Identification of the binding site for fibrinogen recognition peptide gamma 383-395 within the I-alpha(M)-domain of integrin alpha(M)beta(2)

The leukocyte integrin alpha (M)beta (2) (Mac-1, CD11b/CD18) is a cell surf ace adhesion receptor for fibrinogen. The interaction between fibrinogen an d alpha (M)beta (2) mediates a range of adhesive reactions during the immun e-inflammatory response. The sequence gamma (383)TMKIIPFNRLTIG(395), P2-C, within the gamma -module of the D-domain of fibrinogen, is a recognition si te for alpha (M)beta (2) and alpha (x)beta (2). We have now identified the complementary sequences within the alpha I-M-domain of the receptor respons ible for recognition of P2-C, The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structural ly similar I-domains of alpha (M)beta (2), alpha (X)beta (2), and alpha (L) beta (2) i.e. the alpha I-M-. and alpha I-X-domains bind P2-C, and the alph a I-L-domain did not bind this ligand. The Lys(245)-Arg(261) sequence, whic h forms a loop betaD-alpha5 and an adjacent helix alpha5 in the three-dimen sional structure of the alpha I-M-domain, was identified as the binding sit e for P2-C. This conclusion is supported by the following data: 1) mutant c ell lines in which the alpha I-M-domain segments (245)KFG and Glu(253)-Arg( 261) were switched to the homologous alpha I-L-domain segments failed to su pport adhesion to P2-C; 2) synthetic peptides duplicating the Lys(245)-Tyr( 252) and Glu(253)-Arg(261) sequences directly bound the D fragment and P2-C derivative, gamma 384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys2 45-Arg261 segment, Phe(246), Asp(254), and Pro(257), resulted in the loss o f the binding function of the recombinant alpha I-M-domains; and 4) graftin g the alpha (M) (Lys(245)-Arg(261)) Segment into the alpha I-L-domain conve rted it to a P2-C-binding protein. These results demonstrate that the alpha (M)(Lys(245)-Arg(261)) segment, a site of the major sequence and structure difference among alpha I-M-, alpha I-X-, and alpha I-L-domains, is respons ible for recognition of a small segment of fibrinogen, gamma Thr(383)-Gly(3 95), by serving as ligand binding site.