Mitocryptide-2: Identification of Its Minimum Structure for Specific Activation of FPR2-Possible Receptor Switching from FPR2 to FPR1 by Its Physiological C-terminal Cleavages.

Mitocryptides are a novel family of endogenous neutrophil-activating peptides originating from various mitochondrial proteins. Mitocryptide-2 (MCT-2) is one of such neutrophil-activating peptides, and is produced as an N-formylated pentadecapeptide from mitochondrial cytochrome b. Although MCT-2 is a specific endogenous ligand for formyl peptide receptor 2 (FPR2), the chemical structure within MCT-2 that is responsible for FPR2 activation is still obscure. Here, we demonstrate that the N-terminal heptapeptide structure of MCT-2 with an N-formyl group is the minimum structure that specifically activates FPR2. Moreover, the receptor molecule for MCT-2 is suggested to be shifted from FPR2 to its homolog formyl peptide receptor 1 (FPR1) by the physiological cleavages of its C-terminus. Indeed, N-terminal derivatives of MCT-2 with seven amino acid residues or longer caused an increase of intracellular free Ca2+ concentration in HEK-293 cells expressing FPR2, but not in those expressing FPR1. Those MCT-2 derivatives also induced ß-hexosaminidase secretion in neutrophilic/granulocytic differentiated HL-60 cells via FPR2 activation. In contrast, MCT-2(1-4), an N-terminal tetrapeptide of MCT-2, specifically activated FPR1 to promote those functions. Moreover, MCT-2 was degraded in serum to produce MCT-2(1-4) over time. These findings suggest that MCT-2 is a novel critical factor that not only initiates innate immunity via the specific activation of FPR2, but also promotes delayed responses by the activation of FPR1, which may include resolution and tissue regeneration. The present results also strongly support the necessity of considering the exact chemical structures of activating factors for the investigation of innate immune responses.

Identification of Residues Critical for FPR2 Activation by the Cryptic Peptide Mitocryptide-2 Originating from the Mitochondrial DNA-Encoded Cytochrome b.

Similar to bacteria, synthesis of mitochondrial DNA-encoded proteins requires an N-formylated methionine to initiate translation. Thus, the N-formylated methionine peptides originating from mitochondria should be recognized as danger signals. To date, only one such peptide, denoted as mitocryptide-2 (MCT-2), originating from the N-terminal of the mitochondrial cytochrome b, has been isolated from mammalian tissues. Human neutrophils express FPR1 and FPR2 that detect formyl peptides, and the precise structural determinants for receptor recognition remain to be elucidated. MCT-2 is known to activate neutrophils through FPR2 but not FPR1. The aim of this study was to elucidate the structural determinants of importance for receptor preference and human neutrophil activation in MCT-2 by generating a series of MCT-2 variants. We show that there is an absolute requirement for the N-formyl group and the side chain of Met1 at position 1 of MCT-2 but also the C terminus is of importance for MCT-2 activity. We also uncovered individual side chains that positively contribute to MCT-2 activity as well as those suppressed in the response. The MCT-2 peptide and its two polymorphic variants ([Thr7]MCT-2 and [Ser8]MCT-2) all activated neutrophils, but MCT-2 containing Ile7 and Asn8 was the most potent. We also show that some peptide variants displayed a biased FPR2-signaling property related to NADPH oxidase activation and ß-arrestin recruitment, respectively. In conclusion, we disclose several critical elements in MCT-2 that are required for neutrophil activation and disclose structural insights into how FPR2 recognition of this mitochondrial DNA-derived peptide may increase our understanding of the role of FPR2 in aseptic inflammation.

Mitocryptides from Human Mitochondrial DNA-Encoded Proteins Activate Neutrophil Formyl Peptide Receptors: Receptor Preference and Signaling Properties.

