Atp6v1c1 is an essential component of the osteoclast proton pump and in F-actin ring formation in osteoclasts

Bone resorption relies on the extracellular acidification function of V-ATPase (vacuolar-type proton-translocating ATPase) proton pump(s) present in the plasma membrane of osteoclasts. The exact configuration of the osteoclast-specific ruffled border V-ATPases remains largely unknown. In the present study, we found that the V-ATPase subunit Atp6v1c1 (C1) is highly expressed in osteoclasts, whereas subunits Atp6v1c2a (C2a) and Atp6v1c2b (C2b) are not. The expression level of C1 is highly induced by RANKL [receptor activator for NF-kappaB (nuclear factor kappaB) ligand] during osteoclast differentiation; C1 interacts with Atp6v0a3 (a3) and is mainly localized on the ruffled border of activated osteoclasts. The results of the present study show for the first time that C1-silencing by lentivirus-mediated RNA interference severely impaired osteoclast acidification activity and bone resorption, whereas cell differentiation did not appear to be affected, which is similar to a3 silencing. The F-actin (filamentous actin) ring formation was severely defected in C1-depleted osteoclasts but not in a3-depleted and a3(-/-) osteoclasts. C1 co-localized with microtubules in the plasma membrane and its vicinity in mature osteoclasts. In addition, C1 co-localized with F-actin in the cytoplasm; however, the co-localization chiefly shifted to the cell periphery of mature osteoclasts. The present study demonstrates that Atp6v1c1 is an essential component of the osteoclast proton pump at the osteoclast ruffled border and that it may regulate F-actin ring formation in osteoclast activation.     

Src kinase activity is essential for osteoclast function

Deletion of the c-src gene impairs osteoclast bone resorbing activity, causing osteopetrosis. Although it has been concluded that restoring only the Src adaptor function at least partly rescues the cell attachment and skeletal phenotypes, the contribution of Src kinase activity remains controversial. Src forms a complex with Pyk2 and Cbl after adhesion-induced stimulation of alpha(V)beta(3) integrin. To demonstrate the importance of the Pyk2-Src association in osteoclasts and to distinguish the contributions of the Src adaptor and kinase activities in cytoskeletal organization and osteoclast function, we expressed mutants of Src and Pyk2 in osteoclasts using adenovirus vectors. Eliminating the Src-binding site on Pyk2 (Pyk2(Y402F)) markedly inhibited bone resorption by osteoclast-like cells, whereas kinase-dead Pyk2 had little effect. Kinase-dead Src, unlike kinase-dead Pyk2, markedly inhibited the bone-resorbing activity of wild type osteoclasts and failed to significantly restore bone-resorbing activity to Src(-/-) osteoclast-like cells. Activation of Src kinase by overexpressing kinase-dead Csk failed to reverse the inhibitory effect of Pyk2(Y402F), suggesting that osteoclastic bone resorption requires both c-Src kinase activity and the targeting of Src kinase by Pyk2. Src-catalyzed phosphorylation of Cbl on Tyr-731 is reported to induce the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signaling pathway that is critical for osteoclast function. Expressing the Cbl(Y731F) mutant in osteoclasts markedly reduced their bone resorbing activity, suggesting that phosphorylation of Cbl(Y731) and the subsequent recruitment and activation of phosphatidylinositol 3-kinase may be critical signaling events downstream of Src in osteoclasts.     

