Abstract
The N-formyl peptide receptor is a G protein-coupled transmembrane receptor involved in stimulating a variety of differential responses in neutrophils including chemotaxis, degranulation, superoxide production, transcriptional activation, and actin reorganization. Although it is known that N-formyl-Met-Leu-Phe induces actin reorganization, the sequence of events from the receptor to the actin cytoskeleton is not well characterized. To study the signaling pathway from the N-formyl peptide receptor to the actin cytoskeleton, we developed a model system utilizing microinjection techniques with a nonhematopoietic cell line. An expression vector coding for the N-formyl peptide receptor was microinjected into porcine aortic endothelial cells and stimulated with N-formyl-Met-Leu-Phe to induce actin reorganization and membrane ruffling. The receptor-mediated signal was blocked by pertussis toxin and by a dominant negative Rac-N17, indicating the involvement of G(i)alpha subunit and the small guanosine triphosphatase Rac, respectively. Moreover, Gbetagamma subunits and membrane targeted forms of phosphatidylinositol (PI) 3-kinase alpha were sufficient to induce similar actin reorganization, and coexpression of various mutants of PI 3-kinase with the N-formyl peptide receptor identified a link to class Ia PI-3 kinase-mediated actin reorganization.
Highlights
In granulocytes a short exposure to fMLP induces actin polymerization, membrane ruffling, and cell polarization leading to cell migration toward a concentration gradient [2]
We initiated a study utilizing porcine aortic endothelial (PAE) cells microinjected with cDNA expressing the Formyl peptide receptors (FPR) and various cDNA constructs encoding constitutively active or dominant negative proteins that have been previously shown to play a role in actin cytoskeletal signaling pathways
The N-Formyl Peptide Receptor Induces Actin Reorganization in PAE Cells—FPR transduce multiple signaling cascades that are essential for chemotaxis, actin reorganization, and transcriptional activation
Summary
Microinjections and immunofluorescence microscopy were carried out as described previously [15]. PAE cells grown in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium containing 10% fetal bovine serum were plated on coverslips. Expression vectors encoding FLAG-tagged FPR and/or various tagged cDNAs known to be involved in G protein.
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