Dvl regulates endo- and exocytotic processes through binding to synaptotagmin

© 2006 The Authors

Genes to Cells (2007)

12

, 49–61

Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

49

DOI: 10.1111/j.1365-2443.2006.001030.x

Blackwell Publishing IncMalden, USAGTCGenes to Cells1356-9597© The Author. Journal compilation © by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.XXXXOriginal ArticleDvl and SynaptotagminS Kishida et al.

Dvl regulates endo- and exocytotic processes through binding to synaptotagmin

Shosei Kishida

1,†

, Kozue Hamao

1,†

, Makoto Inoue

2

, Mamoru Hasegawa

2

, Yoshiharu Matsuura

3

, Katsuhiko Mikoshiba

4

, Mitsunori Fukuda

5

and Akira Kikuchi

1,

*

1

Department of Biochemistry, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan

2

DNAVEC Corporation, 1-2-11, Kannondai, Tsukuba, 305-0856, Japan

3

Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamada-oka, Suita, 565-0871, Japan

4

Laboratory of Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1, Hirosawa, Wako, 351-0198, Japan

5

Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai, 980-8578, Japan

Dvl, an important component of the Wnt signalling pathway, is thought to be involved insynaptogenesis. In this study, we investigated whether Dvl regulates neurotransmitter release.Knockdown of Dvl in PC12 cells suppressed K

+

-induced dopamine release, and this phenotypewas restored by expression of Dvl-1. We identified synaptotagmin (Syt) I, which is involvedin neurotransmitter release, as a Dvl-binding protein. Dvl directly bound to the C2B domain ofSyt I. Dvl colocalized with Syt I at the tip of neurites of differentiated PC12 cells and of neuronsin the rat dorsal root ganglion. Dvl and Syt I was located in large dense-core vesicles, whichcontain dopamine. In addition, endocytosis of vesicles containing Syt I was suppressed in Dvlknockdown PC12 cells. Dvl inhibited the binding of Syt I to the complex consisting of syntaxin-1A and SNAP-25. Furthermore, µµµµ

2-adaptin of AP-2, which is known to play a role in endocytosis,formed a complex with Dvl and Syt I. Taken together, these results suggest that Dvl is involvedin endo- and exocytotic processes through the binding to Syt I.

Introduction

The Dvl protein family is a critical component of theWnt signalling pathway (Wharton 2003). All Dvl familymembers, including those in mammals:

Xenopus

and

Drosophila

, contain three highly conserved domains: aDIX domain, a PDZ domain, and a DEP domain. Dvlinterprets the Wnt-generated receptor activation andtransmits its signal to at least two distinct pathways (the

β

-catenin and planar cell polarity (PCP) pathways), andthereby organizes pathway-specific subcellular signallingcomplexes, signal amplification, and dynamic controlthrough feedback regulation (Wharton 2003). Thesefunctions of Dvl in the Wnt signal pathway could bemediated by Dvl-binding proteins, including caseinkinase, Axin, Frat, Nkd, Frodo, Strabismus, and Daam1(Wharton 2003). Dvl may act as a functional switchbetween distinct downstream pathways by different binding

partners in the Wnt signalling pathway. However, someDvl-binding proteins such as Notch (Axelrod

et al.

1996)and Eps8 (Inobe

et al.

1999) function in other signallingpathways. Notch is a transmembrane receptor for alateral inhibitory signalling pathway of Delta. Eps8 is asubstrate of EGF receptor kinase and mediates EGFreceptor intracellular trafficking. Thus, it is possible thatDvl regulates not only Wnt signalling but also othersignalling depending on the binding proteins.

In humans and mice, three

Dvl

genes,

Dvl-1

,

Dvl-2

,and

Dvl-3

have been identified.

Dvl-1

is ubiquitouslyexpressed at the early stages of development (Wharton2003). In the central nervous system Dvl-1 is highlyexpressed in areas of high neuronal density at embryonicand postnatal stages of development. A

Dvl-1

knockoutmouse study showed that

Dvl-1

is not required for earlydevelopment, but

Dvl-1

null mice exhibit behavioralabnormalities and neurological deficits (Lijam

et al.

1997).

Dvl-2

has been shown to be highly expressed inthe outer root sheath and hair precursor cells.

Dvl-2

knockout mice show 50% lethality because of cardio-vascular outflow tract defects and all

Dvl-1/2

double

Communicated by

: Kozo Kaibuchi*

Correspondence

: E-mail: [email protected]

†

These authors contributed equally to this work.

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et al.


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knockout mutant embryos display craniorhachischisis,a completely open neural tube from the midbrain to thetail (Hamblet

et al.

2002). Therefore, there is functionalredundancy between Dvl-1 and Dvl-2 in neural tubeclosure and Dvls are suggested to be required for theformation and/or function of specific neuronal pathways.

It has been shown that Dvl-1 localizes to axonalmicrotubules and regulates microtubule stability throughGSK-3 (Hall

et al.

2000; Ciani & Salinas 2005). Dvl-1activates Rho-kinase and inhibits nerve growth factor(NGF)-dependent neurite extension in PC12 cells(Kishida

et al.

