The zinc- and calcium-binding S100B interacts and co-localizes with IQGAP1 during dynamic rearrangement of cell membranes

The Zn(2+)- and Ca(2+)-binding S100B protein is implicated in multiple intracellular and extracellular regulatory events. In glial cells, a relationship exists between cytoplasmic S100B accumulation and cell morphological changes. We have identified the IQGAP1 protein as the major cytoplasmic S100B target protein in different rat and human glial cell lines in the presence of Zn(2+) and Ca(2+). Zn(2+) binding to S100B is sufficient to promote interaction with IQGAP1. IQ motifs on IQGAP1 represent the minimal interaction sites for S100B. We also provide evidence that, in human astrocytoma cell lines, S100B co-localizes with IQGAP1 at the polarized leading edge and areas of membrane ruffling and that both proteins relocate in a Ca(2+)-dependent manner within newly formed vesicle-like structures. Our data identify IQGAP1 as a potential target protein of S100B during processes of dynamic rearrangement of cell membrane morphology. They also reveal an additional cellular function for IQGAP1 associated with Zn(2+)/Ca(2+)-dependent relocation of S100B.     

Mapping the zinc ligands of S100A2 by site-directed mutagenesis

S100 family proteins are characterized by short individual N and C termini and a conserved central part, harboring two Ca(2+)-binding EF-hands, one of them highly conserved among EF-hand family proteins and the other characteristic for S100 proteins. In addition to Ca(2+), several members of the S100 protein family, including S100A2, bind Zn(2+). Two regions in the amino acid sequences of S100 proteins, namely the helices of the N-terminal EF-hand motif and the very C-terminal loop are believed to be involved in Zn(2+)-binding due to the presence of histidine and/or cysteine residues. Human S100A2 contains four cysteine residues, each of them located at positions that may be important for Zn(2+) binding. We have now constructed and purified 10 cysteine-deficient mutants of human S100A2 by site-directed mutagenesis and investigated the contribution of the individual cysteine residues to Zn(2+) binding. Here we show that Cys(1(3)) (the number in parentheses indicating the position in the sequence of S100A2) is the crucial determinant for Zn(2+) binding in association with conformational changes as determined by internal tyrosine fluorescence. Solid phase Zn(2+) binding assays also revealed that the C-terminal residues Cys(3(87)) and Cys(4(94)) mediated a second type of Zn(2+) binding, not associated with detectable conformational changes in the molecule. Cys(2(22)), by contrast, which is located within the first EF hand motif affected neither Ca(2+) nor Zn(2+) binding, and a Cys "null" mutant was entirely incapable of ligating Zn(2+). These results provide new information about the mechanism and the site(s) of zinc binding in S100A2.     

The effects of Ca2+ binding on the dynamic properties of a designed Ca2+-binding protein

The effects of Ca(2+) binding on the dynamic properties of Ca(2+)-binding proteins are important in Ca(2+) signaling. To understand the role of Ca(2+) binding, we have successfully designed a Ca(2+)-binding site in the domain 1 of rat CD2 (denoted as Ca.CD2) with the desired structure and retained function. In this study, the backbone dynamic properties of Ca.CD2 have been investigated using (15)N spin relaxation NMR spectroscopy to reveal the effect of Ca(2+) binding on the global and local dynamic properties without the complications of multiple interactive Ca(2+) binding and global conformational change. Like rat CD2 (rCD2) and human CD2 (hCD2), residues involved in the recognition of the target molecule CD48 exhibit high flexibility. Mutations N15D and N17D that introduce the Ca(2+) ligands increase the flexibility of the neighboring residues. Ca(2+)-induced local dynamic changes occur mainly at the residues proximate to the Ca(2+)-binding pocket or the residues in loop regions. The beta-strand B of Ca.CD2 that provides two Asp for the Ca(2+) undergoes an S(2) decrease upon the Ca(2+) binding, while the DE-loop that provides one Asn and one Asp undergoes an S(2) increase. Our study suggests that Ca(2+) binding has a differential effect on the rigidity of the residues depending on their flexibility and location within the secondary structure.     

