title: Pseudopodial and β-arrestin-interacting proteomes from migrating breast cancer cells upon PAR2 activation.
abstract: 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.
authors: Department of Biological Sciences/Proteomics Unit, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom.
authors: Nikolaos
authors: N
authors: Parisis
authors: Department of Biological Sciences/Proteomics Unit, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom.
authors: Gergana
authors: G
authors: Metodieva
authors: Department of Biological Sciences/Proteomics Unit, University of Essex, Wivenhoe Park, Colchester, Essex CO4 3SQ, United Kingdom. Electronic address: mmetod@essex.ac.uk.
authors: Metodi V
authors: MV
authors: Metodiev
mesh_terms: D019390:Arrestins / Q000378:metabolism*; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D002465:Cell Movement; D002966:Clathrin / Q000378:metabolism; D019295:Computational Biology; D004705:Endocytosis; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D061986:MCF-7 Cells; D013058:Mass Spectrometry; D009687:Nuclear Proteins / Q000378:metabolism; D000090243:Nucleophosmin; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D011554:Pseudopodia / Q000378:metabolism*; D044464:Receptor, PAR-2 / Q000378:metabolism*; D015398:Signal Transduction; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D002966:Clathrin; D018840:HSP70 Heat-Shock Proteins; D009687:Nuclear Proteins; D020543:Proteome; D044464:Receptor, PAR-2; D000071557:beta-Arrestins; D000090243:Nucleophosmin; D048049:Extracellular Signal-Regulated MAP Kinases