CLIC-1 functions as a chloride channel when expressed and purified from bacteria

CLIC-1 is a member of a family of proteins related to the bovine intracellular chloride channel p64 which has been proposed to function as a chloride channel. We expressed CLIC-1 as a glutathione S-transferase fusion protein in bacteria. The fusion protein was purified by glutathione affinity, and CLIC-1 was released from its fusion partner by digestion with thrombin. After further purification, CLIC-1 was reconstituted into phospholipid vesicles by detergent dialysis. Chloride permeability of reconstituted vesicles was assessed using a valinomycin dependent chloride efflux assay, demonstrating increased vesicular chloride permeability with CLIC-1 compared with control. CLIC-1-dependent chloride permeability was inhibited by indanyloxyacetic acid-94 with an apparent IC(50) of 8.6 micrometer. The single channel properties of CLIC-1 were determined using the planar lipid bilayer technique. We found that CLIC-1 forms a voltage-dependent, Cl-selective channel with a rectifying current-voltage relationship and single channel conductances of 161 +/- 7.9 and 67.5 +/- 6.9 picosiemens in symmetric 300 and 150 mm KCl, respectively. The anion selectivity of this activity is Br approximately Cl > I. The open probability of CLIC-1 channels in planar bilayers was decreased by indanyloxyacetic acid-94 with an apparent IC(50) of 86 micrometer at 50 mV. These data convincingly demonstrate that CLIC-1 is capable of forming a novel, chloride-selective channel in the absence of other subunits or proteins.     

Osteopontin deficiency produces osteoclast dysfunction due to reduced CD44 surface expression

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.     

Inhibition of clathrin-coated vesicle acidification by duramycin

Clathrin-coated vesicles contain a proton translocating ATPase which operates in parallel with a chloride transporter (Xie, X.-S., Stone, D.K., and Racker, E. (1983) J. Biol. Chem. 258, 14834-14838). The polypeptide antibiotic, duramycin, has a dual inhibitory effect on clathrin-coated vesicle acidification. Low amounts of duramycin (5 micrograms/100 micrograms of protein) inhibit by 50% the proton translocation facilitated by chloride translocation. Under these conditions duramycin inhibits also 36Cl uptake when driven by either the electrogenic proton pump or by inward directed K+ movement in the presence of valinomycin. Higher amounts of duramycin (20 micrograms/100 micrograms of protein) are needed to inhibit by 50% the proton pump itself, as evidenced by reduced proton translocation facilitated by an outward potassium movement in the presence of valinomycin. In addition, the amount of duramycin needed to inhibit the proton pump corresponded well with the amount needed to inhibit the ouabain-insensitive, N-ethylmaleimide-sensitive ATPase activity of clathrin-coated vesicles.     

c-Src links a RANK/αvβ3 integrin complex to the osteoclast cytoskeleton

RANK ligand (RANKL), by mechanisms unknown, directly activates osteoclasts to resorb bone. Because c-Src is key to organizing the cell's cytoskeleton, we asked if the tyrosine kinase also mediates RANKL-stimulated osteoclast activity. RANKL induces c-Src to associate with RANK(369-373) in an αvβ3-dependent manner. Furthermore, RANK(369-373) is the only one of six putative TRAF binding motifs sufficient to generate actin rings and activate the same cytoskeleton-organizing proteins as the integrin. While c-Src organizes the cell's cytoskeleton in response to the cytokine, it does not participate in RANKL-stimulated osteoclast formation. Attesting to their collaboration, αvβ3 and activated RANK coprecipitate, but only in the presence of c-Src. c-Src binds activated RANK via its Src homology 2 (SH2) domain and αvβ3 via its SH3 domain, suggesting the kinase links the two receptors. Supporting this hypothesis, deletion or inactivating point mutation of either the c-Src SH2 or SH3 domain obviates the RANK/αvβ3 association. Thus, activated RANK prompts two distinct signaling pathways; one promotes osteoclast formation, and the other, in collaboration with c-Src-mediated linkage to αvβ3, organizes the cell's cytoskeleton.     

Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man

Chloride channels play important roles in the plasma membrane and in intracellular organelles. Mice deficient for the ubiquitously expressed ClC-7 Cl(-) channel show severe osteopetrosis and retinal degeneration. Although osteoclasts are present in normal numbers, they fail to resorb bone because they cannot acidify the extracellular resorption lacuna. ClC-7 resides in late endosomal and lysosomal compartments. In osteoclasts, it is highly expressed in the ruffled membrane, formed by the fusion of H(+)-ATPase-containing vesicles, that secretes protons into the lacuna. We also identified CLCN7 mutations in a patient with human infantile malignant osteopetrosis. We conclude that ClC-7 provides the chloride conductance required for an efficient proton pumping by the H(+)-ATPase of the osteoclast ruffled membrane.     

