Ligand Recognition by Cytoadhesins in Vascular Biology

抄録

Interaction of integrin receptors with ligands is critically involved in a number of physiological processes and in the pathogenesis of many human diseases. However, the mechanism of ligand recognition is still poorly understood. Macromolecular protein ligands such as fibrinogen have several contact sites which, after initial docking into the activated integrin receptor, stabilize the receptor-ligand complex so that it becomes nondissociable. Available data on integrin-ligand interactions on cells indicate that activation does not result in a change in the thermodynamics of this reaction, but does increase the kinetics of the interaction. The ligand binding to integrins has been previously described kinetically to be a two-step reaction, leading through the initially reversible complex to the final irreversible one. The fibrinogen binding to α_IIbβ₃ induces conformational changes in the receptor molecule that lead to platelet aggregation and granular secretion, tyrosine phosphorylation of intracellular proteins, and ion fluxes across the membrane. The spatial distribution of contact sites on the receptor for both fibrinogen binding motifs, RGD and HHLGGAKQAGDV (γ400-411), has been characterized by fluorescence resonance energy transfer measurements. Binding experiments using functional analogues of these fibrinogen sequences, cRGD and cHarGD, and fluorescence resonance energy transfer analysis showed that both peptides simultaneously interacted with distinct sites on α_IIbβ₃ separated by 6.1±0.5nm. However, they were not fully specific and to some extent mutually displaced each other. Binding of cRGD and cHarGD to α_IIbβ₃ resulted in distinct conformational alterations in the receptor and the opposite microenvironmental changes, as indicated by exposure of ligand induced binding site (LIBS) epitopes and electron paramagnetic resonance analysis using 5-doxylstearic acid as a spin probe, thus supporting the concept that both peptide ligands, due to binding to distinct sites, initiate the sending of different signals during outside-in signaling mediated by α_IIbβ₃. These data suggest that during fibrinogen-α_IIbβ₃ complex assembly, the sequential contacts between key sequences of each component of the complex may initiate different waves of conformational changes in the receptor transmitted into the cell. Furthermore, present data suggest that binding of different macromolecular ligands to α_IIbβ₃, such as fibronectin or von Willebrand factor, both containing only the RGD motif, may induce distinct signaling pathways from that initiated by fibrinogen.