A molecular basis for integrin alphaMbeta 2 ligand binding promiscuity.
The leukocyte integrin alpha(M)beta(2) is a highly promiscuous leukocyte receptor capable of binding a multitude of unrelated ligands. To understand the molecular basis for the broad ligand recognition of alpha(M)beta(2), the inter-integrin chimera was created. In the chimeric integrin, the betad-alpha5 loop-alpha5 helix segment comprised of residues Lys(245)-Arg(261) from the alpha(M)I domain of alpha(M)beta(2) was inserted into the framework of alpha(L)beta(2). The construct was expressed in HEK 293 cells, and the ability of generated cells to adhere to fibrinogen and its derivatives was characterized first. Grafting the alpha(M)(Lys(245)-Arg(261)) sequence converted alpha(L)beta(2) into a fibrinogen-binding protein capable of mediating efficient and specific adhesion similar to that of wild-type alpha(M)beta(2). Verifying a switch in the binding specificity of alpha(L)beta(2), the chimeric receptor became competent to support cell migration to fibrinogen. Mutations at positions Phe(246), Asp(254), and Pro(257) within Lys(245)-Arg(261) of alpha(M)beta(2) produced significant decreases in cell adhesion, illustrating the critical role of these residues in ligand binding. The insertion of alpha(M)(Lys(245)-Arg(261)) imparted to the chimeric integrin the ability to recognize many typical alpha(M)beta(2) protein ligands. Furthermore, cells expressing the chimeric receptor, but not alpha(L)beta(2), were able to stick to uncoated plastic, which represents the hallmark of wild-type alpha(M)beta(2). These results suggest that alpha(M)(Lys(245)-Arg(261)) serves as a consensus binding site for interaction with a variety of distinct molecules and, thus, may define the degenerate recognition properties inherent to alpha(M)beta(2).
Yakubenko
Valentin P
VP
Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic Foundation, Ohio 44195, USA.
Lishko
Valeryi K
VK
Lam
Stephen C-T
SC
Ugarova
Tatiana P
TP
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement; D005340:Fibrinogen / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D008024:Ligands; D016177:Macrophage-1 Antigen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D008024:Ligands; D016177:Macrophage-1 Antigen; D011993:Recombinant Fusion Proteins; D005340:Fibrinogen