Thyroid hormone (TH) acts through binding to nuclear protein thyroid hormone receptors (TR), which regulate transcription of specific genes. Clinically significant abnormalities related to TR function are believed present in patients with serious illness, thyroid hormone resistance, and possibly in tumors and obesity. With the availability of cloned cDNAs coding for three forms of human TR (TRalphas and TRbeta), semipurified TR preparations, and cloned TH responsive genes, we are in a position to answer important questions about TH function in human health and disease. While three forms of hTR have been identified, it is possible that other forms are present, and we will screen human cDNA libraries to identify these. We will study the expression of hTR alpha and hTR beta in various human tissues by Northern Blot to determine which forms are expressed in all tissues. The RNAse protection assay will also be used to evaluate the forms of hTR expressed and to quantitate the amount in tissues. To fully evaluate hTR expression, we will develop antibodies to hTRs using synthetic peptides or fusion proteins. With these tools, we can determine hTR mRNA levels, TR protein, and functional TR as (131I)-T3 binding capacity. Function of the hTRs will be determined by co- transfection of hTR and rGH-CAT plasmid constructs into appropriate cell lines. Co-transfection studies will allow us to compare specifically TR alpha and TR beta control of gene transcription, and to evaluate both TRs with different responsive genes. We will clone and sequence portions of the alpha and beta TR genes in order to characterize the genes and to explain the apparent formation of multiple TR isoforms from one primary transcript. mRNA, Southern Blots of genomic DNA, and immunoprecipitable TR protein will be studied in fibroblasts of patients with thyroid hormone resistance. To identify if a specific isoform of TR is related to their illness,-cDNAs will be prepared from their mRNA for specific TRs, amplified, and sequenced. Our studies will identify the forms of functional hTR present in human tissues, their ability to control transcription of specific genes, and the molecular abnormality of TR present in patients with thyroid hormone resistance.
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