Location of the Zn(2+)-binding site on S100B as determined by NMR spectroscopy and site-directed mutagenesis

In addition to binding Ca(2+), the S100 protein S100B binds Zn(2+) with relatively high affinity as confirmed using isothermal titration calorimetry (ITC; K(d) = 94 +/- 17 nM). The Zn(2+)-binding site on Ca(2+)-bound S100B was examined further using NMR spectroscopy and site-directed mutagenesis. Specifically, ITC measurements of S100B mutants (helix 1, H15A and H25A; helix 4, C84A, H85A, and H90A) were found to bind Zn(2+) with lower affinity than wild-type S100B (from 2- to >25-fold). Thus, His-15, His-25, Cys-84, His-85, and perhaps His-90 of S100B are involved in coordinating Zn(2+), which was confirmed by NMR spectroscopy. Previous studies indicate that the binding of Zn(2+) enhances calcium and target protein-binding affinities, which may contribute to its biological function. Thus, chemical shift perturbations observed here for residues in both EF-hand domains of S100B during Zn(2+) titrations could be detecting structural changes in the Ca(2+)-binding domains of S100B that are pertinent to its increase in Ca(2+)-binding affinity in the presence of Zn(2+). Furthermore, Zn(2+) binding causes helix 4 to extend by one full turn when compared to Ca(2+)-bound S100B. This change in secondary structure likely contributes to the increased binding affinity that S100B has for target peptides (i.e., TRTK peptide) in the presence of Zn(2+).     

Ca2+ -dependent interaction of S100A1 with the sarcoplasmic reticulum Ca2+ -ATPase2a and phospholamban in the human heart

The Ca(2+)-binding S100A1 protein displays a specific and high expression level in the human myocardium and is considered to be an important regulator of heart contractility. Diminished protein levels detected in dilated cardiomyopathy possibly contribute to impaired Ca(2+) handling and contractility in heart failure. To elucidate the S100A1 signaling pathway in the human heart, we searched for S100A1 target proteins by applying S100A1-specific affinity chromatography and immunoprecipitation techniques. We detected the formation of a Ca(2+)-dependent complex of S100A1 with SERCA2a and PLB in the human myocardium. Using confocal laser scanning microscopy, we showed that all three proteins co-localize at the level of the SR in primary mouse cardiomyocytes and confirmed these results by immunoelectron microscopy in human biopsies. Our results support a regulatory role of S100A1 in the contraction-relaxation cycle in the human heart.     

Protein kinase A- and C-induced insulin release from Ca2+ -insensitive pools

Insulin secretion is known to depend on an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)). However, recent studies have suggested that insulin secretion can also be evoked in a Ca(2+)-independent manner. In the present study we show that treatment of intact mouse islets and RINm5F cells with protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or protein kinase A (PKA) activator forskolin promoted insulin secretion with no changes of [Ca(2+)](i). Moreover, insulin secretion mediated by PMA or forskolin was maintained even when extracellular or cytosolic Ca(2+) was deprived by treatment of cells with ethylene glycol bis(beta-amino ethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-amino phenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxy methyl ester) (BAPTA/AM) in RINm5F cells. The secretagogue actions of PMA and forskolin were blocked by GF109203X and H89, selective inhibitors for PKC and PKA, respectively. PMA treatment caused translocation of PKC-alpha and PKC- epsilon from cytosol to membrane, implying that selectively PKC-alpha and PKC- epsilon isoforms might be important for insulin secretion. Co-treatment with high K(+) and PMA showed a comparable level of insulin secretion to that of PMA alone. In addition, PMA and forskolin evoked insulin secretion in cells where Ca(2+)-dependent insulin secretion was completed. Our data suggest that PKC and PKA can elicit insulin secretion not only in a Ca(2+)-sensitive manner but also in a Ca(2+)-independent manner from separate releasable pools.     

