BACKGROUND
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 , which are generated by the electron-transporting nadph-oxidase system  <cit> .
the molecular basis for cellular recognition of chemoattractants is their binding to specific cell surface receptors  <cit> .
despite the structural variability of the numerous extracellular ligands, many of them bind to  specific receptors belonging to a large family of pertussis toxin-sensitive, g-protein-coupled receptors .
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  <cit> .
the pattern recognition formyl peptide receptor  family belongs to the gpcr group of chemoattractant receptors, and human neutrophil granulocytes express two members of this family, i.e., fpr <dig> and fpr <dig>  <cit> .
fpr <dig> was originally defined as an orphan receptor, and the gene was cloned from an hl- <dig> cell cdna library by low-stringency hybridization with the fpr <dig> sequence  <cit> .
recently, several fpr2-specific ligands have been identified  <cit> , including mitochondrial and microbial peptides  <cit> , various antimicrobial peptides  <cit> , the acute phase protein serum amyloid a   <cit> , the neurotoxic prion peptide fragment 106- <dig>  <cit> , and synthetic peptides, such as wkymvm  <cit>  and mmk- <dig>  <cit> .
to date, no defined structure has been identified as the determinant for fpr <dig> 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 fpr <dig> and fpr <dig>  <cit> .
the many studies that have been performed on fpr1-induced cell functions and signaling reveal that fpr <dig> 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  generates a second messenger following cleavage of pip <dig>  and this is the starting signal for a transient increase in cytosolic free calcium.
binding of the cleavage product, ip <dig>  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 i  <cit> .
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 i  <cit> .
although our knowledge of the signal transduction pathways utilized by fpr <dig> is currently somewhat limited, the significant homology observed between fpr <dig> and fpr <dig>  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  those induced by the prototype fpr <dig> agonist fmlf  <cit> .
however, fundamental differences between the signaling profiles of these two receptors have been described; the pip2-binding peptide pbp <dig>  <cit>  selectively inhibits a signaling pathway triggered by fpr <dig>  without affecting signaling via fpr <dig>  <cit> .
fpr <dig> has also been shown to trigger a unique type of ca2+ influx across the plasma membrane  <cit> .
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 i that originates from the release of ca2+ from the intracellular stores  <cit> .
these results point to two important differences in the signaling mediated by the two fprs in human neutrophils.
whereas fpr <dig> 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  radical production by neutrophils.
it is noteworthy that the influx of calcium induced by fpr <dig> is apparently insensitive to pbp <dig> treatment  <cit> .
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  <cit> , we show that the fpr <dig> agonist induces an increase in i that involves ca2+ release from intracellular stores, and this signaling pathway is inhibited by the fpr2-specific inhibitor pbp <dig>
RESULTS
the fmlf and wkymvm peptides activate the nadph-oxidase in neutrophils
the nadph-oxidase activities induced by the fpr1-specific peptide agonist fmlf and the fpr2-specific peptide agonist wkymvm were of similar magnitude .
the kinetics of the responses induced by equimolar concentrations  of these peptides were also very similar, with peak activity observed after approximately  <dig> minute.
in accordance with earlier findings  <cit> , the wkymvm-induced response was inhibited by the membrane permeable polyphosphoinositide-binding peptide rhodamine-b-qrlfqvkgrr , when it was added prior to activation.
the neutrophil nadph-oxidase activity was completely inhibited by pbp <dig> when activation was triggered through fpr <dig> , whereas pbp <dig> had no inhibitory effect on the fpr1-mediated  neutrophil response .
a molar ratio of agonist to inhibitor of 1: <dig> was required to inhibit the nadph-oxidase response.
despite the very similar activation patterns of fpr <dig> and fpr <dig>  functional differences between these two highly homologous receptors become apparent in the presence of pbp <dig>
the fmlf and wkymvm peptides mobilize intracellular ca2+ in neutrophils
it is well known that binding of a specific ligand to fpr <dig> results in plc-dependent cleavage of pip <dig>  and that the product, ip <dig>  mobilizes calcium from intracellular storage organelles.
