The objective of this proposal is the elucidation of the structure- function relationships of human thyrotropin (hTSH) and its receptor by developing and characterizing the action of novel analogs with enhanced binding affinity, in vitro and in vivo biological activity ( superactive analogs ). There are extensive studies of functional domains of gonadotropins but only limited studies of those in TSH, primarily from our laboratory, which show significant differences in structure-function relationships. Moreover, until our recent observations, there had not been reports for any glycoprotein hormone or any member of the cystine knot growth factor superfamily of such analogs with major increases in receptor binding affinity or in vitro bioactivity. We have initiated the design of superactive analogs based on evolutionary considerations, homology comparison of sequences from various species, and homology modeling of hTSH. The first generation of superactive hTSH analogs displayed up to 1,000-fold increase in potency and 2-fold increased maximal response. To design a second generation of even more potent analogs we will selectively introduce clusters of charged residues, present in homologous hormones, within the peripheral loops of hTSH, separately and in various combinations. In the current proposal we will: 1) perform systematic multidomain mutagenesis in the peripheral hairpin loops of both hTSH subunits to optimize the potency and efficacy of the analogs; 2) express and purify large amounts of the most active analogs and characterize their biochemical and biological properties; 3) attempt to determine the areas of the hTSH receptor that interact with the positively charged residues within the loops of hTSH superactive analogs; 4) test whether site-specific removal of sialic acid will increase the receptor binding and in vitro activity of selected hTSH analogs. These studies should provide novel insights into structure-function relationships of TSH and a novel approach to the study of TSH receptor activation. Highly active recombinant hTSH analogs should be of great value in several in vitro applications related to receptor binding or activation and should have improved therapeutic benefit in stimulating 131I uptake and thyroglobulin secretion in patients with thyroid cancer and nontoxic goiter. High affinity hTSH analogs may also have unique capabilities for novel therapies of thyroid cancer involving targeted delivery of radionuclides, toxins, tumor suppressor genes or the sodium/iodide symporter gene. Moreover, their design provides a rational strategy for the development of similar analogs for other members of the cystine know growth factor superfamily. Finally, the public health importance of this proposal is underscored by the large number of new cases of unusually aggressive childhood thyroid cancer detected in Europe because of the Chernobyl accident and of new adult cases expected in the U.S. because of nuclear testing in Nevada in the 1950's.
| Zhang, M; Tong, K P; Fremont, V et al. (2000) The extracellular domain suppresses constitutive activity of the transmembrane domain of the human TSH receptor: implications for hormone-receptor interaction and antagonist design. Endocrinology 141:3514-7 |