2004). Further, Dvl interacts with MuSK,a receptor tyrosine kinase, and this interaction is requiredfor Agrin-induced acetylcholine receptor clustering atthe neuromuscular junction (Luo

et al.

2002). Theseresults suggest that Dvl is involved in synaptogenesisincluding the regulation of neurite extension andsynapse development. In addition, Dvl has been shownto localize to intracellular small vesicles by binding tolipids (Kishida

et al.

1999; Capelluto

et al.

2002) and totranslocate to the plasma membranes in a ligand-dependentmanner (Cliffe

et al.

2003). Dvl also forms a complexwith

β

-arrestin, which plays a role in receptor-mediatedendocytosis (Chen

et al.

2001). Thus, Dvl has beensuggested to regulate the trafficking of intracellularvesicles, but the roles of Dvl in neurotransmitter releaseare not well understood.

Synaptotagmin (Syt) is a Ca

2+

-binding protein thatcontains an N-terminal transmembrane region and twoC-terminal C2 domains and is thought to regulatemembrane traffic (Chapman 2002; Südhof 2004). Thereare at least 15 Syt family members in mammalian cells.Syt I is evolutionally conserved and is the best characterizedisoform of Syt. Syt I is enriched in synaptic vesicles andis known to be essential for synaptic vesicle exocytosisand endocytosis in neurons. Soluble N-ethylmaleimide-sensitive fusion factor (NSF) attachment protein receptor(SNARE) proteins are thought to form the minimalmembrane fusion machinery that mediates exocytosis(Weber

et al.

1998). In neurons, synaptobrevin is vesiclemembrane SNARE (v-SNARE), and syntaxin-1A andSNAP-25 are target membrane SNARE (t-SNARE).Syntaxin-1A and SNAP-25 can form a complex on theplasma membrane to bind to synaptobrevin. Ca

2+

promotesthe interaction of Syt I with the syntaxin-1A and SNAP-25 complexes and with the ternary SNARE complex,suggesting that the interaction of Syt I with the SNAREcomplex plays a role in the reversible docking of secretaryvesicles. It has also been reported that Syt I stimulates themembrane fusion of reconstituted v- and t-SNAREproteoliposomes

in vitro

by facilitating SNARE complexformation (Tucker

et al.

2004). Furthermore, Syt I interacts

with

µ

2- and

α

-adaptin of AP-2, which play an essentialrole in endocytosis, and this interaction is important inendocytosis, because perturbation of the interactioninhibits transferrin internalization in living cells (Haucke

et al.

2000). Thus, Syt I is implicated in the regulation ofendocytosis in addition to exocytosis.

In this study, we found that knockdown of Dvl in ratadrenal medulla-derived pheochromocytoma PC12 cellsdecreases K

+

-dependent dopamine release, and further-more identified Syt as a Dvl-binding protein. Here, weshow that Dvl is involved in endo- and exocytotic processesof PC12 cells and that this action of Dvl is mediatedthrough the binding to Syt I.

Results

Involvement of Dvl in dopamine release from PC12 cells

We have demonstrated that Dvl regulates the neuriteextension of PC12 cells (Kishida

et al.

2004), and PC12cells is known to release dopamine by depolarization(Ritchie 1979). To examine whether Dvl is involved inthe regulation of neurotransmitter release, the proteinlevels of Dvl were reduced by RNA interference(RNAi) in PC12 cells. The expression of Dvl-1, Dvl-2,and Dvl-3 was detected by their specific antibodies andall of them were decreased by RNAi (Fig. 1A). In thisexperiment, a small interfering RNA (siRNA) consistingof 21-mer duplexed RNA based on a sequence fromrat Dvl-2 was designed. This sequence has one and twomismatches from Dvl-1 and Dvl-3, respectively. Since ithas been reported that siRNA may target genes withsequence similarity (Jackson

et al.

2003), it appearedthat siRNA for Dvl-2 reduces the protein level of allof Dvls. The expression levels of Syt I, SNAP-25,syntaxin-1A, synaptobrevin, and

β

-actin were not changed(Fig. 1A).

A low concentration (4.7 m

m

) of KCl did not inducedopamine release, while a high concentration (60 m

m

)of KCl stimulated it (Fig. 1B). K

+

-induced dopaminerelease was greatly reduced in Dvl knockdown cells ascompared with control PC12 cells (Fig. 1B). Expressionof Flag-HA-Dvl-1 (wild type) (WT) encoded by aretrovirus vector rescued the inhibition of the dopaminerelease in Dvl knockdown PC12 cells (Fig. 1C,D).Knockdown or expression of Dvl-1 did not affect theneurite extension of PC12 cells maintained with growthmedium used for dopamine release assay (10% fetal calfserum and 5% horse serum) (data not shown). Thus, it islikely that Dvl regulates dopamine release from PC12cells without affecting morphology. These results prompted

Dvl and Synaptotagmin

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us to clarify how Dvl is involved in the release of neuro-transmitter and to search for proteins that interact withDvl-1 using the yeast two-hybrid method.