thus, a transient increase in intracellular free calcium was achieved when neutrophils were treated with the fpr <dig> agonist fmlf .
moreover, a similar response was obtained when neutrophils were treated with the fpr2-specific ligand wkymvm .
in the presence of extracellular calcium, the response is composed of two phases: 1) an initial phase that is dependent upon the release of ca2+ from intracellular stores; and 2) a second phase that is regulated by the stores  <cit>  but relies on the opening of plasma membrane channels.
the pip2-binding peptide pbp <dig> inhibits the neutrophil calcium response to the fpr <dig> agonist wkymvm
the direct transfer of the system used to determine oxygen radical release to a system for determining transient rises in intracellular calcium levels revealed the expected insensitivity to pbp <dig> of the fmlf-induced neutrophil response .
however, the wkymvm-triggered response was only partly inhibited .
this is in accordance with previously published results  <cit> .
it is important to point out that there is no direct link between the rise in i and activation of the nadph-oxidase.
this is clearly shown in desensitized cells in which the cytoskeleton is disrupted by cytochalasin b. these cells produce large quantities of oxygen radicals  that is inhibited by pbp <dig> , but in contrast to what is seen with control  cells  oxidase activation in the desensitized cells is not associated with any rise in i .
the concentrations of intracellular calcium were determined upon stimulation with either cytb  or wkymvm .
cells were first loaded with fura- <dig>  before pre-incubation with wkymvm  or cytb  at 15°c.
the fluorescence levels corresponding to  <dig> mm and  <dig> nm are indicated by the bars.
abscissa, time of study; ordinates, intracellular ca2+ changes given in arbitrary units.
the curves are representative of at least four experiments.
the sensitivity of the calcium measurement system is very low when high concentrations of agonist  are used, and we set out to determine if the insensitivity to pbp <dig> of the wkymvm induced response, is due to the concentration used.
we found that when the concentration of agonist was reduced  to  <dig> nm or  <dig> nm, the response triggered by wkymvm was completely inhibited by pbp <dig> .
lower concentrations of wkymvm did not evoke any ca2+ response.
in contrast to the situation observed for wkymvm, there was no inhibition by pbp <dig> when the concentration of fmlf was reduced to  <dig> nm, which was the lowest concentration to induce a response .
these data clearly show that the pbp <dig> peptide selectively inhibits the calcium response mediated by fpr <dig>
the transient increases in intracellular ca2+ induced by fpr <dig> and fpr <dig> rely on mobilization of ca2+ from intracellular storage organelles
the signals generated by the binding of an fpr2-selective agonist to a neutrophil have been shown to induce opening of the calcium channels in the plasma membrane, without mobilization of the ca2+-storing organelles  <cit> .
accordingly, it has been shown that there is no increase in the level of intracellular free calcium when extracellular calcium is lacking  <cit> .
this contrasts with the signals known to be generated by the fmlf/fpr <dig> ligand-receptor-pairing.
these signals induce the mobilization of ca2+ from intracellular storage organelles, and an increase in free intracellular calcium is also achieved in the absence of extracellular calcium .
to assess the effects of calcium influx on neutrophil responses, the calcium ions in the extracellular medium were removed by treatment with egta immediately before the addition of the receptor-specific agonist.
the concentration of egta used was that required to achieve a maximal inhibition on ionomycin induced the nadph-oxidase in neutrophils , an activity that is dependent on the influx of calcium from extracellular medium across the plasma membrane.
we found that wkymvm induced an increase in intracellular calcium in the absence of extracellular calcium .
similar outcomes were obtained when the fpr <dig> agonist mmk- <dig>  or fmlf  was used.
the kinetics of the wkymvm-induced response resembled those of the other peptides, and we could not confirm that the ca2+ signaling induced by the fpr <dig> agonist induces a calcium influx across the plasma membrane that is independent of the emptying of the intracellular stores.