Cytoplasmic pH is regulated in isolated avian osteoclasts by a Cl-/HCO3- exchanger

Osteoclast resorb bone in an acid compartment formed by the bone-attachment site. The low pH of the resorption compartment provides a lysosome-like milieu suitable for acid proteases to degrade collagen. Solubilization of the hydroxyapatite that makes up bone mineral consumes about 2 moles of protons per moles of calcium dissolved, requiring a massive proton flux to maintain a low pH in the resorption compartment. In order to determine how the osteoclast maintains a physiological cytoplasmic pH while secreting massive amounts of acid, we studied the intracellular pH of osteoclasts using esterified fluorescein derivatives while controlling the electrolyte composition of the medium. The principal finding is that osteoclasts have a high capacity for chloride/bicarbonate exchange which enables them to maintain normal intracellular pH in the face of a large loading of base equivalents. Thus, the overall process of proton secretion during bone resorption is similar to the polarized acid elimination by renal epithelia, involving a proton pump on one surface of the cell, and a Cl-/HCO3- exchange to maintain cytoplasmic pH.     

Characterization of acid flux in osteoclasts from patients harboring a G215R mutation in ClC-7

The chloride-proton antiporter ClC-7 has been speculated to be involved in acidification of the lysosomes and the resorption lacunae in osteoclasts; however, neither direct measurements of chloride transport nor acidification have been performed.Human osteoclasts harboring a dominant negative mutation in ClC-7 (G215R) were isolated, and used these to investigate bone resorption measured by CTX-I, calcium release and pit scoring. The actin cytoskeleton of the osteoclasts was also investigated. ClC-7 enriched membranes from the osteoclasts were isolated, and used to test acidification rates in the presence of a V-ATPase and a chloride channel inhibitor, using a H⁺ and Cl^? driven approach. Finally, acidification rates in ClC-7 enriched membranes from ADOII osteoclasts and their corresponding controls were compared.Resorption by the G215R osteoclasts was reduced by 60% when measured by both CTX-I, calcium release, and pit area when comparing to age and sex matched controls. In addition, the ADOII osteoclasts showed no differences in actin ring formation. Finally, V-ATPase and chloride channel inhibitors completely abrogated the H⁺ and Cl^? driven acidification. Finally, the acid influx was reduced by maximally 50% in the ClC-7 deficient membrane fractions when comparing to controls.These data demonstrate that ClC-7 is essential for bone resorption, via its role in acidification of the lysosomes and resorption lacunae in osteoclasts.     

Downregulation of small GTPase Rab7 impairs osteoclast polarization and bone resorption

During skeletal growth and remodeling the mineralized bone matrix is resorbed by osteoclasts through the constant secretion of protons and proteases to the bone surface. This relies on the formation of specialized plasma membrane domains, the sealing zone and the ruffled border, and vectorial transportation of intracellular vesicles in bone-resorbing osteoclasts. Here we show that Rab7, a small GTPase that is associated with late endosomes, is highly expressed and is predominantly localized at the ruffled border in bone-resorbing osteoclasts. The decreased expression of Rab7 in cultured osteoclasts by antisense oligodeoxynucleotides disrupted the polarization of the osteoclasts and the targeting of vesicles to the ruffled border. These impairments caused a significant inhibition of bone resorption in vitro. The results indicate that the late endocytotic pathway is involved in the osteoclast polarization and bone resorption and underscore the importance of Rab7 in osteoclast function.     

