Abstract
The urokinase-type plasminogen activator receptor (uPAR) sustains cell migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate chemotactic signaling in response to urokinase. We have characterized the early signaling events triggered by the Ser-Arg-Ser-Arg-Tyr (SRSRY) chemotactic uPAR sequence. Cell exposure to SRSRY peptide promotes directional migration on vitronectin-coated filters, regardless of uPAR expression, in a specific and dose-dependent manner, with maximal effect at a concentration level as low as 10 nm. A similar concentration profile is observed in a quantitative analysis of SRSRY-dependent cytoskeletal rearrangements, mostly consisting of filamentous structures localized in a single cell region. SRSRY analogues with alanine substitutions fail to drive F-actin formation and cell migration, indicating a critical role for each amino acid residue. As with ligand-dependent uPAR signaling, SRSRY stimulates protein kinase C activity and results in ERK1/2 phosphorylation. The involvement of the high affinity N-formyl-Met-Leu-Phe receptor (FPR) in this process is indicated by the finding that 100 nm N-formyl-Met-Leu-Phe inhibits binding of D2D3 to the cell surface, as well as SRSRY-stimulated cell migration and F-actin polarization. Moreover, cell exposure to SRSRY promotes FPR-dependent vitronectin release and increased uPAR.alphavbeta5 vitronectin receptor physical association, indicating that alphavbeta5 activity is regulated by the SRSRY uPAR sequence via FPR. Finally, we provide evidence that alphavbeta5 is required for SRSRY-dependent ERK1/2 phosphorylation, whereas it is not required for protein kinase C activation. The data indicate that the ability of uPAR to stimulate cell migration and cytoskeletal rearrangements is retained by the SRSRY peptide alone and that it is supported by cross-talk between FPR and alphavbeta5.
Highlights
The urokinase-type plasminogen activator receptor sustains cell migration through its capacity to promote pericellular proteolysis, regulate integrin function, and mediate chemotactic signaling in response to urokinase
The data indicate that the ability of urokinase-type plasminogen activator receptor (uPAR) to stimulate cell migration and cytoskeletal rearrangements is retained by the SRSRY peptide alone and that it is supported by cross-talk between formyl-Met-Leu-Phe receptor (FPR) and ␣v5
We provide evidence that SRSRYp triggers cross-talk between high affinity fMLP (FPR) and ␣v5 vitronectin receptors, resulting in cytoskeletal rearrangements and cell migration
Summary
Reagents—Recombinant D1 (residues 1– 87), D2 (residues 88 –183), D3 (residues 184 –284), and D2D3 (residues 88 –284) uPAR domains and the pertussis toxin (PTX) were from Calbiochem. Subconfluent 293 and 293/uPAR cells were detached by mild trypsinization, incubated with 10% fetal calf serum-Dulbecco’s modified Eagle’s medium for 1 h at 37 °C in 5% CO2, briefly acid-treated to avoid any interference by serum-derived membrane-bound growth factors as previously described [18], washed with PBS, and counted. Co-immunoprecipitation—For the analysis of uPAR1⁄7␣v complexes, detached and acid-treated 293/uPAR cells were exposed to 100 nM fMLP or diluents at 37 °C in 5% CO2 for 30 min and incubated with SRSRYp or diluents for 1 h. Cells were lysed in radioimmune precipitation assay buffer and cleared by centrifugation at 12,000 rpm, and 400 g/sample were incubated overnight at 4 °C with 5 g/ml VNR147 anti-␣v mAb. The immunoprecipitated proteins recovered by absorption to protein G-Sepharose and separated onto a 10% SDS-PAGE under nonreducing conditions were transferred to nitrocellulose membranes. Western blots were performed by incubating filters with 2 g/ml R4 anti-uPAR mAb or anti-␣v polyclonal Ab for 2 h at 4 °C
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