Identification of Syt as a Dvl-1-binding protein

A 1.8-kb cDNA insert was found to encode a sequencecontaining an open reading frame of Syt XI from 1

×

10

6

initial transformants of a mouse brain cDNA library.There are at least 15 Syt family members in mammalian

cells. To determine which Syt family members interactwith Dvl-1 in intact cells, haemaggulutinin (HA)-taggedDvl-1 (HA-Dvl-1) was expressed with Flag-tagged Syt I(Flag-Syt I), III, IV, VII, IX, and XI in COS cells (Fig. 2B).When the lysates were immunoprecipitated with anti-Flagantibody, HA-Dvl-1 was found in the Flag-Syt I and XIimmune complexes, but not in the Flag-Syt III, IV, VII,and IX immune complexes (Fig. 2B). Neither HA-Dvl-1 nor Flag-Syt I was immunoprecipitated from thelysates with non-immune immunoglobulin (data notshown). HA-Dvl-2 and Myc-Dvl-3 were also observedin the Flag-Syt I and XI immune complexes (Fig. 2C, D).Thus, each Dvl member specifically forms a complexwith Syt I and XI. In addition, HA-Dvl-2 formed acomplex with Flag-Syt IV faintly, but HA-Dvl-1 andMyc-Dvl-3 did not. At present, we do not know thereason for the differences between the bindings of Dvlmembers and Syt IV.

Interaction between Dvl and Syt I

Among the Syt family members, Syt I has been extensivelystudied and is known to play a role in exocytosis andendocytosis of synaptic vesicles (Chapman 2002; Südhof2004). Therefore, we analyzed the biological functionof the binding between Dvl-1 and Syt I. Syt I consists of421 amino acids and has one transmembrane domainand two Ca

2+

binding domains, C2A and C2B (Fig. 2A).To examine which regions of Syt I and Dvl-1 are responsiblefor their complex formation, various deletion mutants ofT7-Syt I and HA-Dvl-1 were generated (Fig. 2A). T7-Syt I-(

∆

C2A) was found in the Myc-Dvl-1 immunecomplex as efficiently as T7-Syt I (WT), while neitherT7-Syt I-(

∆

C2AB) nor T7-Syt I-(

∆

C2B) was detected(Fig. 2E). HA-Dvl-1-(140–670), HA-Dvl-1-(

∆

228–250),and HA-Dvl-1-(

∆

251–336) formed a complex withT7-Syt I to the same extent as HA-Dvl-1 (WT). How-ever, HA-Dvl-1-(281–670) was not observed in theT7-Syt I immune complex and HA-Dvl-1-(

∆

141–227) wasobserved to a lesser extent (Fig. 2F). Thus, the regionscontaining the C2B domain of Syt I and amino acids141–227 of Dvl-1 are important for the interaction ofDvl-1 with Syt I.

To examine whether the interactions of Dvl and Syt Iare direct,

in vitro

binding assays using the purified proteins(Fig. 3A) were performed. Maltose-binding protein (MBP)-Syt I-(C2B), but not MBP-Syt I-(C2A), interacted withglutathione-

S

-transferase (GST)-Dvl-1 (Fig. 3B). C2B isknown to bind to Ca

2+

(Chapman 2002; Südhof 2004).However, the interaction of MBP-Syt I-(C2B) withGST-Dvl-1 was little influenced in the presence of freeCa

2+

(Fig. 3B). Analyses by surface plasmon resonance

Figure 1 Involvement of Dvl in dopamine release from PC12cells. (A) The siRNA for Dvl was stably expressed in PC12 cellsusing retrovirus vector. Lysates (10 µg of proteins) of Dvlknockdown cells were probed with anti-Dvl-1, anti-Dvl-2,anti-Dvl-3, anti-Syt I, anti-SNAP-25, anti-syntaxin-1A, anti-synaptobrevin, and anti-β actin-antibodies. (B) Control PC12cells (�, �) or Dvl knockdown PC12 cells (�, �) were treatedwith the low K+ solution (�, �) or the high K+ solution (�, �)for the indicated periods of time. The results shown are means ±S.E. of three independent experiments. (C) Flag-HA-Dvl-1(WT) was stably expressed in Dvl knockdown PC12 cells usingretrovirus vector. Lysates (10 µg of proteins) of the indicated cellswere probed with anti-Dvl and anti-GSK-3β antibodies. (D)Control PC12 cells (�), Dvl-knockdown cells (�), or Dvlknockdown cells expressing Flag-HA-Dvl-1 (WT) (�) weretreated with the high K+ solution for the indicated periods of time.The results shown are means ± S.E. of five independentexperiments.