CIB1, a ubiquitously expressed Ca2+-binding protein ligand of the InsP3 receptor Ca2+ release channel

A family of Ca(2+)-binding proteins (CaBPs) was shown to bind to the inositol 1,4,5-trisphosphate receptor (InsP(3)R) Ca(2+) release channel and gate it in the absence of InsP(3), establishing them as protein ligands (Yang, J., McBride, S., Mak, D.-O. D., Vardi, N., Palczewski, K., Haeseleer, F., and Foskett, J. K. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7711-7716). However, the neuronally restricted expression of CaBP and its inhibition of InsP(3)R-mediated Ca(2+) signaling when overexpressed (Kasri, N. N., Holmes, A. M., Bultynck, G., Parys, J. B., Bootman, M. D., Rietdorf, K., Missiaen, L., McDonald, F., De Smedt, H., Conway, S. J., Holmes, A. B., Berridge, M. J., and Roderick, H. L. (2004) EMBO J. 23, 312-321; Haynes, L. P., Tepikin, A. V., and Burgoyne, R. D. (2004) J. Biol. Chem. 279, 547-555) have raised questions regarding the functional implications of this regulation. We have discovered the Ca(2+)-binding protein CIB1 (calmyrin) as a ubiquitously expressed ligand of the InsP(3)R. CIB1 binds to all mammalian InsP(3)R isoforms in a Ca(2+)-sensitive manner dependent on its two functional EF-hands and activates InsP(3)R channel gating in the absence of InsP(3). In contrast, overexpression of CIB1 or CaBP1 attenuated InsP(3)R-dependent Ca(2+) signaling, and in vitro pre-exposure to CIB1 reduced the number of channels available for subsequent stimulation by InsP(3). These results establish CIB1 as a ubiquitously expressed activating and inhibiting protein ligand of the InsP(3)R.     

Calcium-dependent translocation of S100A11 requires tubulin filaments

Protein translocation between different subcellular compartments might play a significant role in various signal transduction pathways. The S100 family is comprised of the multifunctional, small, acidic proteins, some of which translocate in the form of vesicle-like structures upon increase in intracellular Ca(2+) levels. Previously, cells were fixed before and after calcium activation in order to examine the possible relocation of S100 proteins. In this study, we were able to track the real-time translocation. We compared the localization of endogenous S100A11 to that of the S100A11-green fluorescent protein. The application of thapsigargin, an agent increasing intracellular Ca(2+) levels, resulted in the relocation of the S100A11. In contrast, addition of EGTA, which specifically binds Ca(2+), either inhibited the ongoing process of translocation or prevented its induction. Since translocation was not affected by treatment with brefeldin A, it appears that S100A11 relocates in an endoplasmic reticulum-Golgi-independent pathway. Furthermore, the depolymerization of actin filaments by amlexanox did not affect the capacity of S100A11 to translocate. However, the time course treatment with demecolcine, which depolymerizes tubulin filaments, resulted in cease of translocation, suggesting that the tubulin network is required for this process.     

STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx

Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.     

The giant protein AHNAK is a specific target for the calcium- and zinc-binding S100B protein: potential implications for Ca2+ homeostasis regulation by S100B

Transformation of rat embryo fibroblast clone 6 cells by ras and temperature-sensitive p53val(135) is reverted by ectopic expression of the calcium- and zinc-binding protein S100B. In an attempt to define the molecular basis of the S100B action, we have identified the giant phosphoprotein AHNAK as the major and most specific Ca(2+)-dependent S100B target protein in rat embryo fibroblast cells. We next characterized AHNAK as a major Ca(2+)-dependent S100B target protein in the rat glial C6 and human U-87MG astrocytoma cell lines. AHNAK binds to S100B-Sepharose beads and is also recovered in anti-S100B immunoprecipitates in a strict Ca(2+)- and Zn(2+)-dependent manner. Using truncated AHNAK fragments, we demonstrated that the domains of AHNAK responsible for interaction with S100B correspond to repeated motifs that characterize the AHNAK molecule. These motifs show no binding to calmodulin or to S100A6 and S100A11. We also provide evidence that the binding of 2 Zn(2+) equivalents/mol S100B enhances Ca(2+)-dependent S100B-AHNAK interaction and that the effect of Zn(2+) relies on Zn(2+)-dependent regulation of S100B affinity for Ca(2+). Taking into consideration that AHNAK is a protein implicated in calcium flux regulation, we propose that the S100B-AHNAK interaction may participate in the S100B-mediated regulation of cellular Ca(2+) homeostasis.     

