The overall objective of this grant proposal is to characterize the receptor binding domain(s) of diphtheria toxin, and to characterize fully and purify the specific diphtheria toxin-binding cell surface protein(s) from highly toxin-sensitive Vero cells. The diphtheria toxin-binding proteins are detected by i) prebinding toxin to extrinsically radiolabeled Vero cells, followed by non- ionic detergent lysis and immunoprecipitation of the formed (receptor-toxin) complexes with anti-toxin coupled to Sepharose, and by ii) chemical cross-linking of radioiodinated diphtheria toxin bound to its receptor on the surface of Vero cells. Monoclonal antibodies will be prepared against the isolated diphtheria toxin- binding protein(s), as well as to the toxin receptor on Vero cells, to determine whether the diphtheria toxin-binding protein(s) corresponds to the toxin receptor. These monoclonal antibodies will also be utilized to investigate the cell surface distribution and mechanism of internalization of the toxin receptor on Vero cells, as well as to investigate the location and physiological state of the receptor under conditions where it lacks toxin-binding capacity (e.g. NaF, vanadate, or TPA treatment). The gene that codes for the toxin receptor will be cloned, sequenced, and will be employed to transfect toxin-resistant mouse L-cells in order to determine whether toxin resistance is due to lack of receptor expression. The results of this proposed project will provide an insight into the possible nature of the physiological cell surface receptor that diphtheria toxin utilizes to gain illicit access to the cell cytosol, will significantly extend our knowledge of toxin: receptor interactions, and will further our understanding on receptor- mediated internalization of such macromolecules as growth factors, hormones, and other exotoxins.
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