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Genes to Cells (2007) 12, 49–61 © 2006 The AuthorsJournal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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Figure 2 Interaction of Dvl with Syt. (A) Schematic representation of deletion mutants of human Dvl-1 and mouse Syt I used in thisstudy. WT, wild type; TM, transmembrane region. (B, C, and D) Lysates (20 µg of proteins) of COS cells expressing Dvl-1 (B), Dvl-2(C), or Dvl-3 (D) with Syt family members were probed with anti-Dvl and anti-Flag antibodies. The same lysates (200 µg of proteins)were immunoprecipitated with anti-Flag antibody, and then the immunoprecipitates were probed with the indicated antibodies. Ab,antibody; Ig, immunoglobulin; IP, immunoprecipitation. (E) Lysates (20 µg of proteins) of COS cells expressing the indicated proteinswere probed with anti-Myc and anti-T7 antibodies. The same lysates (200 µg of proteins) were immunoprecipitated with anti-Mycantibody, and then the immunoprecipitates were probed with the same antibodies. The arrowhead and arrow indicate T7-Syt I (WT)and T7-Syt I-∆C2A, respectively. (F) Lysates (20 µg of proteins) of COS cells expressing the indicated proteins were probed with theanti-HA and anti-T7 antibodies. The same lysates (200 µg of protein) were immunoprecipitated with anti-T7 antibody, and then theimmunoprecipitates were probed with the anti-HA and anti-T7 antibodies. Arrowheads indicate T7-Syt I. The results shown arerepresentative of five independent experiments.


© 2006 The Authors Genes to Cells (2007) 12, 49–61Journal compilation © 2006 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

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(SPR) demonstrated that the static dissociation constant,Kd, is 140 nM for the binding of MBP-Syt I-(C2B) toGST-Dvl-1 (Fig. 3C). Approximately 0.6 mol of GST-Dvl-1 bound to 1 mol of Syt I-(C2AB) (Fig. 3D). Thebinding affinity of GST-Dvl-1-(∆141–227) to Syt I-(C2AB)seemed to be similar to that of GST-Dvl-1, but themaximal binding activity of GST-Dvl-1-(∆141–227) toSyt I-(C2AB) was reduced, which is consistent with theresults in intact cells.

Colocalization of Dvl-1 and Syt I in PC12 cells and DRG neuron

Syt I is highly expressed in the brain and endocrineglands, and is localized to synaptic vesicles and secretorygranules. Next, we examined the interaction betweenDvl and Syt I in PC12 cells to know the functionalsignificance of their binding. When Myc-Dvl-1 wasexpressed in PC12 cells using Sendai virus vector, it wasco-immunoprecipitated with endogenous Syt I (Fig. 4A).It has been shown that the immunoprecipitates byanti-Syt I antibody contain synaptic vesicles or large densecore vesicles (LDCVs) (Lowe et al. 1988). Taken togetherwith the previously reported observations that Dvlassociates with cytoplasmic vesicles (Capelluto et al. 2002),Dvl may colocalize with Syt I on the vesicles. EndogenousSyt I was observed in the peripheral region of PC12 cellsnot treated with NGF, and endogenous Dvl was alsodetected in the peripheral region, where the two proteinswere colocalized (Fig. 4B). Anti-Dvl antibody alsorecognized proteins throughout the cytoplasm (Fig. 4B).In PC12 cells induced to differentiate by NGF, Syt I wasdominantly observed at the termini of extended neurites.Dvl was also detected in the neurite termini andcolocalized with Syt I (Fig. 4B).

Rat dorsal root ganglion neuron is a well establishedcell model for sympathetic peripheral neuron as well asrat hippocampal neuron for CNS neuron (Banker &Goslin 1998). Syt I was enriched at the growth cone regionof rat dorsal root ganglion neuron (Fig. 4C). Furthermore,Syt I was present in the cytoplasm with a punctate pattern(Fig. 4C). Consistent with the observations of PC12 cellstreated with NGF, Dvl and Syt I were colocalized at thetips of the neurite termini. Taken together, these resultsindicate that Dvl and Syt I form a complex in neuro-endocrine and neuronal cells.

Neuroendocrine and neuronal cells contain at leasttwo types of vesicles, LDCVs containing catecholamineand neuropeptide and small synaptic vesicles (synaptic-like microvesicles (SMVs) in PC12 cells) containingacetylcholine (Winkler 1997). To characterize the sub-cellular distribution of Dvl and Syt in PC12 cells, the

Figure 3 Direct binding of Dvl to Syt. (A) GST, GST-Dvl-1(WT), GST-Dvl-1-(∆141–227), MBP, MBP-Syt I-(C2A), MBP-Syt I-(C2B), and Syt I-(C2AB), and syntaxin-1A (0.25 µg ofprotein) were stained with CBB. (B) GST or GST-Dvl-1 (0.6µM) immobilized on glutathione-Sepharose 4B was incubatedwith MBP, MBP-Syt I-(C2A) or MBP-Syt I-(C2B) in thepresence or absence of 100 µM free Ca2+. GST or GST-Dvl-1 wasprecipitated by centrifugation, and the precipitates were stainedwith CBB. The arrowhead indicates the position of MBP-Syt I-(C2A) and MBP-Syt I-(C2B). (C) GST-Dvl-1 were immobilizedonto a CM5 sensor tip surface via the anti-GST antibody, whichwas covalently coupled to the surface by standard amine-couplingchemistry. GST was immobilized onto the surface of the referencecell on the same sensor tip. Four different concentrations (125,250, 500, and 1000 nM) of MBP-Syt I-(C2B) were injected ontothe surface of the sensor tip at 25 °C for 3 min to form thecomplex and then the sensor tip was washed with the buffer for3 min to dissociate the complex. The plotted data shows therelative binding of MBP-Syt I-(C2B) to cell. (D) Syt I-(C2AB) (0.5 µM)immobilized on protein A Sepharose 4B was incubated with theindicated concentrations of GST-Dvl-1 (WT) (�) or GST-Dvl-1-(∆141–227) (�). Syt I-(C2AB) was precipitated by centrifugation,and the precipitates were stained with CBB. The obtained resultswere plotted in the lower panel. The results shown arerepresentative of three independent experiments.

