Elsevier

Matrix Biology

Volumes 60–61, July 2017, Pages 70-85
Matrix Biology

Review
Force regulated conformational change of integrin αVβ3

https://doi.org/10.1016/j.matbio.2016.07.002Get rights and content

Highlights

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    We characterized the bending and unbending conformational changes of a single integrin αVβ3 molecule on a living cell surface in real-time.

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    The bending and unbending conformational changes are regulated by tensile force, dependent on the ligand, and altered by point mutations.

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    Our findings provide insights into how the physiological functions and molecular structure of αVβ3 are coupled at the single molecule level.

Abstract

Integrins mediate cell adhesion to extracellular matrix and transduce signals bidirectionally across the membrane. Integrin αVβ3 has been shown to play an essential role in tumor metastasis, angiogenesis, hemostasis and phagocytosis. Integrins can take several conformations, including the bent and extended conformations of the ectodomain, which regulate integrin functions. Using a biomembrane force probe, we characterized the bending and unbending conformational changes of single αVβ3 integrins on living cell surfaces in real-time. We measured the probabilities of conformational changes, rates and speeds of conformational transitions, and the dynamic equilibrium between the two conformations, which were regulated by tensile force, dependent on the ligand, and altered by point mutations. These findings provide insights into how αVβ3 acts as a molecular machine and how its physiological function and molecular structure are coupled at the single‐molecule level.

Introduction

Integrin αVβ3 is a member of the integrin family and is widely expressed on endothelial cells, osteoclasts and blood cells. It acts as a bridging molecule between the cell and the extracellular environment, and mediates cell adhesions and mechanosignaling [1], [2], [3]. The over-expression of integrin αVβ3 in certain tumor cells facilitates tumor development, angiogenesis, and metastasis [4].

Integrins are heterodimers of non-covalently associated α and β subunits with an extracellular domain, a transmembrane segment, and a cytoplasmic region [5]. Integrins can adopt multiple conformations, including a bent or extended ectodomain, a joined or separated tailpiece and a closed or opened β subunit hybrid domain, which have been visualized by electron microscopy and crystallography [5], [6], [7], [8], [9], [10], [11] and detected by conformation-specific antibodies [12]. Specifically, integrin αVβ3, the subject of the present paper, has been observed in both bent and extended ectodomain conformations [9], [10], [13]. In the bent conformation, the N-terminal headpiece of the integrin (including the β-propeller and thigh domains of the α subunit and βA and hybrid domains of the β subunit) is bent towards and contacts the C-terminal tailpiece (including Calf-1 and Calf-2 domains of the α subunit and I-EGF-1 to I-EGF-4 domains and β tail domain of the β subunit). Upon extension, the headpiece flips upwards around the N-terminus of Calf-1 and I-EGF-1 domains and becomes more aligned with the tailpiece.

Integrin conformation is known to correlate with integrin activity: the bent conformation correlates with low affinity for ligands whereas the extended conformation correlates with high affinity. Integrin activation is accompanied by ectodomain extension, as shown by studies with extracellular activators, such as divalent cations (e.g. Mg2 +, Mn2 +), activating antibodies (e.g. CBR LFA1/2), and ligand-mimicking peptides, and with intracellular activators such as overexpressed talin head domain [7], [13], [14], [15], [16], [17]. Several activation-associated mutations also result in more extended conformations compared to the wild-type (WT) [12]. Molecular dynamics (MD) simulations have suggested that mechanical forces may induce integrin conformational changes [18], [19], [20], [21], [22]. Using a single-molecule force technique, the biomembrane force probe (BFP), we characterized bending and unbending conformational changes of single αLβ2 integrins on a living cell, which has demonstrated a role for mechanical force to regulate integrin conformational changes [23].

Building upon these recent studies, here we used a BFP to investigate bending and unbending conformational changes of single αVβ3 integrins on living cells. Characterization of the conformational change dynamics revealed the effects of mutation, force and ligand engagement.

Section snippets

Real-time observation of single integrin αVβ3 conformational changes on living cells

We used a BFP to characterize the conformational dynamics and binding kinetics of human integrin αVβ3 expressed on mouse lung endothelial cells (mLECs), including WT and two gain-of-function (GOF) mutants (MT), D723R and L138I (see Experimental procedures). D723R, located at the N-terminus of the β3 cytoplasmic domain (Fig. 1A), disrupts interactions between the α- and β-subunit transmembrane domain-tail to induce integrin tail separation and functional activation [24], [25], [26]. L138I, a

Discussion

Integrins play a key role in sensing the mechanical stiffness of the extracellular matrix and external forces applied through the matrix. In both cases, magnitude and time courses of the forces affect cellular signaling pathways and outcomes. Available evidence suggests that multiple components of the physical linkage between the matrix ligands and the F-actin contribute to mechanosensing, to a large part through force-regulated conformation changes. Understanding how forces across integrins

Proteins and reagents

Biotinylated FNIII7–10 was a gift from Dr. Andres Garcia (Georgia Tech, GA). Human plasma fibrinogen was a gift from Dr. Shaun Jackson (The University of Sydney, Australia). The antibody AP3 was a generous gift from Dr. Peter Newman (Blood Center of Wisconsin, WI). The antibody LIBS-2 was purchased from EMD Millipore (Billerica, MA). The anti-mouse β1 blocking Ab HMβ1-1 was purchased from Biolegend (San Diego, CA).

MAL-PEG3500-NHS and Biotin-PEG3500-NHS were purchased from JenKem (Plano, TX).

Lifetime analysis

Lifetime data were categorized into bins of successive force ranges under which lifetimes were measured. The average lifetime in each force bin was collected to plot the lifetime curve as a function of force.

Measurements of molecular stiffness

Stretch method as previously described was used to measure the molecular stiffness [23]. The retraction phase from the BFP “force vs. time” signal was converted to “force vs. displacement” (Fig. 4A), in which the displacement was calculated as the differential displacement between the BFP tracking system and the piezoelectric actuator feedback system. A tensile force pulled on the serially linked cell and the receptor-ligand molecular complex. Therefore, the reciprocal of the system stiffness

Acknowledgements

We thank A. Garcia (Georgia Institute of Technology, Atlanta, GA) for FNIII7–10, P. J. Newman (BloodCenter of Wisconsin, Milwaukee, WI) for antibody AP3, and S. Jackson and L. Ju (The University of Sydney, Sydney, Australia) for fibrinogen.

This work was supported by National Institutes of Health grant R01AI044902 (CZ), Army Research Office DOD W911NF-16-1-0257 (CZ), and the Army Research Office MURI W911NF-14-0403 (MS).

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