Phagocytic neutrophils express formyl peptide receptors (FPRs; FPR1 and FPR2) that distinctly recognize peptides starting with an N-formylated methionine (fMet). This is a hallmark of bacterial metabolism; similar to prokaryotes, the starting amino acid in synthesis of mitochondrial DNA-encoded proteins is an fMet. Mitochondrial cryptic peptides (mitocryptides; MCTs) with an N-terminal fMet could be identified by our innate immune system; however, in contrast to our knowledge about bacterial metabolites, very little is known about the recognition profiles of MCTs. In this study, we determined the neutrophil-recognition profiles and functional output of putative MCTs originating from the N termini of the 13 human mitochondrial DNA-encoded proteins. Six of the thirteen MCTs potently activated neutrophils with distinct FPR-recognition profiles: MCTs from ND3 and ND6 have a receptor preference for FPR1; MCTs from the proteins ND4, ND5, and cytochrome b prefer FPR2; and MCT-COX1 is a dual FPR1/FPR2 agonist. MCTs derived from ND2 and ND4L are very weak neutrophil activators, whereas MCTs from ND1, ATP6, ATP8, COX2, and COX3, do not exert agonistic or antagonistic FPR effects. In addition, the activating MCTs heterologously desensitized IL-8R but primed the response to the platelet-activating factor receptor agonist. More importantly, our data suggest that MCTs have biased signaling properties in favor of activation of the superoxide-generating NADPH oxidase or recruitment of ß-arrestin. In summary, we identify several novel FPR-activating peptides with sequences present in the N termini of mitochondrial DNA-encoded proteins, and our data elucidate the molecular basis of neutrophil activation by MCTs.

Generation of monoclonal antibodies against mitocryptide-2: toward a new strategy to investigate the biological roles of cryptides.

We recently identified a novel family of neutrophil-activating peptides including mitocryptide-1 and mitocryptide-2 (MCT-2) that are endogenously produced from various mitochondrial proteins. Among them, MCT-2 is an N-formylated pentadecapeptide derived from mitochondrial cytochrome b and is found to promote neutrophilic migration and phagocytosis efficiently. Signaling mechanisms of neutrophil activation by MCT-2 have been investigated at the cellular level, and MCT-2 has been demonstrated to be an endogenous specific ligand for formyl peptide receptor-2 (also referred to as formyl peptide receptor-like 1). It was also found that MCT-2 promoted neutrophilic functions via the activation of Gi2 proteins and phosphorylation of ERK1/2 consecutively. However, the physiological production, distribution, and functions of MCT-2 are not yet elucidated. Here, to investigate the roles of MCT-2 in vivo, we generated monoclonal antibodies (mAbs) against human MCT-2 (hMCT-2) that have two different characteristics. One mAb, NhM2A1, not only bound to the region of positions 10-15 of hMCT-2 but also recognized its C-terminal cleavage site that is presumably produced upon enzymatic hydrolysis of cytochrome b, indicating that NhM2A1 specifically interacts with hMCT-2 but not its parent protein. Moreover, we succeeded in acquiring a specific neutralizing mAb, NhM2A5, which blocks the bioactivities of hMCT-2. Specifically, NhM2A5 inhibited hMCT-2-induced ß-hexosaminidase release in neutrophilic/granulocytic differentiated HL-60 cells by binding to the region of positions 5-12 of hMCT-2. Functional analysis using obtained mAbs that specifically recognize hMCT-2 but not its parent protein, cytochrome b, and that neutralize bioactivities of hMCT-2 is expected to reveal the physiological roles of MCT-2, which are presently very difficult to investigate. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Mitochondrial protein-derived cryptides: Are endogenous N-formylated peptides including mitocryptide-2 components of mitochondrial damage-associated molecular patterns?

Recently, much attention has been paid to "nonclassical" bioactive peptides, which are fragmented peptides simultaneously produced during maturation and degradation of various functional proteins. We identified many fragmented peptides derived from various mitochondrial proteins including mitocryptide-1 and mitocryptide-2 that efficiently activate neutrophils. These endogenous, functionally active, fragmented peptides are referred to as "cryptides." Among them, mitocryptide-2 is an N-formylated cryptide cleaved from mitochondrial cytochrome b that is encoded in mitochondrial DNA (mtDNA). It is known that 13 proteins encoded in mtDNA are translated in mitochondria as N-formylated forms, suggesting the existence of endogenous N-formylated peptides other than mitocryptide-2. Here, we investigated the effects of N-formylated peptides presumably cleaved from mtDNA-encoded proteins other than cytochrome b on the functions of neutrophilic cells to elucidate possible regulation by endogenous N-formylated cryptides. Four N-formylated cryptides derived from cytochrome c oxidase subunit I and NADH dehydrogenase subunits 4, 5, and 6 among 12 peptides from mtDNA-encoded proteins efficiently induced not only migration but also ß-hexosaminidase release, which is an indicator of neutrophilic phagocytosis, in HL-60 cells differentiated into neutrophilic cells. These activities were comparable to or higher than those induced by mitocryptide-2. Although endogenous N-formylated peptides that are contained in mitochondrial damage-associated molecular patterns (DAMPs) have yet to be molecularly identified, they have been implicated in innate immunity. Thus, N-formylated cryptides including mitocryptide-2 are first-line candidates for the contents of mitochondrial DAMPs to promote innate immune responses. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 580-587, 2016.