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Figure 4 Colocalization of Dvl and Syt I in PC12 and neuronal cells. (A) Postnuclear supernatant (20 µg of proteins) of PC12 cellsexpressing Myc-Dvl-1 were probed with anti-Dvl and anti-Syt I antibodies. The same supernatant (200 µg of protein) wasimmunoprecipitated with anti-Syt I antibody or control IgG, and the immunoprecipitates were probed with anti-Dvl and anti-Syt Iantibodies. (B) PC12 cells treated without or with 100 ng/mL NGF for 48 h were stained with anti-Dvl (green) and anti-Syt I (red)antibodies. Right panels show merged images. Bars, 10 µm. (C) Rat dorsal root ganglion neuron was stained with the anti-Dvl (green)and anti-Syt I (red) antibodies. DIC, differential interference contrast. Bar, 10 µm. (D) Upper left panel, dopamine content in eachfraction. The post nuclear supernatants of PC12 cells treated with (�) or without (�) NGF for 48 h were fractionated on 0.6–1.6 Mcontinuous sucrose gradients. Upper right panel, the expression levels of Dvl and Syt I in the lysates of PC12 cells treated with or withoutNGF. Lower panel, the aliquots of each fraction were probed with anti-Dvl, anti-Syt I, and anti-synaptophysin (Syp) antibodies. Fractions12–16, in which dopamine was abundant, contained large dense-core vesicles (LDCV). Fractions 4–10, in which synaptophysin waspresent contained synaptic-like microvesicles (SMV). (E) Myc-Dvl-1 (WT) or HA-Dvl-1-(∆141–227) was expressed in wild-type PC12cells using Sendai virus vector. The lysates (10 µg of proteins) of the indicated cells were probed with anti-Dvl and anti-GSK-3β antibodies.(F) Control PC12 cells (�) and PC12 cells expressing Myc-Dvl-1 (WT) (�) or Myc-Dvl-1-(∆141–227) (�) using Sendai virus vectorwere treated with high K+ solution for the indicated periods of time. The results shown are means ± S.E. of five independent experiments.



55

post-nuclear supernatants were fractionated by sedi-mentation through sucrose density gradients (Fig. 4D).Dopamine, which is present in LDCVs, was detected infractions 12–16, and treatment of PC12 cells with NGFfor 48 h increased the dopamine content (Fig. 4D). SytI was also detected in fractions 12–16. NGF increasedthe total amount of Syt I and the amount of Syt I whichoverlapped with the fractions containing dopamine andLDCVs (Fig. 4D). Synaptophysin (Syp), which is amarker of SMVs, was detected in fractions 4–10. Takentogether, these results demonstrate that Syt I is mainlylocalized to LDCVs (Fig. 4D).

Most Dvl was detected in the soluble fractions (fractions1 and 2) and a small fraction of Dvl was present infractions 4–10 and 12–16 (Fig. 4D). Although NGF didnot change the total amount of Dvl (Fig. 4D, upper rightpanel), the amount of Dvl that was detected in fractions10–16 was increased. This may reflect the increment inDvl complexed with Syt I according to the synthesis ofSyt I by NGF. Thus, Dvl could be localized to bothLDCVs and SMVs in addition to the cytosolic fractions.We collected the fractions containing LDCVs, and LDCVswere immunoisolated by anti-Syt I antibody. However,endogenous Dvl was not detected in the immunoisolatedLDCVs (data not shown). Since it was hard to show thephysical interaction between Dvl and Syt I on LDCVs atthe endogenous level, we next tried to prove the functionalbinding of Dvl and Syt I on LDCVs.

To examine whether the interaction of Dvl with SytI is important for dopamine release, we expressedMyc-Dvl-1 (WT) or HA-Dvl-1-(∆141–227) in wild-typePC12 cells using a Sendai virus vector (Fig. 4E).Over-expression of Dvl-1 (WT) did not affect K+-induceddopamine release, but that of HA-Dvl-1-(∆141–227)impaired dopamine release (Fig. 4F). Taken together,these results indicate that Dvl is necessary for dopaminerelease by binding to Syt I and that Dvl-1-(∆141–227)acts as a dominant-negative factor for it.