therefore, the pattern of responses triggered through fpr <dig> is identical to that mediated through fpr <dig>
light emission was measured with or without egta, and a representative experiment of at least five performed is shown.
abscissa, time of study ; ordinate, superoxide production, given as light emission and expressed as cpm × 10- <dig>
discussion
human neutrophils express the formyl peptide receptor family members fpr <dig> and fpr <dig>  which share a high degree of amino acid sequence identity.
despite the similarities between the two receptors, the signaling events that follow receptor occupancy and activation differ.
these differences may be related either to a specific agonist or distinct difference between the receptors  <cit> .
we have previously shown that a receptor agonist that binds to both fpr <dig> and fpr <dig> can trigger receptor-specific signals, and depending on the particular triggering agonist, both receptors can either inhibit the functional repertoire of the neutrophils or activate the same repertoire  <cit> .
one of the early signals generated by activated neutrophil gpcrs  is the rapid rise of i, which affects different cellular functions.
for a resting neutrophil, the free cytosolic calcium concentration is maintained at a very low level  relative to the level in the extracellular fluid .
to maintain this concentration gradient, cells are equipped with ion pumps that transport ca2+ out of the cells as well as to the intracellular calcium storing organelles, which contain high levels of the calcium-binding protein calreticulin.
it is generally accepted that the main pathway for the control of rapid changes of i in neutrophils is that represented by the capacitive model, which starts with the production of inositol- <dig> ,5-trisphosphate .
the main pathway for ip <dig> production involves plc-mediated hydrolysis of membrane pip2; the generated ip <dig> releases calcium from intracellular stores by binding to specific receptors on the intracellular storage organelles.
the depletion of the intracellular ca2+ stores regulates the opening of store-operated ca2+ channels  in the plasma membrane, thereby providing a rich source of capacitive entry of calcium ions, originating from the extracellular space  <cit> .
with respect to the different signaling properties of the fprs, a recent report has demonstrated that the regulatory mechanism that leads to an increase in i is unique to fpr <dig>  binding of an agonist to either of the two fprs results in a signaling cascade, which in turn leads to an increase in intracellular calcium through the influx of ions across the plasma membrane.
whereas fpr <dig> influences the plasma membrane channels through the emptying of intracellular stores, as described above, fpr <dig> appears to generate a signal that acts directly on the ion channels in the plasma membrane so as to facilitate the influx of ca2+ independently of the filling status of the storage organelles  <cit> .
although we could not verify this difference in signaling profile, we show that the signals from fpr <dig> and fpr <dig> initiate an increase in cytosolic free calcium derived from the emptying of intracellular stores.
the increase in i was achieved when the calcium in the extracellular milieu was depleted through the addition of egta; these data are in accordance with recent results from experiments in which a much higher concentration  of agonist was used  <cit> .
with respect to the different signaling properties of the two receptors, we recently showed that fpr <dig> and fpr <dig> have different sensitivities to pbp <dig>  <cit> .
pbp <dig> inhibits fpr2-triggered activation of the neutrophil nadph-oxidase.
however, when this system was directly transferred to a calcium measurement system, we found that the cytosolic increase in free ca2+ was largely insensitive to pbp <dig>
in the present study, we confirm the difference between fpr1-dependent and fpr2-dependent activation of the neutrophil nadph-oxidase.
moreover, we show that pbp <dig> inhibits not only the oxidase activity, but also the fpr2-induced increase in i. the transient rise in i peaks at agonist concentrations that are much lower than those used in the oxidase assay, and the inhibitory effects of pbp <dig> are, thus, disclosed only when the concentration of the fpr <dig> agonist is reduced.
no inhibitory effect of pbp <dig> is seen when fmlf is used as the agonist, irrespective of the concentration of the chemoattractant used to activate the neutrophils.
the molecular mechanism underlying the effects of pbp <dig> on fpr2-mediated induction of cell functions is complex.
