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Sci. Signal., 8 September 2009
Vol. 2, Issue 87, p. ra51
[DOI: 10.1126/scisignal.2000396]

RESEARCH

Proteomic Analysis of Integrin-Associated Complexes Identifies RCC2 as a Dual Regulator of Rac1 and Arf6

Jonathan D. Humphries1,2*, Adam Byron1,2*, Mark D. Bass1,2, Sue E. Craig1,2, John W. Pinney2{dagger}, David Knight2, and Martin J. Humphries1,2{ddagger}

1 Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester M13 9PT, UK.
2 Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK.

* These authors contributed equally to this work.

{dagger} Present address: Centre for Bioinformatics, Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK.

Abstract: The binding of integrin adhesion receptors to their extracellular matrix ligands controls cell morphology, movement, survival, and differentiation in various developmental, homeostatic, and disease processes. Here, we report a methodology to isolate complexes associated with integrin adhesion receptors, which, like other receptor-associated signaling complexes, have been refractory to proteomic analysis. Quantitative, comparative analyses of the proteomes of two receptor-ligand pairs, {alpha}4β1–vascular cell adhesion molecule–1 and {alpha}5β1–fibronectin, defined both core and receptor-specific components. Regulator of chromosome condensation–2 (RCC2) was detected in the {alpha}5β1–fibronectin signaling network at an intersection between the Rac1 and adenosine 5'-diphosphate ribosylation factor 6 (Arf6) subnetworks. RCC2 knockdown enhanced fibronectin-induced activation of both Rac1 and Arf6 and accelerated cell spreading, suggesting that RCC2 limits the signaling required for membrane protrusion and delivery. Dysregulation of Rac1 and Arf6 function by RCC2 knockdown also abolished persistent migration along fibronectin fibers, indicating a functional role for RCC2 in directional cell movement. This proteomics workflow now opens the way to further dissection and systems-level analyses of adhesion signaling.

{ddagger} To whom correspondence should be addressed. E-mail: martin.humphries{at}manchester.ac.uk

Citation: J. D. Humphries, A. Byron, M. D. Bass, S. E. Craig, J. W. Pinney, D. Knight, M. J. Humphries, Proteomic Analysis of Integrin-Associated Complexes Identifies RCC2 as a Dual Regulator of Rac1 and Arf6. Sci. Signal. 2, ra51 (2009).

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