Spatiotemporal resolution of Ca2+ signaling events by real time imaging of single B cells

Antigen-induced B cell activation requires mobilization of the Ca(2+) second messenger. This process is associated with the subcellular relocalization of signal effector proteins of the B cell antigen receptor such as the adaptor protein SLP65. Here we describe a broadly applicable live cell imaging method to simultaneously visualize intracellular Ca(2+) flux profiles and the translocation of cytosolic signaling proteins to the plasma membrane in real time. Our approach delineated the kinetic hierarchy of Ca(2+) signaling events in B cells and revealed a timely ordered contribution of various organelles to the overall Ca(2+) signal. The developed experimental setup provides a useful tool to resolve the spatiotemporal signaling dynamics in various receptor signaling systems.     

A novel S100 target conformation is revealed by the solution structure of the Ca2+-S100B-TRTK-12 complex

The Alzheimer-linked neural protein S100B is a signaling molecule shown to control the assembly of intermediate filament proteins in a calcium-sensitive manner. Upon binding calcium, a conformational change occurs in S100B exposing a hydrophobic surface for target protein interactions. The synthetic peptide TRTK-12 (TRTKIDWNKILS), derived from random bacteriophage library screening, bears sequence similarity to several intermediate filament proteins and has the highest calcium-dependent affinity of any target molecule for S100B to date (K(d) <1 microm). In this work, the three-dimensional structure of the Ca(2+)-S100B-TRTK-12 complex has been determined by NMR spectroscopy. The structure reveals an extended, contiguous hydrophobic surface is formed on Ca(2+)-S100B for target interaction. The TRTK-12 peptide adopts a coiled structure that fits into a portion of this surface, anchored at Trp(7), and interacts with multiple hydrophobic contacts in helices III and IV of Ca(2+)-S100B. This interaction is strikingly different from the alpha-helical structures found for other S100 target peptides. By using the TRTK-12 interaction as a guide, in combination with other available S100 target structures, a recognition site on helix I is identified that may act in concert with the TRTK-12-binding site from helices III and IV. This would provide a larger, more complex site to interact with full-length target proteins and would account for the promiscuity observed for S100B target protein interactions.     

A structural basis for S100 protein specificity derived from comparative analysis of apo and Ca(2+)-calcyclin

Calcyclin is a homodimeric protein belonging to the S100 subfamily of EF-hand Ca(2+)-binding proteins, which function in Ca(2+) signal transduction processes. A refined high-resolution solution structure of Ca(2+)-bound rabbit calcyclin has been determined by heteronuclear solution NMR. In order to understand the Ca(2+)-induced structural changes in S100 proteins, in-depth comparative structural analyses were used to compare the apo and Ca(2+)-bound states of calcyclin, the closely related S100B, and the prototypical Ca(2+)-sensor protein calmodulin. Upon Ca(2+) binding, the position and orientation of helix III in the second EF-hand is altered, whereas the rest of the protein, including the dimer interface, remains virtually unchanged. This Ca(2+)-induced structural change is much less drastic than the "opening" of the globular EF-hand domains that occurs in classical Ca(2+) sensors, such as calmodulin. Using homology models of calcyclin based on S100B, a binding site in calcyclin has been proposed for the N-terminal domain of annexin XI and the C-terminal domain of the neuronal calcyclin-binding protein. The structural basis for the specificity of S100 proteins is discussed in terms of the variation in sequence of critical contact residues in the common S100 target-binding site.     