Involvement of Dvl in endocytosis

It is believed that endocytosis is coupled with exocytosisand this coupling is involved in the secretion of neuroen-docrine cells (Südhof 2004). Antibody against lumenaldomain of Syt I on the synaptic vesicles was used as amarker to study recycling and endocytosis of synapticvesicles (Matteoli et al. 1992). The internalization of theantibody was increased in time-dependent and K+-dependent manners (Fig. 5A,B). The internalized anti-body was observed not only into neurite tip but also intoother portions of neurite and cell body (Fig. 5A). Theseresults indicate that the internalization of Syt I-containing

vesicles is coupled with exocytosis. When Dvl wasreduced by RNAi in PC12 cells, the internalization ofSyt I-containing vesicles was suppressed as comparedwith control PC12 cells (Fig. 5A,B). When Dvl-1 wasover-expressed in PC12 cells using a Sendai virus vector,the internalization of anti-lumenal Syt I antibody was notaffected, whereas over-expression of Dvl-1-(∆141–227) inPCl2 cells decreased it (Fig. 5C). Thus, the decrease inthe internalization of Syt I in Dvl knockdown PC12 cellsmight be a consequence of the blockade of exocytosis.

Formation of a complex between Dvl and exocytotic or endocytoic machineries

Finally, we found how Dvl is involved in transmitterrelease at the molecular level. We examined the effects ofDvl on the formation of a complex between Syt I and t-SNARE (syntaxin-1A and SNAP-25), which is importantfor neurotransmitter release. It has been reported that theC2AB domain of Syt I binds to t-SNARE, and that theaffinity of the binding of Syt I to SNAP25 is about 200nM in the presence of Ca2+ (Gerona et al. 2000). In vitrobiochemical studies using recombinant proteins showedthat syntaxin-1A binds to immobilized Syt I-(C2AB)(Fig. 6A). Stoichiometric binding was observed uponaddition of Ca2+, with about 0.8 mol of syntaxin-1A boundto 1 mol of Syt I-(C2AB) (data not shown). GST-Dvl-1(WT) inhibited the binding of Syt I-(C2AB) and syntaxin-1A in a dose-dependent manner, but GST-Dvl-1-(∆141–227) inhibited it to a lesser extent (Fig. 6A). A binary SNAREcomplex including syntaxin-1A and SNAP-25 bound toMBP-Syt I-(C2AB) (Fig. 6B). GST-Dvl-1 inhibited thebinding of lower concentrations (0.13 and 0.25 µM) ofSNARE to MBP-Syt I-(C2AB), but it had little affect onthe binding of higher concentrations (1 µM) of SNAREto MBP-Syt I-(C2AB). These results indicate that Dvlcompetes with t-SNARE for the binding to Syt I in vitro.

Since Syt I binds to µ2-adaptin of AP-2, which playsa critical role in endocytosis (Haucke et al. 2000), we alsoexamined a complex between Dvl, Syt I, and µ2-adaptin.When HA-µ2 was expressed with Myc-Dvl-1 or T7-SytI, HA-µ2 formed a complex with Myc-Dvl-1 in additionto T7-Syt I (Fig. 6C). When these proteins are expressedsimultaneously, the amount of Dvl immunoprecipitatedwith µ2 was increased (Fig. 6C). Therefore, Dvl maybind to a complex of Syt I and µ2 more efficiently thanto either of them.

DiscussionIn this study, we have for the first time demonstrated thatDvl is involved in neurotransmitter release and that this

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action is mediated by the binding to Syt I. Syts constitutea family of membrane traffic proteins that are characterizedby an N-terminal transmembrane domain, a variable linker,and two C-terminal C2-domains (Chapman 2002;Südhof 2004). Based on their sequences and properties,at least 15 Syts can be grouped into six classes: class 1(Syts I and II), class 2 (Syt VII), class 3 (Syts III, V, VI, andX), class 4 (Syts IV and XI), class 5 (Syts IX), and class6 (Syts VIII, XII, XIII, XIV, and XV). We found thateach of Dvl-1, -2, and -3 bound to Syts I and XI, but notto Syts III, IV, VII, and IX. Amino acids 141–227 ofDvl-1 were required for the binding to the C2B domainof Syt I. Since the corresponding regions of Dvl-2 and-3 have 83 and 90% similarities, respectively, with aminoacids 141–227 of Dvl-1, this region is important for thebinding of the Dvl protein family with Syts. Dvlcolocalized with Syt I at the neurite termini in PC12cells induced to differentiate by NGF and in dorsal rootganglion neurons. Furthermore, sucrose density gradient-

subcellular fractionation indicated that Dvl is present inthe fractions containing the large dense-core vesicles,which also contains Syt I, in addition to the cytosol. Thus,Dvl binds to Syt I, which is located to secretory vesicles.

