Thyroid and steroid hormones play many important roles, mediated by nuclear receptors, in regulation of cellular homeostasis, differentiation, and proliferation. Derangements in the production of these hormones are manifested by numerous disorders. A question central to understanding nuclear receptor function is how the ligand induces changes in receptor conformation that engage the transcription activation functions and allow the receptor to interact with components of the transcription apparatus. Our laboratory has recently defined the X-ray crystallographic structure of the ligand binding domain (LBD) of the rat thyroid hormone receptor (TR)alpha and human TRbeta, which contain hormone activated transcription functions. Pilot studies in the Sponsor's laboratory also show that in the presence of thyroid hormone (triiodothyronine (T3) receptor TRs interact with the factor RIP14O thought to interact with proximal promoter proteins. The strategy for this proposal is to use the X-ray structures as guides to engineer 23 different mutants in residues that lie on the receptor surface. These mutations will sample all the surfaces of the LBD and will replace the native residues with bulky charged residues, either Arg or Glu. After confirming that the mutations leave hormone and DNA binding intact we will determine which mutations block hormone dependent binding of RIP 140. In addition, the mutant hTRbetas will be tested for their ability to activate transcription of reporter genes in transfected cells. We will also examine whether influences of the mutations on binding of RIP 140 to hTRbeta are correlated with transcriptional activity. It is hypothesized that effects on RIP 140 interactions are only one way that mutations can affect TR function. These studies should shed light on the mechanisms TR function, that should be relevant for understanding the actions of nuclear receptors and transcriptional regulators in general.