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Abstract

Fibronectin (FN) is an essential glycoprotein of the extracellular matrix; binds integrins, syndecans, collagens, and growth factors; and is assembled by cells into complex fibrillar networks. The RGD motif in FN facilitates cell binding- and fibrillogenesis through binding to alpha 5 beta 1 and alpha v-class integrins. However, whether RGD is the sole binding site for alpha v-class integrins is unclear. Most notably, substituting aspartate with glutamate (RGE) was shown to eliminate integrin binding in vitro, while mouse genetics revealed that FNRGE preserves alpha v-class integrin binding and fibrillogenesis. To address this conflict, we employed single-cell force spectroscopy, engineered cells, and RGD motif-deficient mice (Fn1(Delta RGD/Delta RGD)) to search for additional alpha v-class integrin-binding sites. Our results demonstrate that alpha 5 beta 1 and alpha v-class integrins solely recognize the FN-RGD motif and that av-class, but not alpha 5 beta 1, integrins retain FN-RGE binding. Furthermore, Fn1(Delta RGD/Delta RGD) tissues and cells assemble abnormal and dysfunctional FNARGD fibrils in a syndecan-dependent manner. Our data highlight the central role of FN Delta RGD and the functionality of FN-RGE for alpha v-class integrins.