In addition to the ability to bind to Syt I, the followingevidence indicated that Dvl might contribute to dopaminerelease from PC12 cells. When Dvl was reduced byRNAi, depolarization-dependent dopamine release inPC12 cells was decreased. Moderate expression of Dvl-1 mediated by retrovirus vector in Dvl knockdown cellsrescued the dopamine release to the WT level. Over-expression of Dvl-1 mediated by Sendai virus vector didnot affect K+-induced dopamine release, suggesting thatendogenous Dvl is not a limiting factor for dopaminerelease. In contrast, over-expression of Dvl-1-(∆141–227),which has a reduced ability to bind to Syt I, inhibiteddopamine release. The DIX domain of Dvl mediates theoligomerisation of Dvl (Kishida et al. 1999). If theoligomerisation of wild-type Dvl is necessary for

Figure 5 Involvement of Dvl in endocytosisof vesicles containing Syt I. (A) Inhibitionof Syt I recycling in Dvl knockdown PC12cells. Control PC12 cells or Dvl knockdownPC12 cells treated with NGF for 48 h wereincubated with the high K+ solution containinganti-lumenal Syt I antibody for 10 or 20min or with the low K+ solution containinganti-lumenal Syt I antibody for 20 min.The panels show the typical images of theinternalization of anti-lumenal Syt I antibody(red) and differential interference contrast(DIC) image (grey scale) of the cells. ScaleBars, 10 µm. (B) The average intensity ofthe fluorescence (50 cells per each group)derived from internalized anti-lumenal SytI antibody after stimulation with the highK+ solution was quantified. (�), controlPC12 cells; (�), Dvl knockdown PC12cells. (C) Control PC12 cells (�) and PC12cells expressing Dvl-1 (�) or PC12 cellsexpressing Dvl-1-(∆141–227) (�) usingSendai virus vector were incubated withanti-lumenal Syt I antibody in the high K+

solution for the indicated periods of time,and the amount of the internalized antibodywas quantified. The results shown are means± S.E. of three independent experiments.



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dopamine release, Dvl-1-(∆141–227) may inhibit thefunction of endogenous Dvl by forming a complex withit in a dominant-negative manner. Furthermore, consistentwith the concept that endocytosis is coupled with exocytosis(Südhof 2004), K+-dependent internalization of Syt Iwas also suppressed in Dvl knockdown or Dvl-1-(∆141–227)-expressing PC12 cells. Taken together, these resultssuggest that Dvl is involved in the depolarization-dependent dopamine release from PC12 cells by bindingto Syt I.

Wnt-3a induces the accumulation of β-catenin andactivates Rho in PC12 cells (Kishida et al. 2004). However,purified Wnt-3a did not affect K+-induced dopaminerelease (data not shown). Therefore, the activation of theβ-catenin and Rho pathways do not affect dopaminerelease from PC12 cells. Dvl inhibits NGF-dependentneurite extension of PC12 cells (Kishida et al. 2004).However, it is unlikely that regulation of dopaminerelease by Dvl is due to effects on neurite outgrowth,because Dvl inhibited dopamine release from PC12 cellswithout NGF treatment. Dvl may have a role in regulatingthe neurotransmitter release independently of the Wnt-3asignalling pathway. Wnt-7a is expressed by granule cellsduring synaptogenesis with mossy fibers and increases

synapsin I clustering in mossy fiber axons (Ciani & Salinas2005). Therefore, some Wnt ligands may regulate neuro-transmitter release through Dvl.

How does Dvl regulate neurotransmitter releasethrough binding to Syt I? Syt I is involved in endo–exocytosis by binding to Ca2+, t-SNARE, and adaptorprotein AP-2 (Shao et al. 1997; Haucke et al. 2000). Ourin vitro data showed that Dvl inhibits the interaction ofSyt I with t-SNARE. Since syntaxin-1A binds to theC2A domain of Syt I (Shao et al. 1997) and Dvl binds tothe C2B domain of Syt I, the association of Syt I withDvl may cause conformational change and reduce theaffinity of Syt I for syntaxin-1A. Although we cannotdraw conclusions about the actions of Dvl on theSNARE complex because of the difficulty of examiningthe interaction of Dvl, Syt I, and t-SNARE at theendogenous level, these observations suggest that Dvlmay inhibit the ability of Syt I to stimulate the dockingor fusion between vesicles and membranes. Alternatively,Dvl may function in releasing Syt I from t-SNARE.

The C2B domain of Syt I has been shown to bind toµ2- and α-adaptin, both of which are the subunits of theendocytotic adaptor complex AP-2, and this interactionis required for clathrin-dependent endocytosis (Haucke

Figure 6 Formation of a complex betweenDvl and exocytotic or endocytotic machi-neries. (A) Syt I-(C2AB) (0.5 µM)complexed with the anti-Syt I antibodyand protein A Sepharose was incubatedwith syntaxin-1A (3 µM) and the indicatedconcentrations of GST-Dvl-1 (WT) (�) orGST-Dvl-1-(∆141–227) (�). The obtainedresults were plotted in the lower panel.(B) MBP-Syt I-(C2AB) (0.5 µM) immo-bilized with amylose resin was incubatedwith the indicated concentrations of SNAREcomplex in the absence or presence ofGST-Dvl-1 (WT). The obtained resultswere plotted in the lower panel. (�, �),SNAP25; (�, �), syntaxin-1A; (�, �), inthe presence of GST-Dvl-1; (�, �), in theabsence of Dvl-1. The results shown arerepresentative of four independent experi-ments. (C) Lysates (200 µg of protein) ofCOS cells expressing Myc-Dvl-1, HA-µ2,and T7-Syt I as indicated were immuno-precipitated with anti-HA antibody, andthe immunoprecipitates were probedwith the anti-Dvl, anti-HA, and anti-T7antibodies. The arrowhead and arrowindicate HA-µ2 and T7-Syt I, respectively.

