I. Structure, function and regulation of the human beta1 thyroid hormone receptor (h-TRbeta1). A. Identification of two regions in the hormone binding domain which interact with T3. We developed monoclonal antibodies (mAb) to map the regions of h- TRbeta1 which interact with the thyroid hormone, 3,3',5-triiodo-L- thyronine (T3), by screening clones which inhibited the binding of T3. Two such mAbs, C3 and C4, were obtained. Analysis of the binding data indicates that binding of T3 to h-TRbeta1 is competitively inhibited by both mAbs. We mapped the epitopes of C3 and C4 to Glu248-Val256 and the C-terminal Glu457-Asp461, respectively. Thus, we have identified two T3 interaction sites in h-TRbeta1. These two sites could be involved in relaying the hormonal signal to regulate the expression of the T3 target genes. B. Structural modification of h-TRbeta1 by T3-dependent phosphorylation. We evaluated the effects of T3 on the phosphorylation of h-TR1 and found that T3 increased the phosphorylation of h-TRbeta1 10-fold. T3- dependent phosphorylation alters the protease susceptibility of h- TRbeta1, indicating structural modification of h-TRbeta1 by T3- dependent phosphorylation. The T3-dependent phosphorylation-induced structural modification could provide a viable molecular mechanism for T3-induced activation of the transcriptional activity of h-TRbeta1. II. The molecular basis of resistance to thyroid hormones (RTH). A transgenic model of RTH has been developed. Transgenic mice expressing high levels of mutant mRNA had pituitary resistance to T3. These mice displayed decreased weights in association with reductions in white adipose mass and a behavioral phenotype characterized by a significant attenuation of the normal increases in locomotor activity induced by the dopamine reuptake blocker methylphenidate. These mice will be useful in the study of fat cell biology and hyperactivity. III. Thyroid hormones and tumorigenesis. We studied the interaction of h-TRbeta1 with the tumor suppressor p53. We found that h-TRbeta1 physically interacts with p53 leading to the inhibition of the binding of h-TRbeta1 to DNA. The transcriptional activity of h-TRbeta1 is repressed by the wild type p53, but is activated by the mutant p53, suggesting that one of the mechanisms by which p53 regulates cell proliferation could be mediated by h-TRbeta1.
