Cytokeratin 1 and gC1qR mediate high molecular weight kininogen binding toendothelial cells

High molecular weight kininogen (NK) attaches to endothelial cells by separ ate sites on the heavy and light chains and requires 15-50 mu M zinc. Previ ously identified binding proteins include gC1qR, cytokeratin 1, and the uro kinase plasminogen activator receptor; however, their relative contribution s to binding are not yet clarified. We have prepared affinity columns to wh ich were coupled either cleaved HK or peptide LDCNAEVYVVPWEKKIYPTVNCQPLMGM derived from heavy-chain domain 3. Endothelial cell membranes were solubili zed and chromatographed in the presence or absence of zinc ion, the bound p roteins were eluted, and active fractions were identified by dot blot using biotinylated HK, SDS/PAGE, and Western blot analysis. The peptide containi ng column eluate revealed but one band at 68 kDa if zinc ion was present wh ich was identified as cytokeratin 1 by amino acid sequencing of an internal peptide, The HK affinity column revealed bands at 68 kDa (cytokeratin 1), 33 kDa (gC1qR), and 66 kDa (unidentified). HK or domain 3-derived peptide b ound to the 68 kDa band; prekallikrein and Factor XII did not. HK or Factor XII bound to the 33-kDa band if zinc was present while no binding to the 6 6 kDa band was observed. Antibody to cytokeratin 1 inhibited HK binding to endothelial cells by 30%, antibody to gC1qR inhibited RR binding to endothe lial cells by 72%, and a mixture of both inhibited binding by 86%. Our data suggest HK binding by interaction of the heavy-chain domain 3 with cytoker atin 1 and the light chain with gC1qR. (C) 1999 Academic Press .