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Vol. 17, Issue 2, 966-976, February 2006

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MAGI-1 Is Required for Rap1 Activation upon Cell-Cell Contact and for Enhancement of Vascular Endothelial Cadherin-mediated Cell Adhesion

Department of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan

Submitted July 19, 2005; Revised November 28, 2005; Accepted November 29, 2005
Monitoring Editor: Martin A. Schwartz

Rap1 is a small GTPase that regulates adherens junction maturation. It remains elusive how Rap1 is activated upon cell-cell contact. We demonstrate for the first time that Rap1 is activated upon homophilic engagement of vascular endothelial cadherin (VE-cadherin) at the cell-cell contacts in living cells and that MAGI-1 is required for VE-cadherin-dependent Rap1 activation. We found that MAGI-1 localized to cell-cell contacts presumably by associating with -catenin and that MAGI-1 bound to a guanine nucleotide exchange factor for Rap1, PDZ-GEF1. Depletion of MAGI-1 suppressed the cell-cell contact-induced Rap1 activation and the VE-cadherin-mediated cell-cell adhesion after Ca²⁺ switch. In addition, relocation of vinculin from cell-extracellular matrix contacts to cell-cell contacts after the Ca²⁺ switch was inhibited in MAGI-1-depleted cells. Furthermore, inactivation of Rap1 by overexpression of Rap1GAPII impaired the VE-cadherin-dependent cell adhesion. Collectively, MAGI-1 is important for VE-cadherin-dependent Rap1 activation upon cell-cell contact. In addition, once activated, Rap1 upon cell-cell contacts positively regulate the adherens junction formation by relocating vinculin that supports VE-cadherin-based cell adhesion.

Abbreviations used: AJ, adherens junction; CFP, cyan fluorescent protein; ECM, extracellular matrix; EGFP, enhanced green fluorescent protein; FRET, fluorescence resonance energy transfer; GEF, guanine nucleotide exchange factor; GAP, GTPase activating protein; HAEC, human aortic endothelial cell; HUVEC, human umbilical vascular endothelial cell; JAM, junctional adhesion molecule; MAGI-1, MAGUK with inverted domain structure-1; GFP, green fluorescent protein; PBS, phosphate-buffered saline; PDZ, PSD95/DiscLarge/ZO-1; PECAM-1, platelet and endothelial cell adhesion molecule-1; siRNAs, small interfering RNAs; TJ, tight junction; VE-cadherin, vascular endothelial cadherin; VEC-Fc, recombinant VE-cadherin ectodomain-Fc chimera; YFP, yellow fluorescent protein.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Naoki Mochizuki (nmochizu{at}ri.ncvc.go.jp).

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