Abstract
Acidic extracellular pH is characteristic of the cell microenvironment in several important physiological and pathological contexts. Although it is well established that acidic extracellular pH can have profound effects on processes such as cell adhesion and migration, the underlying molecular mechanisms are largely unknown. Integrin receptors physically connect cells to the extracellular matrix, and are thus likely to modulate cell responses to extracellular conditions. Here, we examine the role of acidic extracellular pH in regulating activation of integrin αvβ3. Through computational molecular dynamics simulations, we find that acidic extracellular pH promotes opening of the αvβ3 headpiece, indicating that acidic pH can thereby facilitate integrin activation. This prediction is consistent with our flow cytometry and atomic force microscope-mediated force spectroscopy assays of integrin αvβ3 on live cells, which both demonstrate that acidic pH promotes activation at the intact cell surface. Finally, quantification of cell morphology and migration measurements shows that acidic extracellular pH affects cell behavior in a manner that is consistent with increased integrin activation. Taken together, these computational and experimental results suggest a new and complementary mechanism of integrin activation regulation, with associated implications for cell adhesion and migration in regions of altered pH that are relevant to wound healing and cancer.
Highlights
Binding between cells and the extracellular matrix (ECM) is critical to complex processes such as cell adhesion and migration
From the definition of pKa, these residues would be protonated at a pH below the threshold pKa of 6.2 for a majority of integrins (.50%), computationally approximating an acidic pH on that order; we refer to this state hereafter as ‘‘acidic pH.’’ Multi-Conformation Continuum Electrostatics (MCCE) results indicated seven amino acids with pKa values that were elevated from their expected solution values, and were protonated in our simulations (Fig. 2A–B, Table 1)
We have used molecular dynamics (MD) simulations, flow cytometry experiments, atomic force microscope (AFM) force spectroscopy, and cell-level morphology and migration experiments to assess the effect of acidic extracellular pH on integrin avb3 conformation and avb3 CHO-B2 cell behavior
Summary
Binding between cells and the extracellular matrix (ECM) is critical to complex processes such as cell adhesion and migration. Integrins undergo large-scale conformational changes in order to attain a high-affinity configuration during the process of integrin activation These receptors are currently understood to exist in equilibrium among three main conformational states (Fig. 1). In the low-affinity state, the extracellular leg domains are bent and the headpiece is closed, with an acute angle between the I-like and hybrid domains. This conformation generally exhibits little to no binding to biological ligands [5,6,7,8], but can bind to small RGD peptides in solution [9]. The third conformation, with extended legs and a closed headpiece, is expected to be of intermediate affinity [5]
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