Pseudopodial and β-arrestin-interacting proteomes from migrating breast cancer cells upon PAR2 activation

Metastatic cancer cells form pseudopodia (PD) to facilitate their migration. The proteinase-activated receptor-2 (PAR-2) transduces migratory signals from proteases, and it forms protein complexes with β-arrestin and other signalling molecules that are enriched in pseudopodia. More generally, however, pseudopodial regulation is poorly understood. Here, we purified the pseudopodial proteomes of breast cancer cells after activation of the endogenous PAR-2 and we combined gel-based approaches with label-free high-resolution mass spectrometry to identify proteins that accumulate at the pseudopodia upon PAR-2-mediated migration. We identified > 410 proteins in the cell body and > 380 in the pseudopodia upon PAR2 activation, of which 93 were enriched in the pseudopodia. One of the pathways strongly enriched in the PD was the clathrin-mediated endocytosis signalling pathway, highlighting the importance of the scaffolding function of β-arrestin in PAR-2 signalling via its endocytosis. We therefore immunoprecipitated β-arrestins, and with mass spectrometry we identified 418 novel putative interactors. These data revealed novel β-arrestin functions that specifically control PAR-2-regulated signalling in migrating breast cancer cells but also showed that some β-arrestin functions are universal between GPCRs and cell types. In conclusion, this study reveals novel proteins and signalling pathways potentially important for migration of breast cancer cells.

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► Pseudopodia (PD) purification of migrating breast cancer cells & mass spectrometry.
► PD-enriched proteins presented with the signalling pathways upon PAR-2 activation.
► β-arrestins immunoprecipitation upon PAR-2 activation in breast cancer cells.
► Identification of > 400 novel candidate β-arrestin interactors by Orbitrap Velos.
► Novel PAR-2-regulated functions for β-arrestins during cell migration are revealed.