Detection of Ca(2+)-binding proteins by electrophoretic migration in the presence of Ca2+ combined with 45Ca2+ overlay of protein blots.

When high affinity Ca(2+)-binding proteins like calmodulin, or proteins with a high Ca(2+)-binding capacity like calsequestrin, underwent sodium dodecyl sulfate-gel electrophoresis in Laemmli systems, their electrophoretic migration rates were much higher in gels containing 1 mM Ca2+ than in gels containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Replacement of EGTA by Ca2+ in the gel, combined with the blotting of electrophoretically separated proteins on polyvinylidene difluoride membranes and subsequent 45Ca2+ overlay, proved a very effective means of detecting Ca(2+)-binding proteins. This combined approach is important since artifacts occur in both techniques when used separately. We found that the usual procedure of adding Ca2+ to the sample before electrophoresis without including it in the gel itself (C.B. Klee, T. H. Crouch, and M. H. Krinks, 1979, Proc. Natl. Acad. Sci. USA 76, 6270-6273) permitted the detection of only very high affinity Ca(2+)-binding proteins.     

Responses of Ca2+-binding proteins to localized,transient changes in intracellular [Ca2+

In smooth muscle cells, various transient, localized [Ca(2+)] changes have been observed that are thought to regulate cell function without necessarily inducing contraction. Although a great deal of effort has been put into detecting these transients and elucidating the mechanisms involved in their generation, the extent to which these transient Ca(2+) signals interact with intracellular Ca(2+)-binding molecules remains relatively unknown. To understand how the spatial and temporal characteristics of an intracellular Ca(2+) signal influence its interaction with Ca(2+)-binding proteins, mathematical models of Ca(2+) diffusion and regulation in smooth muscle cells were used to study Ca(2+) binding to prototypical proteins with one or two Ca(2+)-binding sites. Simulations with the models: (1) demonstrate the extent to which the rate constants for Ca(2+)-binding to proteins and the spatial and temporal characteristics of different Ca(2+) transients influence the magnitude and time course of the responses of these proteins to the transients; (2) predict significant differences in the responses of proteins with one or two Ca(2+)-binding sites to individual Ca(2+) transients and to trains of transients; (3) demonstrate how the kinetic characteristics determine the fidelity with which the responses of Ca(2+)-sensitive molecules reflect the magnitude and time course of transient Ca(2+) signals. Overall, this work demonstrates the clear need for complete information about the kinetics of Ca(2+) binding for determining how well Ca(2+)-binding molecules respond to different types of Ca(2+) signals. These results have important implications when considering the possible modulation of Ca(2+)- and Ca(2+)/calmodulin-dependent proteins by localized intracellular Ca(2+) transients in smooth muscle cells and, more generally, in other cell types.     

S100P is a novel interaction partner and regulator of IQGAP1

Ca(2+)-binding proteins of the S100 family participate in intracellular Ca(2+) signaling by binding to and regulating specific cellular targets in their Ca(2+)-loaded conformation. Because the information on specific cellular targets of different S100 proteins is still limited, we developed an affinity approach that selects for protein targets only binding to the physiologically active dimer of an S100 protein. Using this approach, we here identify IQGAP1 as a novel and dimer-specific target of S100P, a member of the S100 family enriched in the cortical cytoskeleton. The interaction between S100P and IQGAP1 is strictly Ca(2+)-dependent and characterized by a dissociation constant of 0.2 μM. Binding occurs primarily through the IQ domain of IQGAP1 and the first EF hand loop of S100P, thus representing a novel structural principle of S100-target protein interactions. Upon cell stimulation, S100P and IQGAP1 co-localize at or in close proximity to the plasma membrane, and complex formation can be linked to altered signal transduction properties of IQGAP1. Specifically, the EGF-induced tyrosine phosphorylation of IQGAP1 that is thought to function in assembling signaling intermediates at IQGAP1 scaffolds in the subplasmalemmal region is markedly reduced in cells overexpressing S100P but not in cells expressing an S100P mutant deficient in IQGAP1 binding. Furthermore, B-Raf binding to IQGAP1 and MEK1/2 activation occurring downstream of IQGAP1 in EGF-triggered signaling cascades are compromised at elevated S100P levels. Thus, S100P is a novel Ca(2+)-dependent regulator of IQGAP1 that can down-regulate the function of IQGAP1 as a signaling intermediate by direct interaction.     

