title: Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6.
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(4)beta(1)-vascular cell adhesion molecule-1 and alpha(5)beta(1)-fibronectin, defined both core and receptor-specific components. Regulator of chromosome condensation-2 (RCC2) was detected in the alpha(5)beta(1)-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.
authors.affiliation: Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Manchester, UK.
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Humphries
authors.forename: Adam
authors.initials: A
authors.lastname: Byron
authors.forename: Mark D
authors.initials: MD
authors.lastname: Bass
authors.forename: Sue E
authors.initials: SE
authors.lastname: Craig
authors.forename: John W
authors.initials: JW
authors.lastname: Pinney
authors.forename: David
authors.initials: D
authors.lastname: Knight
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Humphries
mesh_terms: D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D002465:Cell Movement / Q000502:physiology; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D005353:Fibronectins; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D039041:Integrin alpha4beta1 / Q000235:genetics / Q000378:metabolism*; D039081:Integrin alpha5beta1 / Q000235:genetics / Q000378:metabolism*; D020014:K562 Cells; D040901:Proteomics; D015398:Signal Transduction / Q000502:physiology*; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: D000089982:ADP-Ribosylation Factor 6; D002868:Chromosomal Proteins, Non-Histone; D005353:Fibronectins; D020662:Guanine Nucleotide Exchange Factors; D039041:Integrin alpha4beta1; D039081:Integrin alpha5beta1; C107953:RAC1 protein, human; C477650:RCC2 protein, human; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human; D020830:rac1 GTP-Binding Protein