Identification of a novel recognition sequence for integrin αMβ2 within the γ-chain of fibrinogen

The interaction of leukocyte integrin alpha(M)beta(2) (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alpha(M)beta(2) and blocks the interaction of fibrinogen with the receptor and that Asp(199) within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp(199)-Gly(200) to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alpha(M)beta(2) recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma 377-395, inhibited adhesion of alpha(M)beta(2)-transfected cells to immobilized D-100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized Pa directly supported efficient adhesion of the alpha(M)beta(2)-expressing cells, including activated and non-activated monocytoid cells. The I domain of alpha(M)beta(2) was implicated in recognition of P2, as the biotinylated recombinant alpha(M)I domain specifically bound to both P2 and P1 peptides, Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma 377-386 (P2-N) and gamma 383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma 190-202 and gamma 377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alpha(M)beta(2).