FPRL-1 induces modifications of migration-associated proteins in human neutrophils.
Human polymorphonuclear neutrophils (PMNs) are an important cell population of the innate immune system, which migrates following concentration gradients of chemokines or chemoattractants to locations of infection and inflammation in order to eliminate invading microorganisms and cell debris. For both migration and adhesion of PMNs to various tissues, the dynamic remodeling of the cytoskeleton is key prerequisite. In this context, the formyl peptide receptor-like 1 (FPRL-1) is an important chemoattractant receptor expressed on PMNs. In this study, we show that a short stimulation of FPRL-1 with either a synthetic peptide ligand (W-peptide) or a natural ligand (sCKbeta8-1) changes the protein pattern of PMNs as assessed by 2-D-DIGE. MS analysis of selected deregulated protein species resulted in the identification of proteins that are involved in the remodeling process of the actin- and tubulin-based cytoskeleton, such as L-plastin, moesin, cofilin, and stathmin. Subsequent validation experiments performed either by Western blotting or phosphoprotein-specific gel staining (Pro-Q Diamond) revealed that L-plastin is phosphorylated, whereas moesin, cofilin, and stathmin are dephosphorylated in PMNs upon FPRL-1 stimulation. These findings suggest that FPRL-1 signaling targets proteins that regulate the motility of PMNs and moreover show that 2-D-DIGE is a technique capable of detecting and quantifying differently modified (e.g., phosphorylated) protein variants.
Boldt
Karsten
K
Boehringer Ingelheim Pharma GmbH & Co. KG, Department of Respiratory Research, Genomics Group, Biberach an der Riss, Germany.
Rist
Wolfgang
W
Weiss
Stefanie M
SM
Weith
Andreas
A
Lenter
Martin C
MC
D002465:Cell Movement; D002469:Cell Separation; D003598:Cytoskeletal Proteins / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D008024:Ligands; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D009504:Neutrophils / Q000166:cytology* / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D044042:Receptors, Formyl Peptide / Q000378:metabolism*; D044067:Receptors, Lipoxin / Q000378:metabolism*
D003598:Cytoskeletal Proteins; C105101:FPR2 protein, human; D008024:Ligands; D010455:Peptides; D044042:Receptors, Formyl Peptide; D044067:Receptors, Lipoxin; D020929:Mitogen-Activated Protein Kinase Kinases