphosis, and its effect is mediated by thyroid hormone receptors (TRs), To investig ate the function of Xenopus TRs, we have recently developed a thyroid hormone dependent in vivo transcription system by introducing TRs and RXRs (9-cis-retinoic acid receptors) into Xenopus oocytes. Interesting ly, using this system, we have found that the TR alpha B cloned previo usly is defective in transcriptional activation compared with TR alpha A, In vitro DNA binding experiments show that TR alpha B . RXR hetero dimers have drastically reduced affinity for a thyroid hormone respons e element, Site-directed mutagenesis shows that two of the seven amino acid residues that differ between TR alpha A and TR alpha B are respo nsible for the defect in TR alpha B function. These two residues affec t the DNA binding by both TR . RXR heterodimers and TR homodimers, In contrast, heterodimer formation with RXRs is not affected as demonstra ted by coimmunoprecipitation and dominant-transcriptional inhibition e xperiments, By cDNA and genomic DNA sequence analysis, we have demonst rated that the residues, which affect TR alpha B function when mutated , are identical between the wild type TR alpha B and TR alpha A, Thus, our experiments have discovered the first amphibian TR mutant, The DN A binding and transcription activation functions of the mutant are dis cussed in relation to the recently published TR crystal structure.