Stepped Changes of Monovalent Ligand-binding Force during Ligand-induced Clustering of Integrin αIIBβ3

Abstract

Recent evidence demonstrated that conformational changes of the integrin during receptor activation affected its binding to extracellular matrix; however, experimental assessment of ligand-receptor binding following the initial molecular interaction has rarely been carried out at a single-molecule resolution. In the present study, laser tweezers were used to measure the binding force exerted by a live Chinese hamster ovary cell that expressed integrin ␣ IIb ␤ 3 (CHO ␣ IIb ␤ 3 ), to the bead carrier coated with the snake venom rhodostomin that served as an activated ligand for integrin ␣ IIb ␤ 3 . A progressive increase of total binding force over time was noticed when the bead interacted with the CHO ␣ IIb ␤ 3 cell; such an increase was due mainly to the recruitment of more integrin molecules to the bead-cell interface. When the binding strength exerted by a single ligandreceptor pair was derived from the "polyvalent" measurements, surprisingly, a stepped decrease of the "monovalent binding force" was noted (from 4.15 to 2.54 piconewtons (pN)); such decrease appeared to occur during the ligand-induced integrin clustering process. On the other hand, the mutant rhodostomin defective in clustering integrins exhibited only one (1.81 pN) unit binding strength.Since the early recognition of integrin as an integral membrane complex involved in the trans-membrane association between the extracellular matrix and the cell, integrin heterodimers have been found to play important roles in controlling various steps that regulate processes as diverse as proliferation, development and differentiation, cell migration, and carcinogenesis (1). The initiation of the integrin-mediated activities involves at least two steps, namely, receptor activation and clustering. Taking platelet cells as an example, the integrin ␣ IIb ␤ 3 proteins on the membrane of a resting platelet bind only loosely to fibrinogen (FBN).3 Stimulation (or activation) of the platelets with agonists (such as ADP or thrombin) induces an inside-out signaling process that confers on integrin ␣ IIb ␤ 3 a stronger binding to FBN, resulting in the onset of platelet aggregation. Subsequent integrin clustering triggers complex intracellular signaling pathways that regulate the extent of the irreversible platelet aggregation and clot retraction (2). Using advanced structural biology techniques, several recent reports have provided convincing evidence that indicates the adoption of different molecular conformations by integrin heterodimers when the inside-out signaling pathway is invoked (3-5). Such structural changes are thought to be the mechanisms underlying increased ligand binding by an integrin during the activation process. Whether or not ligand binding affinity is further modulated during the subsequent integrin clustering process has remained largely elusive (6).To focus this research on the ligand-receptor interaction during the receptor clustering, we utilized snake venom rhodostomin from Agkistrodon rhodostoma as the ligand for integrin ␣ IIb ␤ 3 . Rhodostomin...

Targeted molecular dynamics reveals overall common conformational changes upon hybrid domain swing‐out in β3 integrins

“…This suggests that releasing the restraints imposed by the contacts that were broken during the early time points facilitates the later conformational changes and thus it may partially explain the multistep mechanism observed for ligand binding to integrins, which has been supported by several biophysical techniques 37,68-74. In this model, ligand binding to β3 integrins follows a self priming mechanism in which initial ligand interaction with the RGD site results in changes in the receptor and the ligand that produce higher affinity binding, with perhaps multiple intermediate affinity states, and additional sites of interaction between ligand and receptor 37,68…”

Localized Lipid Packing of Transmembrane Domains Impedes Integrin Clustering

“…Bidirectional integrin signaling involves conformational changes in the hetero-dimer, integrin clustering, and the assembly of a large intracellular adhesion complex [9], [17], [18]. Integrin clustering is defined as the interaction of hetero-dimers to shape lateral assemblies that eventually lead to focal complex formation [13], [15].…”

Membrane Targeting and Coupling of NHE1-IntegrinαIIbβ3-NCX1 by Lipid Rafts following Integrin-Ligand Interactions Trigger Ca2+ Oscillations

“…Immunofluorescence Staining-Immunofluorescence staining was done according to the methods described previously (14,15). Antibody probes and concentrations used in this study included antibodies made against integrin ␣ IIb (1:200) (Chemicon, Temecula, CA), NHE-1 (1:100), NCX-1 (1:100), CD45 (1:100) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and calveolin-1 (1:200) (Sigma).…”

“…We have reported previously that plating CHO ␣ IIb ␤ 3 cells on Fg or rho substrates could elicit transport of integrin ␣ IIb ␤ 3 proteins from intracellular vesicular pool to the cell membrane (14); it appears that NHE1 and NCX1 can also undergo such targeting from intracellular vesicular compartments to the plasma membrane. A major presence of ion exchanger in the intracellular vesicles found in this study is rather new.…”