I. Structure and activity of the human (beta1 thyroid hormone receptor (h- TRbeta1). A. The structure of thyroid hormone binding domain (HBD): To understand the molecular basis of the thyroid hormone-dependent gene regulating activity of h-TRbeta1, the structure of HBD was studied. Truncated h- TRbeta1 fragments were analyzed by circular dichroism (CD). The spectra are compatible with the sequence analysis which predicts that HBD contains alternating stretches of alpha-helix and beta-sheet. A decrease in secondary structure in fragments in which the predicted beta-strand 1 or alpha-helix 8 was deleted, was accompanied by loss of hormone binding activity. These results are consistent with an alpha/beta barrel structure for the HBD of h-TRbeta1. Based on these results, we suggest a new model for h-TRbeta1, consisting of the known DNA binding domain, linked by an alpha-helical hinge to the HBD, with the tertiary structure of an alpha/beta barrel. B. The essential role of phosphorylation in retinoid X receptor (RXR)- dependent activation of h-TRbeta1: RXR is one of the thyroid hormone receptor accessory proteins (TRAPS). We found that in vitro phosphorylation of TR enhanced the binding of TR to RXR(3 on several TREs by 5 to 10-fold. In vivo, phosphorylation increased the RXR-dependent enhancement of TR transcriptional activity by 2 to 3-fold. Thus, phosphorylation is essential for modulating the activity of h-TRbeta1 by RXRbeta. II. The molecular basis of generalized resistance to thyroid hormone (GRTH): To understand the molecular basis of GRTH, we have studied the thyroid hormone and DNA binding characteristics in vitro and the function in vivo of the mutant receptors. The affinity in the binding of the mutant receptors to T3 correlates well with the degree of impairment of their transactivation function in HeLa cells. All of the mutant h-TRbeta1s are able to inhibit the function of transfected wild-type h-TRbeta1, indicating the h-TRbeta1 mutants inhibit the function of normal TR by a dominate negative mechanism. In vitro DNA studies indicate that the mutant receptors show an increased tendency to form homodimer in several TREs. Furthermore, excess amounts of transfected RXR(3 could not reverse the dominant negative potency of mutant TRs in vivo. Therefore, competition for DNA-binding most likely mediates the dominant negative potency in patients with GRTH. These findings should help provide a more rational basis for therapeutic management of GRTH.
