Analysis of the ternary complex formation of human urokinase with the separated two domains of its receptor.
Human urokinase-type-plasminogen-activator receptor (uPAR) is a glycolipid-anchored membrane glycoprotein comprising three structurally similar domains. We have succeeded in direct observation of the ternary complex formation of single-chain urokinase (scuPA) or its N-terminal fragment (ATF) with the separated domain-1 (N-terminal domain) and domain-(2+3) (internal and C-terminal domain) of human uPAR, by means of gel-filtration HPLC analysis. This complex was found to consist of the three components in an equimolar ratio (thus referred to as the three-part complex). To determine the nature of the interaction between these components, cross-linking experiments involving various kinds of cross-linkers and competitive binding assay on ELISA were performed. These experiments have shown that each uPAR domain can bind directly to scuPA at low affinity, and that both these domains contribute to the high-affinity binding between scuPA and uPAR in a synergistic manner. It can be considered that the synergistic effect of domain-1 and domain-(2+3) on scuPA binding would result from a conformational change, and that this steric event might trigger the signal transduction reported for scuPA/uPAR binding.
Oda
M
M
Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd, Japan. masayuki.oda@kyowa.co.jp
Shiraishi
A
A
Hasegawa
M
M
D001386:Azides / Q000378:metabolism; D001667:Binding, Competitive; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry*; D055293:Receptors, Urokinase Plasminogen Activator; D015398:Signal Transduction / Q000502:physiology; D013388:Succinimides / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry*
D001386:Azides; D003432:Cross-Linking Reagents; C524898:PLAUR protein, human; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D013388:Succinimides; C074882:sulfosuccinimidyl-2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate; D014568:Urokinase-Type Plasminogen Activator