Successful acquisition of a neutralizing monoclonal antibody against a novel neutrophil-activating peptide, mitocryptide-1.

Mitocryptide-1 (MCT-1) is a novel neutrophil-activating peptide derived from mitochondrial cytochrome c oxidase subunit VIII, and its physiological role and involvement in various diseases have not yet been elucidated. Generating neutralizing antibodies against the function of MCT-1 is of particular importance for investigating its physiological and pathophysiological roles, because MCT-1 is a fragmented peptide of its mother protein and hence it is very difficult to manipulate its expression level genetically without affecting expression of the mother protein. Here, we report the successful generation of a neutralizing monoclonal antibody (MAb) against MCT-1. This MAb, designated NM1B1, which specifically bound to the region of positions 9-22 of MCT-1, showed concentration-dependent inhibition of MCT-1-induced migration and ß-hexosaminidase release in neutrophilic/granulocytic differentiated HL-60 cells. Thus, NM1B1, as a neutralizing MAb against MCT-1, could elucidate not just the physiological regulatory mechanisms of MCT-1 but also its pathophysiological involvement in various inflammatory diseases in vivo.

A new class of aggregation inhibitor of amyloid-ß peptide based on an O-acyl isopeptide.

Inhibition of amyloid ß peptide (Aß) aggregation is a potential therapeutic approach to treat Alzheimer's disease. We report that an O-acyl isopeptide of Aß1-42 (1) containing an ester bond at the Gly(25)-Ser(26) moiety inhibits Aß1-42 fibril formation at equimolar ratio. Inhibitory activity was retained by an N-Me-ß-Ala(26) derivative (2), in which the ester of 1 was replaced with N-methyl amide to improve chemical stability at physiological pH. Inhibition was verified by fluorescence anisotropy, Western blot, and atomic force microscopy. This report suggests a new class of Aß aggregation inhibitor based on modification of Aß1-42 at Gly(25)-Ser(26).

Comparative properties of Aß1-42, Aß11-42, and [Pyr¹¹]Aß11-42 generated from O-acyl isopeptides.

The use of water-soluble O-acyl isopeptides enabled us to investigate the biochemical properties of Aß11-42 species, by preparing highly concentrated stock solutions after a pretreatment. Aß11-42 and [Pyr(11)]Aß11-42 showed comparable aggregation capability and cytotoxicity, suggesting that the pyroglutamate modification at Glu(11) does not have a crucial role in these events. However, given that Aß11-42 is converted to [Pyr(11)]Aß11-42 by a glutamyl cyclase in vivo, the potential aggregative and cytotoxic nature of [Pyr(11)]Aß11-42 that was observed in the present study provides valuable insights into the pathological functions of pyroglutamate-modified Aß species in Alzheimer's disease.

Isolation and identification of novel neutrophil-activating cryptides hidden in mitochondrial cytochrome C.

Although it is known that neutrophils infiltrate damaged sites immediately after tissue injury, the endogenous factors that induce their acute transmigration and activation have not been thoroughly investigated. For the candidates of those factors, we recently discovered two novel neutrophil-activating cryptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial proteins. In addition, many unknown neutrophil-activating peptides other than MCT-1 and MCT-2 were also observed during their purification. Here, we isolated and purified a novel neutrophil-activating peptide from porcine hearts, which we showed by structural analyses to have an identical primary structure to porcine mitochondrial cytochrome c (68-85). We named this novel functional octadecapeptide as mitocryptide-CYC (MCT-CYC). Structure-activity relationships of cytochrome c on ß-hexosaminidase (ß-HA) release from neutrophilic-differentiated HL- 60 cells demonstrated that peptides derived from the C-terminal part of cytochrome c induced ß-HA release and that cytochrome c (70-85) was the most potent cryptide among them. Since cytochrome c is known to be involved in the apoptotic process, our results suggest that cryptides, including MCT-CYC, derived from mitochondrial cytochrome c are possible factors that induce scavenging of toxic debris produced from apoptotic cells by neutrophils.

