Cadherin interaction probed by atomic force microscopy

W Baumgartner

P Hinterdorfer

W Ness

A Raab

D Vestweber

H Schindler

D Drenckhahn

^{Institute of Anatomy, University of Würzburg, Koellikerstrasse 6, D-97070 Würzburg, Germany;}^{Institute for Biophysics, University of Linz, A-4040 Linz, Austria; and}^{Institute of Cell Biology, University of Münster, D-48149 Münster, Germany}

^{To whom reprint requests should be addressed. E-mail:}ed.grubzreuw-inu.liam@510tana.

Communicated by Martin Lindauer, University of Würzburg, Würzburg, Germany

Received 1999 Nov 17; Accepted 2000 Feb 7.

Abstract

Single molecule atomic force microscopy was used to characterize structure, binding strength (unbinding force), and binding kinetics of a classical cadherin, vascular endothelial (VE)-cadherin, secreted by transfected Chinese hamster ovary cells as cis-dimerized full-length external domain fused to Fc-portion of human IgG. In physiological buffer, the external domain of VE-cadherin dimers is a ≈20-nm-long rod-shaped molecule that collapses and dissociates into monomers (V-shaped structures) in the absence of Ca^{. Trans-interaction of dimers is a low-affinity reaction (}K_D = 10^–10^M,k_off = 1.8 s^,k_on = 10^–10^M^·s^{) with relatively low unbinding force (35–55 pN at retrace velocities of 200–4,000 nm·s}^{). Higher order unbinding forces, that increase with interaction time, indicate association of cadherins into complexes with cumulative binding strength. These observations favor a model by which the inherently weak unit binding strength and affinity of cadherin trans-interaction requires clustering and cytoskeletal immobilization for amplification. Binding is regulated by low-affinity Ca}^{binding sites (}K_D = 1.15 mM) with high cooperativity (Hill coefficient of 5.04). Local changes of free extracellular Ca^{in the narrow intercellular space may be of physiological importance to facilitate rapid remodeling of intercellular adhesion and communication.}

Keywords: cell adhesion, VE-cadherin, binding strength, binding kinetics

Abstract

Adhesive contacts between neighboring cells play a crucial role in various aspects of tissue organization, differentiation, and function. The important biological and medical aspects of such stable intercellular adhesions are well established (1).

In cellular monolayers that form permeability barriers, such as the simple epithelial lining of the intestine or the vascular endothelium covering the inner surface of blood vessels, adhesion between cells is mainly accomplished by Ca^{-dependent adhesion molecules named cadherins (}1, 2). The predominant cadherin of most epithelia is E-cadherin, whereas endothelial cells adhere to each other by vascular endothelial-cadherin (VE-cadherin) (3). Cadherins are type I single membrane-spanning cell surface proteins that require free extracellular Ca^{for homophilic interaction of their N-terminal extracellular domains with cadherins of adjoining cells (}4).

The rod-shaped external domain of classical cadherins is composed of five tandemly repeated ≈110-aa-long subdomains. The outermost N-terminal subdomain (subdomain 1) including the linker region to subdomain 2 can associate laterally to form cis-interacting parallel dimers (5, 6). These cis-dimers are assumed to represent the functional units required for adhesive activity (7, 8). In this model, adhesive bonds between cadherins of interacting cell membranes are predicted to result from binding between subdomains 1 of oppositely oriented cis-dimers to form trans-interacting antiparallel tetramers that are termed adhesion dimers. The binding constants for adhesion dimer formation and the force transmitted by adhesion dimers are still unknown. X-ray crystallographic studies of subdomain 1 of N-cadherin suggest that adhesion dimers might associate laterally into zipper-like supramolecular clusters providing cumulative adhesive strength (5). Adhesion dimers and zipper-like associations were not observed in crystals of E-cadherin subdomains 1–2 (6, 9).

In the present study, we applied atomic force microscopy (AFM) (10) as a powerful molecular approach to probe specific trans-interaction forces and conformational changes of recombinant VE-cadherin strand dimers in aqueous physiological conditions (11–15). Elementary trans-interactions observed between strand dimers revealed Ca^{-dependent highly specific molecular recognition properties that provide a basis for modeling cadherin-mediated intercellular adhesion.}

Acknowledgments

This study was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 487, B5).

Acknowledgments

Abbreviations

VE-cadherin	vascular endothelial cadherin
CHO	Chinese hamster ovary
AFM	atomic force microsopy
PEG	polyethylene glycol
v_r, retrace velocity.

Abbreviations

Footnotes

Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073/pnas.070052697.

Article and publication date are at www.pnas.org/cgi/doi/10.1073/pnas.070052697

Footnotes

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