pbp <dig> displays clearly higher specificity for fpr <dig> than for fpr <dig>  although this pattern of sensitivity is not unique to this receptor  <cit> .
the biochemical and biophysical characterizations of the ten strategically organized basic and hydrophobic amino acids in pbp <dig>  which are also present in the gelsolin molecule, suggest that it may interact with a broad range of negatively charged phosphomonoesters and hydrophobic acyl chains on anionic phospholipids.
this implies that in addition to its blocking/competing activities, which involve proteins that are regulated by cellular phosphoinositides, pbp <dig> may affect the functions of bioactive and signaling lipids.
although the precise signal transduction step that is disrupted by pbp <dig> remains to be elucidated, the receptor-specific and signal-selective effects of this peptide on neutrophil functions suggest that it has potential applications as a tool to manipulate and define how g-protein-coupled receptors produce and integrate the signals generated from activated receptors, as well as to probe new signaling functions of polyphosphoinositides.
CONCLUSIONS
the neutrophil fpr family members fpr <dig> and fpr <dig> share 69% amino acid identity and mediate almost indistinguishable cellular responses.
therefore, these two fprs use the same basic signaling pathways.
accordingly, we show that ca2+ signaling mediated via fpr <dig> follows the same route as fpr1-mediated signaling, which involves initial emptying of the intracellular stores.
however, we found that the fpr2-mediated oxidase activity and ca2+ signaling pathway are inhibited by the fpr2-specific inhibitor pbp <dig>  whereas there is no inhibitory effect of pbp <dig> when the high affinity fpr <dig> agonist fmlf is used, irrespective of the concentration of the chemoattractant used to activate the neutrophils.
these data clearly demonstrate that there is a fundamental difference between the two very closely related receptor members, in that one is pbp10-sensitive  and the other  is pbp10-insensitive.

 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, which are generated by the electron-transporting nadph-oxidase system. the molecular basis for cellular recognition of chemoattractants is their binding to specific cell surface receptors. despite the structural variability of the numerous extracellular ligands, many of them bind to specific receptors belonging to a large family of pertussis toxin-sensitive, g-protein-coupled receptors. 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. the pattern recognition formyl peptide receptor family belongs to the gpcr group of chemoattractant receptors, and human neutrophil granulocytes express two members of this family, i.e., fpr and fpr. fpr was originally defined as an orphan receptor, and the gene was cloned from an hl- cell cdna library by low-stringency hybridization with the fpr sequence. recently, several fpr2-specific ligands have been identified, including mitochondrial and microbial peptides, various antimicrobial peptides, the acute phase protein serum amyloid a, the neurotoxic prion peptide fragment 106-, and synthetic peptides, such as wkymvm and mmk-. to date, no defined structure has been identified as the determinant for fpr 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 fpr and fpr. the many studies that have been performed on fpr1-induced cell functions and signaling reveal that fpr 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 generates a second messenger following cleavage of pip and this is the starting signal for a transient increase in cytosolic free calcium. binding of the cleavage product, ip 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 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 i. although our knowledge of the signal transduction pathways utilized by fpr is currently somewhat limited, the significant homology observed between fpr and fpr 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 those induced by the prototype fpr agonist fmlf. however, fundamental differences between the signaling profiles of these two receptors have been described; the pip2-binding peptide pbp selectively inhibits a signaling pathway triggered by fpr without affecting signaling via fpr. fpr 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 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 fpr 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 radical production by neutrophils. it is noteworthy that the influx of calcium induced by fpr is apparently insensitive to pbp 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, we show that the fpr agonist induces an increase in i that involves ca2+ release from intracellular stores, and this signaling pathway is inhibited by the fpr2-specific inhibitor pbp  the fmlf and wkymvm peptides activate the