Characterization of the interaction between heterodimeric {alphanubeta}6 integrin and urokinase Plasminogen Activator Receptor (uPAR) using functional proteomics

Seong B Ahn, Abidali Mohamedali, Samyuktha Anand, Harish R Cheruku, Debra Birch, Sowmya Gopichandran, David Cantor, Shoba Ranganathan, David W Inglis, Ronald Frank, Michael Agrez, Edouard C Nice, Mark S Baker

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Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin alphavbeta6 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 characterise the uPARalphavbeta6 interaction. Proximity ligation assays (PLA) confirmed the uPARalphavbeta6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with with de novo beta6 subunit expression. PLA studies were then validated using peptide arrays which also identified potential physical sites of uPAR interaction with alphavbeta6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., alphav, beta1, beta3 and beta6 alone). Our data suggests that interaction with uPAR requires expression of the complete alphabeta heterodimer (e.g., alphavbeta1) not individual subunits (i.e., alphav, beta1, beta3 or beta6). 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 alphavbeta6 and uPAR are located in uPAR domains II and III.
Original languageEnglish
Pages (from-to)5956 - 5964
Number of pages9
JournalJournal of Proteome Research
Issue number12
Publication statusPublished - 2014

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