Syk, c-Src, the alphavbeta3 integrin, and ITAM immunoreceptors, in concert, regulate osteoclastic bone resorption

In this study, we establish that the tyrosine kinase Syk is essential for osteoclast function in vitro and in vivo. Syk^−/− osteoclasts fail to organize their cytoskeleton, and, as such, their bone-resorptive capacity is arrested. This defect results in increased skeletal mass in Syk^−/− embryos and dampened basal and stimulated bone resorption in chimeric mice whose osteoclasts lack the kinase. The skeletal impact of Syk deficiency reflects diminished activity of the mature osteoclast and not impaired differentiation. Syk regulates bone resorption by its inclusion with the αvβ3 integrin and c-Src in a signaling complex, which is generated only when αvβ3 is activated. Upon integrin occupancy, c-Src phosphorylates Syk. αvβ3-induced phosphorylation of Syk and the latter's capacity to associate with c-Src is mediated by the immunoreceptor tyrosine-based activation motif (ITAM) proteins Dap12 and FcRγ. Thus, in conjunction with ITAM-bearing proteins, Syk, c-Src, and αvβ3 represent an essential signaling complex in the bone-resorbing osteoclast, and, therefore, each is a candidate therapeutic target.     

Structure and function of V-ATPases in osteoclasts: potential therapeutic targets for the treatment of osteolysis

Excessive activity of osteoclasts becomes manifest in many common lytic bone disorders such as osteoporosis, Paget's disease, bone aseptic loosening and tumor-induced bone destruction. Vacuolar proton pump H+-adenosine triphosphatases (V-ATPases), located on the bone-apposed plasma membrane of the osteoclast, are imperative for the function of osteoclasts, and thus are a potential molecular target for the development of novel anti-resorptive agents. To date, the V-ATPases core structure has been well modeled and consists of two distinct functional domains, the V1 (A, B1, B2, C1, C2, D, E1, E2, F, G1, G2, G3, and H subunits) and V0 (a1, a2, a3, a4, d1, d2, c, c' e1, e2 subunits) as well as the accessory subunits ac45 and M8-9. However, the exact configuration of osteoclast specific V-ATPases remains to be established. Inactivation of subunit a3 leads to osteopetrosis in both mice and man because of non-functional osteoclasts that are capable of acidifying the extracellular resorption lacuna. On the other hand, inactivation of subunits c, d1 and ac45 results in early embryonic lethality, indicating that certain subunits, such as a3, are more specific to osteoclast function than others. In osteoclasts, V-ATPases also cooperate with chloride channel protein CLC-7 to acidify the resorption lacuna. In addition, development of V-ATPases inhibitors such as bafilomycin A1, SB 242784 and FR167356 that selectively target osteoclast specific V-ATPases remains a challenge. Understanding the molecular and cellular mechanisms by which specific subunits of V-ATPase regulate osteoclast function might facilitate the development of novel and selective inhibitors for the treatment of lytic bone disorders. This review summarizes recent research developments in V-ATPases with particular emphasis on osteoclast biology.     

Isolation and characterization of a cDNA clone encoding a novel peptide (OSF) that enhances osteoclast formation and bone resorption

Using an expression cloning approach, we identified and cloned a novel intracellular protein produced by osteoclasts that indirectly induces osteoclast formation and bone resorption, termed OSF. Conditioned media from 293 cells transiently transfected with the 0.9 kb OSF cDNA clone stimulated osteoclast-like cell formation in both human and murine marrow cultures in the presence or absence 10(-9) M 1,25-dihydroxyvitamin D3. In addition, conditioned media from 293 cells transfected with the OSF cDNA clone enhanced the stimulatory effects of 1,25-(OH)2D3 on bone resorption in the fetal rat long bone assay. In situ hybridization studies using antisense oligomers showed expression of OSF mRNA in highly purified osteoclast-like cells from human giant cell tumors of the bone. Northern blot analysis demonstrated ubiquitous expression of a 1.3 kb mRNA that encodes OSF in multiple human tissues. Sequence analysis showed the OSF cDNA encoded a 28 kD peptide that contains a c-Src homology 3 domain (SH3) and ankyrin repeats, suggesting that it was not a secreted protein, but that it was potentially involved in cell signaling. Consistent with these data, immunoblot analysis using rabbit antisera against recombinant OSF demonstrated OSF expression in cell lysates but not in the culture media. Furthermore, recombinant OSF had a high affinity for c-Src, an important regulator of osteoclast activity. Taken together, these data suggest that OSF is a novel intracellular protein that indirectly enhances osteoclast formation and osteoclastic bone resorption through the cellular signal transduction cascade, possibly through its interactions with c-Src or other Src-related proteins.     