nadph-oxidase in neutrophils the nadph-oxidase activities induced by the fpr1-specific peptide agonist fmlf and the fpr2-specific peptide agonist wkymvm were of similar magnitude. the kinetics of the responses induced by equimolar concentrations of these peptides were also very similar, with peak activity observed after approximately minute. in accordance with earlier findings, the wkymvm-induced response was inhibited by the membrane permeable polyphosphoinositide-binding peptide rhodamine-b-qrlfqvkgrr, when it was added prior to activation. the neutrophil nadph-oxidase activity was completely inhibited by pbp when activation was triggered through fpr, whereas pbp had no inhibitory effect on the fpr1-mediated neutrophil response. a molar ratio of agonist to inhibitor of 1: was required to inhibit the nadph-oxidase response. despite the very similar activation patterns of fpr and fpr functional differences between these two highly homologous receptors become apparent in the presence of pbp the fmlf and wkymvm peptides mobilize intracellular ca2+ in neutrophils it is well known that binding of a specific ligand to fpr results in plc-dependent cleavage of pip and that the product, ip mobilizes calcium from intracellular storage organelles. thus, a transient increase in intracellular free calcium was achieved when neutrophils were treated with the fpr agonist fmlf. moreover, a similar response was obtained when neutrophils were treated with the fpr2-specific ligand wkymvm. in the presence of extracellular calcium, the response is composed of two phases: 1) an initial phase that is dependent upon the release of ca2+ from intracellular stores; and 2) a second phase that is regulated by the stores but relies on the opening of plasma membrane channels. the pip2-binding peptide pbp inhibits the neutrophil calcium response to the fpr agonist wkymvm the direct transfer of the system used to determine oxygen radical release to a system for determining transient rises in intracellular calcium levels revealed the expected insensitivity to pbp of the fmlf-induced neutrophil response. however, the wkymvm-triggered response was only partly inhibited. this is in accordance with previously published results. it is important to point out that there is no direct link between the rise in i and activation of the nadph-oxidase. this is clearly shown in desensitized cells in which the cytoskeleton is disrupted by cytochalasin b. these cells produce large quantities of oxygen radicals that is inhibited by pbp, but in contrast to what is seen with control cells oxidase activation in the desensitized cells is not associated with any rise in i. the concentrations of intracellular calcium were determined upon stimulation with either cytb or wkymvm. cells were first loaded with fura- before pre-incubation with wkymvm or cytb at 15°c. the fluorescence levels corresponding to mm and nm are indicated by the bars. abscissa, time of study; ordinates, intracellular ca2+ changes given in arbitrary units. the curves are representative of at least four experiments. the sensitivity of the calcium measurement system is very low when high concentrations of agonist are used, and we set out to determine if the insensitivity to pbp of the wkymvm induced response, is due to the concentration used. we found that when the concentration of agonist was reduced to nm or nm, the response triggered by wkymvm was completely inhibited by pbp. lower concentrations of wkymvm did not evoke any ca2+ response. in contrast to the situation observed for wkymvm, there was no inhibition by pbp when the concentration of fmlf was reduced to nm, which was the lowest concentration to induce a response. these data clearly show that the pbp peptide selectively inhibits the calcium response mediated by fpr the transient increases in intracellular ca2+ induced by fpr and fpr rely on mobilization of ca2+ from intracellular storage organelles the signals generated by the binding of an fpr2-selective agonist to a neutrophil have been shown to induce opening of the calcium channels in the plasma membrane, without mobilization of the ca2+-storing organelles. accordingly, it has been shown that there is no increase in the level of intracellular free calcium when extracellular calcium is lacking. this contrasts with the signals known to be generated by the fmlf/fpr ligand-receptor-pairing. these signals induce the mobilization of ca2+ from intracellular storage organelles, and an increase in free intracellular calcium is also achieved in the absence of extracellular calcium. to assess the effects of calcium influx on neutrophil responses, the calcium ions in the extracellular medium were removed by treatment with egta immediately before the addition of the receptor-specific agonist. the concentration of egta used was that required to achieve a