ISOLATION AND CHARACTERIZATION OF THE KININOGEN-BINDING PROTEIN P-33 FROM ENDOTHELIAL-CELLS - IDENTITY WITH THE GC1Q RECEPTOR

Kininogens, the precursor proteins of the vasoactive kinins, bind spec ifically, reversibly, and saturably to platelets, neutrophils, and end othelial cells. Two domains of the kininogens expose major cell bindin g sites: domain D3 that is shared by H- and L-kininogen and domain D5( H) that is exclusively present in H-kininogen. Previously we have mapp ed the kininogen cell binding sites to 27 residues of D3 (''LDC27'') a nd 20 residues of D5(H) (''HKH20''), respectively (Herwald, H., Hasan, A. A. K., Godovae-Zimmermann, J., Schmaier, A. H., and Muller-Esterl, W. (1995) J. Biol. Chem. 270, 14634-14642; Hasan, A. A. K., Cines, D. B., Herwald, H., Schmaier, A. H., and Muller-Esterl, W. (1995) J. Bio l. Chen. 270, 19256-19261). The corresponding kininogen acceptor site( s) exposed by the cell surfaces are still poorly defined. Using a non- ionic detergent, Nonidet P-40, we have been able to solubilize kininog en binding sites from an endothelial cell line, EA.hy926, in their fun ctionally active form. Affinity chromatography of the solubilized kini nogen binding sites on HKH20, a synthetic peptide representing the D5( H) cell binding site, allowed us to isolate a 33-kDa protein (''p33'') that binds specifically and reversibly to H-kininogen with a K-D (app arent dissociation constant) of 9 +/- 2 nM. Preparative SDS electropho resis followed by NH2-terminal amino acid sequence analysis identified the kininogen-binding protein p33 as the gC1q receptor (''gC1qR''), a n extrinsic membrane protein that interacts with the globular domains of the complement component C1q. The purified p33 binds C1q with moder ate affinity, K-D = 240 +/- 10 nM. Recombinant expression of the corre sponding cDNA in Escherichia coli demonstrated that p33 binds H-kinino gen, but not L-kininogen. Peptide HKH20 but not peptide LDC27 inhibite d binding of H-kininogen to the recombinant p33 in a concentration-dep endent manner, indicating that H-kininogen binds to p33 via domain D5( H). Recombinant p33 efficiently inhibited the binding of H-kininogen t o EA.hy926 cells. Factor XII, but not prekallikrein, competed with H-k ininogen binding to p33. These findings suggest that an endothelial bi nding protein mediates the assembly of critical components of the kini n-generating pathway on the surface of endothelial cells, thereby link ing the early events of kinin formation and complement activation.