Development and in vitro characterization of human recombinant thyrotropin.
We have gained insight into the molecular mechanism of human thyrotropin (hTSH) action through cloning of the human TSHbeta subunit gene, development of recombinant TSH and novel analogues and chimeras produced by site-directed as well as cassette mutagenesis. A variety of loss of function mutations have shown several key domains in both the alpha- and beta-subunits that are important for high-affinity ligand interaction with the receptor. In contrast the specificity of receptor interaction was shown to be determined primarily by areas within the hTSH-beta "seat-belt" region. We have also designed various gain of function mutants (superagonists) using evolutionary considerations, homology modeling, and sequence comparisons within the cystine knot growth factor superfamily. Such superagonists resulted from increasing the positive charge by introduction of lysine or arginine residues or neutralization of negatively charged residues of the peripheral hairpin loops of each subunit in various combinations. Certain superagonists increased receptor binding, in vitro and in vivo bioactivity 100- to 1000-fold, more than that achieved previously for any other known protein ligand. In vivo metabolic clearance and biologic activity could be separately modulated by alteration of TSH carbohydrate structure including production of chimeras that added sites of O-glycosylation and/or covalently linked the alpha- and beta-subunits. These data suggest that electrostatic interactions resulting from net positive charge in TSH and net negative charge in its receptor play an important role in high-affinity TSH receptor binding and signal transduction. Insights gained from the design of such novel recombinant TSH analogues and chimeras should have many diagnostic and therapeutic applications. These include the design of improved in vitro assays for thyrotropic factors as well as the design of second generation recombinant TSH analogues for the detection and treatment of thyroid cancer.
Weintraub
B D
BD
Department of Medicine, University of Maryland School of Medicine and the Institute of Human Virology, Medical Biotechnology Center, Baltimore 21201-1068, USA.
Szkudlinski
M W
MW
D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011989:Receptors, Thyrotropin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013972:Thyrotropin / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*
D011989:Receptors, Thyrotropin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013972:Thyrotropin