RANK ligand-induced elevation of cytosolic Ca2+ accelerates nuclear translocation of nuclear factor kappa B in osteoclasts

RANK ligand (RANKL) induces activation of NFkappaB, enhancing the formation, resorptive activity, and survival of osteoclasts. Ca(2+) transduces many signaling events, however, it is not known whether the actions of RANKL involve Ca(2+) signaling. We investigated the effects of RANKL on rat osteoclasts using microspectrofluorimetry and patch clamp. RANKL induced transient elevation of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) to maxima 220 nm above basal, resulting in activation of Ca(2+)-dependent K(+) current. RANKL elevated [Ca(2+)](i) in Ca(2+)-containing and Ca(2+)-free media, and responses were prevented by the phospholipase C inhibitor. Suppression of [Ca(2+)](i) elevation using the intracellular Ca(2+) chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) abolished the ability of RANKL to enhance osteoclast survival. Using immunofluorescence, NFkappaB was found predominantly in the cytosol of untreated osteoclasts. RANKL induced transient translocation of NFkappaB to the nuclei, which was maximal at 15 min. or BAPTA delayed nuclear translocation of NFkappaB. Delays were also observed upon inhibition of calcineurin or protein kinase C. We conclude that RANKL acts through phospholipase C to release Ca(2+) from intracellular stores, accelerating nuclear translocation of NFkappaB and promoting osteoclast survival. Such cross-talk between NFkappaB and Ca(2+) signaling provides a novel mechanism for the temporal regulation of gene expression in osteoclasts and other cell types.     

New perspectives on S100 proteins: a multi-functional Ca 2+ -, Zn 2+ - and Cu 2+ -binding protein family

S100 proteins (16 members) show a very divergent pattern of cell- and tissue-specific expression, of subcel-lular localizations and relocations, of post-translational modifications, and of affinities for Ca 2+ , Zn 2+ , and Cu 2+ , consistent with their pleiotropic intra- and extracellular functions. Up to 40 target proteins are reported to interact with S100 proteins and for S100A1 alone 15 target proteins are presently known. Therefore it is not surprising that many functional roles have been proposed and that several human disorders such as cancer, neurodegenerative diseases, cardiomyopathies, inflammations, diabetes, and allergies are associated with an altered expression of S100 proteins. It is not unlikely that their biological activity in some cases is regulated by Zn 2+ and Cu 2+ , rather than by Ca 2+ Despite the numerous putative functions of S100 proteins, their three-dimensional structures of, e.g., S100B, S100A6, and S100A7 are surprisingly similar. They contain a compact dimerization domain whose conformation is rather insensitive to Ca 2+ binding and two lateral a-helices III and III, which project outward of each subunit when Ca 2+ is bound. Target docking depends on the two hydrophobic patches in front of the paired EF-hand generated by the binding of Ca 2+. The selec-tivity in target binding is assured by the central linker between the two EF-hands and the C-terminal tail. It appears that the S100-binding domain in some target proteins contains a basic amphiphilic a-helix and that the mode of interaction and activation bears structural similarity to that of calmodulin.© Kluwer Academic Publishers     

Ca2+-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca(2+) channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca(2+)-binding proteins are of particular importance as sensors of presynaptic Ca(2+), and a multiple of them are indeed utilized in the signaling of Ca(2+) channels. However, despite its conserved structure, CaM is the only known EF-hand Ca(2+)-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca(2+) channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca(2+)-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca(2+)-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca(2+), PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca(2+) channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.