Quantitative analysis of C4b dimer binding to distinct sites on the C3b/C4b receptor (CR1)

The complement receptor CR1 (CD35) is a transmembrane protein composed in its extracellular portion of short consensus repeats (SCR 1-30) organized into four long homologous repeats (LHR-A, LHR-B, LHR-C, and LHR-D). Each LHR, except LHR-D, contains a binding site for C3b and/or C4b within its first four SCR. The binding reaction between CR1 and soluble dimers of C4b (C4b₂) was analyzed using the native receptor on human erythrocytes and full-length recombinant CR1 expressed in stably transfected Chinese hamster ovary (CHO) cells. CR1 mutants expressed similarly were used to determine the SCR of LHR- A required for C4b₂ binding and the potential of C3b binding sites in CR1 to bind C4b₂. Erythrocyte CR1, CHO cells expressing full-length recombinant CR1 (ABCD), constructs ACD, and SCR(1-4)D each bound C4b₂ with similar affinities (K(d), ~4 x 10^-7 M). Construct SCR(1-2)D bound C4b₂ with lower affinity (K(d), 1.4 x 10^-6 M) indicating that SCR(1-4) are required for a fully functional C4b₂ binding site. Construct SCR(15-18)D, which contains a C3b site, also bound C4b₂ with lower affinity (K(d) 1.2 x 10^-6 M) than its binding to C3b dimers. Constructs SCR(15-16)D and D did not bind C4b₂. Each CR1 construct that bound C4b₂ functioned as a cofactor for factor I-mediated cleavage to C4d.