Neutrophil granulocytes are crucial for the outcome of the "battle" between the innate immune system and invading micro-organisms, and are key cells in the damaged tissues at sites of infection and inflammation. Neutrophil responses to endogenous and exogenous chemoattractants include locomotory responses, up-regulation of adhesion molecules, secretion of granule constituents, and production of reactive oxygen species (ROS), which are generated by the electron-transporting NADPH-oxidase system [1–3]. The molecular basis for cellular recognition of chemoattractants is their binding to specific cell surface receptors [4–8]. Despite the structural variability of the numerous extracellular ligands, many of them bind to (and activate) specific receptors belonging to a large family of pertussis toxin-sensitive, G-protein-coupled receptors (GPCRs). These receptors share a high degree of amino acid sequence similarity, and although they are activated by different agonists, they transduce downstream signals that have many common features. Nevertheless, it is clear that there are also important differences between the receptor-ligand pairs in terms of functional repertoires [9, 10]. The pattern recognition formyl peptide receptor (FPR) family belongs to the GPCR group of chemoattractant receptors, and human neutrophil granulocytes express two members of this family, i.e., FPR1 and FPR2 [4, 11]. FPR2 was originally defined as an orphan receptor, and the gene was cloned from an HL-60 cell cDNA library by low-stringency hybridization with the FPR1 sequence [12–14]. Recently, several FPR2-specific ligands have been identified [4, 11], including mitochondrial and microbial peptides [15, 16], various antimicrobial peptides , the acute phase protein serum amyloid A (SAA) [18, 19], the neurotoxic prion peptide fragment 106-126 , and synthetic peptides, such as WKYMVM  and MMK-1 . To date, no defined structure has been identified as the determinant for FPR2 binding and activation, although the close relationship between structural variation and function is illustrated by the fact that exchange of the C-terminal L-methionine residue in WKYMVM for the D-isomeric form expands the binding specificity to encompass both FPR2 and FPR1 .
The many studies that have been performed on FPR1-induced cell functions and signaling reveal that FPR1 signaling has all the characteristics of a pertussis toxin-sensitive GPCR. The activated receptor initiates a chain of signaling events, starting with dissociation of the receptor-associated G-protein, and subsequently, activation of a number of downstream signaling pathways. In one of these pathways, activation of phosphoinositide-specific phospholipase C (PLC) generates a second messenger following cleavage of PIP2, and this is the starting signal for a transient increase in cytosolic free calcium. Binding of the cleavage product, IP3, to its receptor located on storage organelles results in the release of Ca2+ from these intracellular organelles and elevation consequent increase in the concentration of free calcium ions in the cytoplasm [Ca2+]i . Emptying of the storage organelles leads to the entry of extracellular Ca2+ through store-operated calcium channels in the plasma membrane, thereby prolonging the increase in [Ca2+]i [25, 26].
Although our knowledge of the signal transduction pathways utilized by FPR2 is currently somewhat limited, the significant homology observed between FPR1 and FPR2 (69% at the amino acid level) suggests that these two receptors share signal transduction features. Accordingly, we have previously shown that the functional responses induced by the FPR2-specific agonist WKYMVM is largely similar to (even indistinguishable from) those induced by the prototype FPR1 agonist fMLF . However, fundamental differences between the signaling profiles of these two receptors have been described; the PIP2-binding peptide PBP10  selectively inhibits a signaling pathway triggered by FPR2, without affecting signaling via FPR1 . FPR2 has also been shown to trigger a unique type of Ca2+ influx across the plasma membrane . It has been suggested that a channel in the plasma membrane opens without involvement of the intracellular storage organelles. Thus, the influx of Ca2+ across the plasma membrane is not preceded by an increase in [Ca2+]i that originates from the release of Ca2+ from the intracellular stores . These results point to two important differences in the signaling mediated by the two FPRs in human neutrophils. Whereas FPR2 triggers a unique calcium signal, which is independent of the intracellular Ca2+ store-influenced Ca2+ channels, and allows for the direct influx of extracellular Ca2+, a PIP2-binding peptide inhibits FPR2-induced (but not FPR1-induced) radical production by neutrophils. It is noteworthy that the influx of calcium induced by FPR2 is apparently insensitive to PBP10 treatment . The mechanism underlying these differences is puzzling and it is thus of importance to verify or falsify the observations.
In the present study, we characterize the neutrophil responses to the FPR2-specific agonist WKYMVM. We confirm the difference between FPR1-dependent and FPR2-dependent activation of the neutrophil NADPH-oxidase. However, in apparent discrepancy with previously published results [28, 29], we show that the FPR2 agonist induces an increase in [Ca2+]i that involves Ca2+ release from intracellular stores, and this signaling pathway is inhibited by the FPR2-specific inhibitor PBP10.