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

Regulator of chromosome condensation–2 is a component of fibronectin-activated signaling pathways that regulate cell migration. Integrin Interactors Integrins mediate cell-cell adhesion, as well as cell adhesion to the extracellular matrix. Identification of the intracellular signaling networks associated with integrins is of interest because integrins are involved in processes such as invasion of tumor cells during metastasis and leukocyte infiltration during inflammation. Humphries et al. developed a method of isolating protein complexes associated with α5β1 integrin, which binds to fibronectin, and with α4β1 integrin, which binds to vascular cell adhesion molecule–1. Although a subset of proteins was detected in both the α5β1 and α4β1 networks, there were several receptor-specific proteins. In particular, regulator of chromosome condensation–2 (RCC2) was identified as a component of the α5β1 integrin–associated signaling network. RCC2 promoted fibronectin-dependent migration by inhibiting two different subnetworks (Rac1 and Arf6). These techniques provide the means to investigate the composition and function of adhesion complexes under different physiological conditions. 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, α4β1–vascular cell adhesion molecule–1 and α5β1–fibronectin, defined both core and receptor-specific components. Regulator of chromosome condensation–2 (RCC2) was detected in the α5β1–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.