title: Characterization of the interaction between heterodimeric αvβ6 integrin and urokinase plasminogen activator receptor (uPAR) using functional proteomics.
abstract: Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvβ6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial-mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR·αvβ6 interaction. Proximity ligation assays (PLA) confirmed the uPAR·αvβ6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo β6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvβ6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, β1, β3, and β6 alone). Our data suggest that interaction with uPAR requires expression of the complete αβ heterodimer (e.g., αvβ6), not individual subunits (i.e., αv, β1, β3, or β6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvβ6 and uPAR are located in uPAR domains II and III.
authors.affiliation: Australian School of Advanced Medicine, Faculty of Human Sciences, ‡Department of Chemistry and Biomolecular Sciences, and §Department of Engineering, Faculty of Science, Macquarie University , Sydney, NSW 2109, Australia.
authors.forename: Seong Beom
authors.initials: SB
authors.lastname: Ahn
authors.forename: Abidali
authors.initials: A
authors.lastname: Mohamedali
authors.forename: Samyuktha
authors.initials: S
authors.lastname: Anand
authors.forename: Harish R
authors.initials: HR
authors.lastname: Cheruku
authors.forename: Debra
authors.initials: D
authors.lastname: Birch
authors.forename: Gopichandran
authors.initials: G
authors.lastname: Sowmya
authors.forename: David
authors.initials: D
authors.lastname: Cantor
authors.forename: Shoba
authors.initials: S
authors.lastname: Ranganathan
authors.forename: David W
authors.initials: DW
authors.lastname: Inglis
authors.forename: Ronald
authors.initials: R
authors.lastname: Frank
authors.forename: Michael
authors.initials: M
authors.lastname: Agrez
authors.forename: Edouard C
authors.initials: EC
authors.lastname: Nice
authors.forename: Mark S
authors.initials: MS
authors.lastname: Baker
mesh_terms: D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D058750:Epithelial-Mesenchymal Transition; D006801:Humans; D016023:Integrins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D040901:Proteomics; D055293:Receptors, Urokinase Plasminogen Activator / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; D016023:Integrins; D055293:Receptors, Urokinase Plasminogen Activator; C073878:integrin alphavbeta6
keywords: colorectal cancer; functional proteomics; ovarian cancer; peptide array; proximity ligation assay; uPAR; αvβ6 integrin