Proteomic profile of osteoclast membrane proteins: identification of Na+/H+ exchanger domain containing 2 and its role in osteoclast fusion

Osteoclast formation and bone resorption are multiple processes that involve the participation of specialized membrane structures and their associated proteins. In this study, we used an MS to analyze the profile of proteins associated with osteoclast membranes and focused on the function of channel proteins in osteoclast differentiation and function. We filtered out with a SEQUEST score greater than 10 and a peptide hit number of more than 2, resulting in the identification of 499 proteins that were commonly found in both macrophages and osteoclasts, 96 proteins selectively found in osteoclasts, and 179 proteins selectively found in macrophages. The proteins that were selectively found in osteoclasts were classified based on their localizations: plasma membrane (17%), ER/Golgi and lysosome/endosome (15%), mitochondrion (18%), nucleus (13%), cytosol (19%), and unknown (18%). Proteins associated with osteoclast function such as v-ATPase, IGF2R, TRAP, and cathepsin K were found in osteoclasts as previously shown. We found several ion channel proteins such as Ank and Nhedc2 and signaling molecules such as Dock5 and RAB-10 in osteoclasts. Inhibition of the Na(+)/H(+) exchanger family by amiloride suppressed RANKL-induced osteoclast fusion and bone resorption. In addition, shRNA for Nhedc2 inhibited osteoclast differentiation. Our results provide a proteomic profile of osteoclast membrane proteins and identify Nhedc2, which is probably associated with proton transport in osteoclasts, as a regulator of osteoclast function.     

Bone resorption and bone remodelling in juvenile carp, Cyprinus carpio L.

The present study considers the important role of bone resorption for bone growth in general, and aims to clarify if and how bone resorption contributes to the skeletal development of carp, Cyprinus carpio L., a teleost species with ‘normal’ osteocyte‐containing (cellular) bone. To ensure the identification of osteoclasts and sites of bone resorption independently from the morphology of the bony cells, bones were studied by histological procedures, and by demonstration of the enzymes which serve as osteoclast markers, viz. tartrate resistant acid phosphatase (TRAP), ATPase and a vacuolar proton pump. Two types of bone‐resorbing cells were observed in juvenile carp: (1) multinucleated giant cells displaying morphological and biochemical attributes which are known from mammalian osteoclasts; and (b) flat cells which lack a visible ruffled border and for which identification requires the performance of enzyme histochemical procedures. Bone resorption performed by osteoclasts mainly occurs at endosteal bone surfaces. To a lesser extent, bone resorption also takes place at periosteal bone surfaces, but without an apparent connection to bone growth. The latter observation, and the occurrence of bone remodelling, suggest that the endoskeleton of juvenile carp might be involved in mineral metabolism. Morphological differences and biochemical similarities to bone resorption in teleosts with acellular bone are discussed.     

TRAFD1 (FLN29) Interacts with Plekhm1 and Regulates Osteoclast Acidification and Resorption