Self-assembly pathways of E22Δ-type amyloid ß peptide mutants generated from non-aggregative O-acyl isopeptide precursors.

The recently identified E22Δ-type amyloid ß peptide (Aß) mutants are reported to favor oligomerization over fibrillization and to exhibit more-potent synaptotoxicity than does wild-type (WT) Aß. Aß(E22Δ) mutants can thus be expected to serve as tools for clarifying the impact of Aß oligomers in Alzheimer's disease (or Alzheimer's-type dementia). However, the biochemical and biophysical properties of Aß(E22Δ) have not been conclusively determined. Here, we evaluated the self-assembly pathways of Aß(E22Δ) mutants generated from water-soluble, non-aggregative O-acyl isopeptide precursors. Circular dichroism spectroscopy, Western blot analysis, and thioflavin-T fluorescence intensity and cellular toxicity assays suggest that the self-assembly pathways of Aß(E22Δ) differed from those of Aß(WT). Aß1-40(E22Δ) underwent a rapid random coil→ß-sheet conformational change in its monomeric or low-molecular-weight oligomeric states, whereas Aß1-40(WT) self-assembled gradually without losing its propensity to form random coil structures. The Aß1-42(E22Δ) monomer formed ß-sheet-rich oligomers more rapidly than did Aß1-42(WT). Additionally, the Aß1-42(E22Δ) oligomers appear to differ from Aß1-42(WT) oligomers in size, shape, or both. These results should provide new insights into the functions of Aß(E22Δ) mutants.

'Click peptide' using production of monomer Aß from the O-acyl isopeptide: application to assay system of aggregation inhibitors and cellular cytotoxicity.

The O-acyl isopeptide of Aß1-42 (1), possessing an ester bond at the Gly(25)-Ser(26) sequence, is a water-soluble and non-aggregative precursor molecule and is capable of production of monomer Aß1-42. The SDS-PAGE result showed that the Aß1-42, produced from 1, adopted monomeric state at first and then self-assembled to oligomer. The oligomeric state was stabilized by nordihydroguaiaretic acid. The Thioflavin-T (ThT) fluorescence intensity derived from Aß1-42 (generated from 1) was suppressed by various aggregation inhibitors. Finally, 1 could generate Aß1-42 via the O-to-N acyl migration under cellular medium conditions and the produced Aß1-42 exhibited cytotoxicity against PC12 cells. These results suggest that the click peptide system, which enables us to predominantly produce monomer Aß1-42 under physiological conditions, would be adoptable to various biochemical and biophysical experiments including cellular system to investigate the functions of Aß1-42.

Mitocryptide-2, a neutrophil-activating cryptide, is a specific endogenous agonist for formyl-peptide receptor-like 1.

Peptides simultaneously produced during maturation and degradation of peptidergic hormones and functional proteins have recently become a great interest because they display unpredictably different biological roles than the parent proteins. Namely, we discovered two novel functional cryptic peptides, mitocryptide-1 (MCT-1) and mitocryptide-2 (MCT-2), hidden in mitochondrial cytochrome c oxidase and cytochrome b, that efficiently induced neutrophilic migration and activation at nanomolar concentrations. We named these functional "cryptic" peptides hidden in protein structures as "cryptides." In this study, we investigated the receptor molecules and cellular signaling mechanisms for neutrophil-activating N-formylated cryptide MCT-2. In order to identify the receptor molecules, we established HEK-293 cells stably expressing either formyl-peptide receptor (FPR) or its homologue FPR-like 1 (FPRL1), because neutrophilic cells express these receptor molecules which recognize N-formylated peptides. We observed that MCT-2 directly bound to FPRL1 and promoted an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), and neither interacted with nor activated FPR, demonstrating that MCT-2 is a specific agonist for FPRL1. Moreover, MCT-2 induced not only [Ca(2+)](i) increase and phosphorylation of extracellular signal-regulated protein kinases 1 and 2, but also ß-hexosaminidase release in neutrophilic/granulocytic cells differentiated from HL-60 cells. Such signaling events were diminished by pretreatment with pertussis toxin, indicating that MCT-2-promoted neutrophilic function is a consequence of G(i)- or G(o)-type G protein-dependent intracellular signaling events via FPRL1 activation. These findings suggest that MCT-2, a cryptide derived from mitochondrial cytochrome b, is a specific endogenous agonist for FPRL1 which is proposed to play key roles in inflammatory responses but whose physiological agonists are equivocal.

