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.
| Language | English |
|---|---|
| Pages | 5956-5964 |
| Number of pages | 9 |
| Journal | Journal of Proteome Research |
| Volume | 13 |
| Issue number | 12 |
| DOIs | |
| Publication status | Published - 5 Dec 2014 |
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Characterization of the interaction between heterodimeric αvβ6 integrin and urokinase plasminogen activator receptor (uPAR) using functional proteomics. / Ahn, Seong Beom; Mohamedali, Abidali; Anand, Samyuktha; Cheruku, Harish R.; Birch, Debra; Sowmya, Gopichandran; Cantor, David; Ranganathan, Shoba; Inglis, David W.; Frank, Ronald; Agrez, Michael; Nice, Edouard C.; Baker, Mark S.
In: Journal of Proteome Research, Vol. 13, No. 12, 05.12.2014, p. 5956-5964.Research output: Contribution to journal › Article › Research › peer-review
TY - JOUR
T1 - Characterization of the interaction between heterodimeric αvβ6 integrin and urokinase plasminogen activator receptor (uPAR) using functional proteomics
AU - Ahn, Seong Beom
AU - Mohamedali, Abidali
AU - Anand, Samyuktha
AU - Cheruku, Harish R.
AU - Birch, Debra
AU - Sowmya, Gopichandran
AU - Cantor, David
AU - Ranganathan, Shoba
AU - Inglis, David W.
AU - Frank, Ronald
AU - Agrez, Michael
AU - Nice, Edouard C.
AU - Baker, Mark S.
PY - 2014/12/5
Y1 - 2014/12/5
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84915747345&partnerID=8YFLogxK
U2 - 10.1021/pr500849x
DO - 10.1021/pr500849x
M3 - Article
VL - 13
SP - 5956
EP - 5964
JO - Journal of Proteome Research
T2 - Journal of Proteome Research
JF - Journal of Proteome Research
SN - 1535-3893
IS - 12
ER -