maximal inhibition on ionomycin induced the nadph-oxidase in neutrophils, an activity that is dependent on the influx of calcium from extracellular medium across the plasma membrane. we found that wkymvm induced an increase in intracellular calcium in the absence of extracellular calcium. similar outcomes were obtained when the fpr agonist mmk- or fmlf was used. the kinetics of the wkymvm-induced response resembled those of the other peptides, and we could not confirm that the ca2+ signaling induced by the fpr agonist induces a calcium influx across the plasma membrane that is independent of the emptying of the intracellular stores. therefore, the pattern of responses triggered through fpr is identical to that mediated through fpr light emission was measured with or without egta, and a representative experiment of at least five performed is shown. abscissa, time of study; ordinate, superoxide production, given as light emission and expressed as cpm × 10- discussion human neutrophils express the formyl peptide receptor family members fpr and fpr which share a high degree of amino acid sequence identity. despite the similarities between the two receptors, the signaling events that follow receptor occupancy and activation differ. these differences may be related either to a specific agonist or distinct difference between the receptors. we have previously shown that a receptor agonist that binds to both fpr and fpr can trigger receptor-specific signals, and depending on the particular triggering agonist, both receptors can either inhibit the functional repertoire of the neutrophils or activate the same repertoire. one of the early signals generated by activated neutrophil gpcrs is the rapid rise of i, which affects different cellular functions. for a resting neutrophil, the free cytosolic calcium concentration is maintained at a very low level relative to the level in the extracellular fluid. to maintain this concentration gradient, cells are equipped with ion pumps that transport ca2+ out of the cells as well as to the intracellular calcium storing organelles, which contain high levels of the calcium-binding protein calreticulin. it is generally accepted that the main pathway for the control of rapid changes of i in neutrophils is that represented by the capacitive model, which starts with the production of inositol-,5-trisphosphate. the main pathway for ip production involves plc-mediated hydrolysis of membrane pip2; the generated ip releases calcium from intracellular stores by binding to specific receptors on the intracellular storage organelles. the depletion of the intracellular ca2+ stores regulates the opening of store-operated ca2+ channels in the plasma membrane, thereby providing a rich source of capacitive entry of calcium ions, originating from the extracellular space. with respect to the different signaling properties of the fprs, a recent report has demonstrated that the regulatory mechanism that leads to an increase in i is unique to fpr binding of an agonist to either of the two fprs results in a signaling cascade, which in turn leads to an increase in intracellular calcium through the influx of ions across the plasma membrane. whereas fpr influences the plasma membrane channels through the emptying of intracellular stores, as described above, fpr appears to generate a signal that acts directly on the ion channels in the plasma membrane so as to facilitate the influx of ca2+ independently of the filling status of the storage organelles. although we could not verify this difference in signaling profile, we show that the signals from fpr and fpr initiate an increase in cytosolic free calcium derived from the emptying of intracellular stores. the increase in i was achieved when the calcium in the extracellular milieu was depleted through the addition of egta; these data are in accordance with recent results from experiments in which a much higher concentration of agonist was used. with respect to the different signaling properties of the two receptors, we recently showed that fpr and fpr have different sensitivities to pbp. pbp inhibits fpr2-triggered activation of the neutrophil nadph-oxidase. however, when this system was directly transferred to a calcium measurement system, we found that the cytosolic increase in free ca2+ was largely insensitive to pbp in the present study, we confirm the difference between fpr1-dependent and fpr2-dependent activation of the neutrophil nadph-oxidase. moreover, we show that pbp inhibits not only the oxidase activity, but also the fpr2-induced increase in i. the transient rise in i peaks at agonist concentrations that are much lower than those used in the oxidase assay, and the inhibitory effects of pbp are, thus, disclosed only when the concentration of the fpr agonist is reduced. no inhibitory effect of pbp is seen when fmlf