Mitocryptide-2: purification, identification, and characterization of a novel cryptide that activates neutrophils.

Neutrophils are a class of leukocytes involved in innate immunity by monitoring and scavenging invading microorganisms and toxic substances. The actions of neutrophils in damaged tissues are still not well understood, particularly in the early stage of inflammation, and as-yet-unknown neutrophil-activating substances are proposed to induce their acute transmigration and activation. Here, we isolated and identified from porcine hearts a neutrophil-activating peptide. Structural analyses indicated that the primary structure of this peptide is formyl-Met-Thr-Asn-Ile-Arg-Lys-Ser-His-Pro-Leu-Met-Lys-Ile-Ile-Asn, which is identical to that of the N-terminal pentadecapeptide of porcine mitochondrial cytochrome b; we therefore named the newly isolated peptide "mitocryptide-2" (MCT-2), since we have recently purified and identified mitocryptide-1, a different class of a neutrophil-activating peptide. Synthetic MCT-2 and its human homolog hMCT-2 induced beta-hexosaminidase release in and chemotaxis of HL-60 cells differentiated into neutrophilic/granulocytic cells. The induction of beta-hexosaminidase release, chemotaxis, and the increase in the intracellular free Ca(2+) concentration by hMCT-2 were completely suppressed by pertussis toxin, indicating the involvement of G(i)- or G(o)-type G proteins in the signaling pathways. Moreover, MCT-2 and hMCT-2 also stimulated beta-hexosaminidase secretion in human neutrophils isolated from peripheral blood in a concentration-dependent manner. Additionally, these peptides partially competed with [(3)H]formyl-Met-Leu-Phe binding to HL-60 cells differentiated into neutrophilic/granulocytic cells, presenting the possibility that the receptor for MCT-2 and hMCT-2 is one of the formyl peptide receptors. These results demonstrate that MCT-2 and its human homolog hMCT-2 are cryptides that activate neutrophils, thus suggesting the presence of regulatory mechanisms involving such mitocryptides in innate immunity.

"Click peptide": pH-triggered in situ production and aggregation of monomer Abeta1-42.

The intense and uncontrollable self-assembling nature of amyloid beta peptide (Abeta) 1-42 is known to cause difficulties in preparing monomeric Abeta1-42; this results in irreproducible or discrepant study outcomes. Herein, we report novel features of a pH click peptide of Abeta1-42 that was designed to overcome these problems. The click peptide is a water-soluble precursor peptide of Abeta1-42 with an O-acyl isopeptide structure between the Gly25-Ser26 sequence. The click peptide adopts and retains a monomeric, random coil state under acidic conditions. Upon change to neutral pH (pH click), the click peptide converts to Abeta1-42 promptly (t(1/2) approximately 10 s) and quantitatively through an O-to-N intramolecular acyl migration. As a result of this quick and irreversible conversion, monomer Abeta1-42 with a random coil structure is produced in situ. Moreover, the oligomerization, amyloid fibril formation and conformational changes of the produced Abeta1-42 can be observed over time. This click peptide strategy should provide a reliable experimental system to investigate the pathological role of Abeta1-42 in Alzheimer's disease.

Controlled production of amyloid beta peptide from a photo-triggered, water-soluble precursor "click peptide".

