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.

Integrin αvβ6 sets the stage for colorectal cancer metastasis

“…The αvβ6•uPAR interaction originally proposed by Saldanha et al and Sowmya et al has now been confirmed by Ahn et al using two orthogonal techniques: proximity ligation assays (PLA) and overlapping peptide array blots [81]. PLA is an emerging method that allows direct detection of protein•protein interactions in cellulo due to the close proximity of interacting proteins (30-40 nm) (refer to Weibrecht et al for a comprehensive review [91]).…”

“…Active plasmin is generated from plasminogen by uPA upon binding its cognate receptor, uPAR [80]. Under these conditions, uPA is relatively Bshielded^from the specific serpin inhibitors (PAI-1, PAI-2 and protease nexin), as are other proteases on the cell surface in the periplasmic space [81]. Once active, plasmin is capable of driving extensive ECM component degradation (e.g.…”

“…Once active, plasmin is capable of driving extensive ECM component degradation (e.g. fibronectin, fibrin, laminin and the protein core of proteoglycans [80,81]) and the activation of the pro-MMPs-1, pro-MMPs-3, and pro-MMPs-9, which subsequently degrade additional ECM components [82,83]. As β6 expression positively correlates with MMP-9 secretion and activity, this has been suggested to mediate the potential for CRC cells to colonise to, and survive within, liver metastases [36].…”

“…PLA is an emerging method that allows direct detection of protein•protein interactions in cellulo due to the close proximity of interacting proteins (30-40 nm) (refer to Weibrecht et al for a comprehensive review [91]). Ahn et al incubated paraformaldehyde-fixed OVCA 429, SW480 and HT29 CRC cell with antibodies 6.4B4 and R4 against αvβ6 and uPAR, respectively [81]. R4 and IgG1 isotype had been conjugated with the PLUS oligonucleotide probe whilst 6.4B4 and its corresponding isotype had been conjugated with the MINUS oligonucleotide probe [81].…”

“…Ahn et al incubated paraformaldehyde-fixed OVCA 429, SW480 and HT29 CRC cell with antibodies 6.4B4 and R4 against αvβ6 and uPAR, respectively [81]. R4 and IgG1 isotype had been conjugated with the PLUS oligonucleotide probe whilst 6.4B4 and its corresponding isotype had been conjugated with the MINUS oligonucleotide probe [81]. When these proximity probes are sufficiently close together, the corresponding oligonucleotides hybridise and ligate, forming a circular DNA molecule [91,92] which is amplified by rolling circle amplification detectable by confocal microscopy with fluorescently labelled complementary oligonucleotides [91].…”