Plekhm1 is a large, multi-modular, adapter protein implicated in osteoclast vesicle trafficking and bone resorption. In patients, inactivating mutations cause osteopetrosis, and gain-of-function mutations cause osteopenia. Investigations of potential Plekhm1 interaction partners by mass spectrometry identified TRAFD1 (FLN29), a protein previously shown to suppress toll-like receptor signaling in monocytes/macrophages, thereby dampening inflammatory responses to innate immunity. We mapped the binding domains to the TRAFD1 zinc finger (aa 37-60), and to the region of Plekhm1 between its second pleckstrin homology domain and its C1 domain (aa 784-986). RANKL slightly increased TRAFD1 levels, particularly in primary osteoclasts, and the co-localization of TRAFD1 with Plekhm1 also increased with RANKL treatment. Stable knockdown of TRAFD1 in RAW 264.7 cells inhibited resorption activity proportionally to the degree of knockdown, and inhibited acidification. The lack of acidification occurred despite the presence of osteoclast acidification factors including carbonic anhydrase II, a3-V-ATPase, and the ClC7 chloride channel. Secretion of TRAP and cathepsin K were also markedly inhibited in knockdown cells. Truncated Plekhm1 in ia/ia osteopetrotic rat cells prevented vesicle localization of Plekhm1 and TRAFD1. We conclude that TRAFD1, in association with Plekhm1/Rab7-positive late endosomes-early lysosomes, has a previously unknown role in vesicle trafficking, acidification, and resorption in osteoclasts.     

Osteoclasts secrete non-bone derived signals that induce bone formation

Bone turnover is a highly regulated process, where bone resorption in the normal healthy individual always is followed by bone formation in a manner referred to as coupling. Patients with osteopetrosis caused by defective acidification of the resorption lacuna have severely decreased resorption, in face of normal or even increased bone formation. This suggests that osteoclasts, not their resorptive activity, are important for sustaining bone formation. To investigate whether osteoclasts mediate control of bone formation by production of bone anabolic signals, we collected conditioned media (CM) from human osteoclasts cultured on either bone or plastic, and tested their effects on bone nodule formation by osteoblasts. Both types of CM were shown to dose-dependently induce bone nodule formation, whereas non-conditioned osteoclast culture medium had no effects. These data show that osteoclasts secrete non-bone derived factors, which induce preosteoblasts to form bone-like nodules, potentially explaining the imbalanced coupling seen in osteopetrotic patients.     

Phosphatidylinositol 3,4,5-trisphosphate directs association of Src homology 2-containing signaling proteins with gelsolin

Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130(Cas), focal adhesion kinase, integrin alpha(v)beta(3), vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, c-Src, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of alpha(v)beta(3)-regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.     

Synapsin phosphorylation by SRC tyrosine kinase enhances SRC activity in synaptic vesicles

Synapsins are synaptic vesicle-associated phosphoproteins implicated in the regulation of neurotransmitter release. Synapsin I is the major binding protein for the SH3 domain of the kinase c-Src in synaptic vesicles. Its binding leads to stimulation of synaptic vesicle-associated c-Src activity. We investigated the mechanism and role of Src activation by synapsins on synaptic vesicles. We found that synapsin is tyrosine phosphorylated by c-Src in vitro and on intact synaptic vesicles independently of its phosphorylation state on serine. Mass spectrometry revealed a single major phosphorylation site at Tyr(301), which is highly conserved in all synapsin isoforms and orthologues. Synapsin tyrosine phosphorylation triggered its binding to the SH2 domains of Src or Fyn. However, synapsin selectively activated and was phosphorylated by Src, consistent with the specific enrichment of c-Src in synaptic vesicles over Fyn or n-Src. The activity of Src on synaptic vesicles was controlled by the amount of vesicle-associated synapsin, which is in turn dependent on synapsin serine phosphorylation. Synaptic vesicles depleted of synapsin in vitro or derived from synapsin null mice exhibited greatly reduced Src activity and tyrosine phosphorylation of other synaptic vesicle proteins. Disruption of the Src-synapsin interaction by internalization of either the Src SH3 or SH2 domains into synaptosomes decreased synapsin tyrosine phosphorylation and concomitantly increased neurotransmitter release in response to Ca(2+)-ionophores. We conclude that synapsin is an endogenous substrate and activator of synaptic vesicle-associated c-Src and that regulation of Src activity on synaptic vesicles participates in the regulation of neurotransmitter release by synapsin.