In biological experiments, poor solubility and uncontrolled assembly of amyloid beta peptide (Abeta) 1-42 pose significant obstacles to establish an experiment system that clarifies the function of Abeta1-42 in Alzheimer's disease (AD). Herein, as an experimental tool to overcome these problems, we developed a water-soluble photo-"click peptide" with a coumarin-derived photocleavable protective group that is based on an O-acyl isopeptide method. The click peptide had nearly 100-fold higher water solubility than Abeta1-42 and did not self-assemble, as the isomerized structure in its peptide backbone drastically changed the conformation that was derived from Abeta1-42. Moreover, the click peptide afforded Abeta1-42 quickly under physiological conditions (pH 7.4, 37 degrees C) by photoirradiation followed by an O-N intramolecular acyl migration. Because the in situ production of intact Abeta1-42 from the click peptide could improve the difficulties in handling Abeta1-42 caused by its poor solubility and highly aggregative nature, this click peptide strategy would provide a reliable experiment system for investigating the pathological function of Abeta1-42 in AD.

Arginine methylation of FOXO transcription factors inhibits their phosphorylation by Akt.

Forkhead box O (FOXO) transcription factors, the key regulators of cell survival, are negatively controlled through the PI3K-Akt signaling pathway. Phosphorylation of FOXO by Akt leads to cytoplasmic localization and subsequent degradation via the ubiquitin-proteasome system. Here we show a paradigm of FOXO1 regulation by the protein arginine methyltransferase PRMT1. PRMT1 methylated FOXO1 at conserved Arg248 and Arg250 within a consensus motif for Akt phosphorylation; this methylation directly blocked Akt-mediated phosphorylation of FOXO1 at Ser253 in vitro and in vivo. Silencing of PRMT1 by small interfering RNA enhanced nuclear exclusion, polyubiquitination, and proteasomal degradation of FOXO1. PRMT1 knockdown led to a decrease in oxidative-stress-induced apoptosis depending on the PI3K-Akt signaling pathway. Furthermore, stable expression of enzymatic inactive PRMT1 mutant increased resistance to apoptosis, whereas this effect was reversed by expression of phosphorylation-deficient FOXO1. Our findings predict a role for arginine methylation as an inhibitory modification against Akt-mediated phosphorylation.

Discovery of mitocryptide-1, a neutrophil-activating cryptide from healthy porcine heart.

Although neutrophils are known to migrate in response to various chemokines and complement factors, the substances involved in the early stages of their transmigration and activation have been poorly characterized to date. Here we report the discovery of a peptide isolated from healthy porcine hearts that activated neutrophils. Its primary structure is H-Leu-Ser-Phe-Leu-Ile-Pro-Ala-Gly-Trp-Val-Leu-Ser-His-Leu-Asp-His-Tyr-Lys-Arg-Ser-Ser-Ala-Ala-OH, and it was indicated to originate from mitochondrial cytochrome c oxidase subunit VIII. This peptide caused chemotaxis at concentrations lower than that inducing beta-hexosaminidase release. Such responses were observed in neutrophilic/granulocytic differentiated HL-60 cells but not in undifferentiated cells, and G(i2)-type G proteins were suggested to be involved in the peptide signaling. Moreover the peptide activated human neutrophils to induce beta-hexosaminidase secretion. A number of other amphipathic neutrophil-activating peptides presumably originating from mitochondrial proteins were also found. The present results suggest that neutrophils monitor such amphipathic peptides including the identified peptide as an initiation signal for inflammation at injury sites.

Cryptide signaling: Amphiphilic peptide-induced exocytotic mechanisms in mast cells.

Amphiphilic peptides with positive charges such as substance P (SP) and mastoparan (MP) are known to induce exocytosis in rat peritoneal mast cells. To elucidate whether and how intracellular Ca(2+) signaling is involved in the peptide-induced exocytosis, here we investigated the relationships between an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)) and exocytosis caused by SP and MP. SP and MP induced exocytosis coinciding with an initial rapid and transient [Ca(2+)](i) increase, but not with a sustained increase. These stimulations were abolished by pertussis toxin, indicating the involvement of the G(i)-family of G proteins in the peptide signaling. Moreover, the [Ca(2+)](i) increase was shown to accelerate and potentiate exocytosis, suggesting that the transient increase in [Ca(2+)](i) positively modified exocytotic secretion. However, it was indicated that the signal of [Ca(2+)](i) increase was not sufficient for the peptide-induced exocytosis, suggesting the participation of alternative mechanisms other than Ca(2+) signaling in the pathway.