Amino acid sequences within the alpha subunit of integrin alpha M beta 2 (Mac-1) critical for specific recognition of C3bi.
Phagocytosis of opsonized particles by neutrophils and monocytes plays a central role in host defense mechanisms against foreign pathogens. This process depends on the interaction between C3bi, a degradation product derived from activation of the complement system, and the alpha M beta 2 (CD11b/CD18, Mac-1) receptor, the major integrin on neutrophils. Previous studies had established a central role for the I domain, a stretch of approximately 200 amino acids within the alpha M subunit in the binding of C3bi, as well as many other alpha M beta 2 ligands. The present study was undertaken to establish the molecular basis of C3bi recognition by alpha M beta 2. The strategy employed the use of a series of mutant receptors in which short segments of the I domain of alpha M were switched to the corresponding segments of alpha L, which is structurally very similar but does not bind C3bi. We report three major findings: (1) The C3bi binding pocket is composed of three regions, P147-R152, P201-K217, and K245-R261 of alpha M, which surround the cation binding site within the MIDAS motif of the I domain. (2) Within the latter segment, K245 plays a critical role in mediating C3bi binding to alpha M beta 2. Mutation of K245 to Ala significantly reduced C3bi binding but had no effect on binding of another alpha M beta 2 I domain ligand, NIF. (3) Blocking of C3bi binding to alpha M beta 2 by monoclonal antibodies is achieved through two different mechanisms: direct competition for the ligand binding site or induction of conformational changes. Overall, these studies support the hypothesis that many of the ligands of alpha M beta 2 bind to overlapping but not identical sites within the I domain. Although the same short structural segments within the I domain may be involved in binding, different amino acids within these segments may contact different ligands.
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Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, The Cleveland Clinic Foundation, Ohio 44195, USA.
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D001667:Binding, Competitive / Q000276:immunology; D002460:Cell Line; D003179:Complement C3b / Q000276:immunology / Q000378:metabolism*; D003169:Complement Inactivator Proteins / Q000494:pharmacology; D005998:Glycine / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D015801:Helminth Proteins / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology; D008239:Lysine / Q000378:metabolism; D016177:Macrophage-1 Antigen / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D015801:Helminth Proteins; D016177:Macrophage-1 Antigen; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C086581:NIF protein, Ancylostoma caninum; D010649:Phenylalanine; D003179:Complement C3b; D008239:Lysine; D005998:Glycine