is used as the agonist, irrespective of the concentration of the chemoattractant used to activate the neutrophils. the molecular mechanism underlying the effects of pbp on fpr2-mediated induction of cell functions is complex. pbp displays clearly higher specificity for fpr than for fpr although this pattern of sensitivity is not unique to this receptor. the biochemical and biophysical characterizations of the ten strategically organized basic and hydrophobic amino acids in pbp which are also present in the gelsolin molecule, suggest that it may interact with a broad range of negatively charged phosphomonoesters and hydrophobic acyl chains on anionic phospholipids. this implies that in addition to its blocking/competing activities, which involve proteins that are regulated by cellular phosphoinositides, pbp may affect the functions of bioactive and signaling lipids. although the precise signal transduction step that is disrupted by pbp remains to be elucidated, the receptor-specific and signal-selective effects of this peptide on neutrophil functions suggest that it has potential applications as a tool to manipulate and define how g-protein-coupled receptors produce and integrate the signals generated from activated receptors, as well as to probe new signaling functions of polyphosphoinositides.  the neutrophil fpr family members fpr and fpr share 69% amino acid identity and mediate almost indistinguishable cellular responses. therefore, these two fprs use the same basic signaling pathways. accordingly, we show that ca2+ signaling mediated via fpr follows the same route as fpr1-mediated signaling, which involves initial emptying of the intracellular stores. however, we found that the fpr2-mediated oxidase activity and ca2+ signaling pathway are inhibited by the fpr2-specific inhibitor pbp whereas there is no inhibitory effect of pbp when the high affinity fpr agonist fmlf is used, irrespective of the concentration of the chemoattractant used to activate the neutrophils. these data clearly demonstrate that there is a fundamental difference between the two very closely related receptor members, in that one is pbp10-sensitive and the other is pbp10-insensitive. 
the aims of this study were to determine whether fpr triggers a unique signal that allows direct influx of extracellular calcium without the emptying of intracellular calcium stores, and whether the gelsolin-derived pip2-binding peptide, pbp selectively inhibits fpr2-mediated transient rise in intracellular ca2+. the transient rise in intracellular ca2+ induced by agonists for fpr or fpr in human neutrophils occurred also in the presence of a chelator of ca2+. pbp inhibited not only fpr2-induced oxidase activity, but also the transient rise in intracellular ca2+. pbp inhibits selectively the signals generated by fpr both with respect to nadph-oxidase activity and the transient rise in intracellular ca2+ induced by agonist exposure. the molecular basis for neutrophil recognition of chemotactic peptides is their binding to specific g-protein-coupled cell surface receptors. the high degree of homology between these two receptors suggests that they share many functional and signal transduction properties, although they exhibit some differences with respect to signaling. ca2+ signaling mediated via fpr follows the same route as fpr which involves initial emptying of the intracellular stores. human neutrophils express two pattern recognition gpcrs, fpr and fpr which belong to the family of formyl peptide receptors.
 the molecular basis for neutrophil recognition of chemotactic peptides is their binding to specific g-protein-coupled cell surface receptors. human neutrophils express two pattern recognition gpcrs, fpr and fpr which belong to the family of formyl peptide receptors. the high degree of homology between these two receptors suggests that they share many functional and signal transduction properties, although they exhibit some differences with respect to signaling. the aims of this study were to determine whether fpr triggers a unique signal that allows direct influx of extracellular calcium without the emptying of intracellular calcium stores, and whether the gelsolin-derived pip2-binding peptide, pbp selectively inhibits fpr2-mediated transient rise in intracellular ca2+.  the transient rise in intracellular ca2+ induced by agonists for fpr or fpr in human neutrophils occurred also in the presence of a chelator of ca2+. pbp inhibited not only fpr2-induced oxidase activity, but also the transient rise in intracellular ca2+.  ca2+ signaling mediated via fpr follows the same route as fpr which involves initial emptying of the intracellular stores. pbp inhibits selectively the signals generated by fpr both with respect to nadph-oxidase activity and the transient rise in intracellular ca2+ induced by agonist exposure. 