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et al. 2000). Thus, Syt I is also known to regulate recyclingor endocytosis of synaptic vesicles (Poskanzer et al. 2003)and to serve as a link between the exocytotic and endo-cytotic events of the synaptic vesicles (Zhang et al. 1994).Dvl has been shown to localize to intracellular vesicles(Capelluto et al. 2002) and to be involved in theinternalization of Frizzled 4, a receptor of Wnt-5a (Chenet al. 2003). Our results show that Dvl binds to µ2-adaptin and that this complex formation is enhanced bythe presence of Syt I also support that Dvl is involved inthe regulation of endocytosis.

Although the exact roles of Dvl-1 in the endo- andexocytosis of Syt I-containing vesicles are not clear atpresent, it is likely that Dvl is involved in multiple stepsof synaptic or synaptic like vesicle cycle (Südhof 1995).First, Dvl may play a role in intracellular translocatingprocess of vesicles filled with transmitters to the releasesites (the active zone), and Dvl may be released from SytI to stimulate the binding of Syt I to t-SNARE beforedocking or fusion. Another possible model is that afterexocytosis, Dvl may inhibit the association of Syt I tothe SNARE complex and binds to the complex betweenSyt I and AP-2, thereby inducing endocytosis. Sincesyntaxin-1A is excluded from recycling synaptic vesiclesat nerve terminals (Mitchell & Ryan 2004), Dvl may beinvolved in the release of syntaxin-1A from Syt I duringendocytosis. In both cases, Dvl acts as a positive regulatorfor dopamine release from PC12 cells. Dvl-1 null miceexhibit behavioral abnormalities and neurologicaldeficits (Lijam et al. 1997). Clarifying the roles of Dvl inendo- and exocytosis would contribute to understandthe molecular mechanism of these phenotypes.

Experimental proceduresMaterials and chemicals

pcDNA-HA-µ2 and pOZ-N were provided by Drs V. Haucke(George-August-University, Göttingen, Germany) and T. Ikura(Tohoku University, Sendai, Japan), respectively. Anti-Myc antibodywas prepared from 9E10 cells. PC12 cells were grown inDulbecco’s modified essential medium (DMEM) supplementedwith 10% fetal bovine serum, 5% horse serum, and 500 ng oftetracycline hydrochloride/mL. Rat dorsal root ganglion neuronswere collected as described (Inoue et al. 1999), and cultured withDMEM containing 10% horse serum and 50 ng of NGF/mL.GST-Syt I-(C2A), -(C2B), -(C2AB), MBP-Syt I-(C2A), and-(C2B) were purified according to manufacturers’ instructionsbesides recombinant protein immobilized to the glutathionesepharose or amylose resin were treated with 10 µg/mL of DNaseand RNase (Roche applied sciences) and washed with 400 mmKCl and 20 mm Tris–HCl (pH 7.5) to remove nucleotide contam-inants (Bhalla et al. 2006). GST-Dvl-1 and GST-Dvl-1-(∆141–

227) were purified from Spodoptera frugiperda (Sf ) 9 cells. Anti-Dvlantibody was prepared as described (Kishida et al. 1999). Anti-Dvl-1, -2, and -3 antibodies were from Santa Cruz Biotechnology,Inc. (Santa Cruz, CA). Anti-T7 antibody was purchased fromNovagen (Madison, WI). Anti-Flag M2, anti-synaptophysin(clone SVP38), and anti-syntaxin-1A (clone HPC-1) antibodieswere from Sigma (Saint Louis, MO). Anti-Syt I and anti-synaptobrevin antibodies were from Stressgen (Victoria BC,Canada). Oyster®550-labelled-antibody against lumenal domain of SytI and anti-SNAP-25 (clone CI71.2) antibodies, and NGF 7.5Swere from Synaptic Systems (Göttingen, Germany) and InvitrogenInc. (Carlsbad, CA), respectively. Other materials were purchasedfrom commercial sources.

Plasmid constructions

pCGN/hDvl-1, pCGN/hDvl-1-(140–670), pCGN/hDvl-1-(∆141–227), pCGN/hDvl-1-(∆228–250), pCGN/hDvl-1-(∆251–336),pCGN/hDvl-1-(281–670), pVIKS/Dvl-1, and pEFBOS-Myc/hDvl-3 were constructed as described (Kishida et al. 1999; Hino etal. 2003). pEF-Flag/Syt I, pEF-Flag/Syt III, pEF-Flag/Syt IV,pEF-Flag/Syt VII, pEF-Flag/Syt IX, pEF-Flag/Syt XI, pEF-T7/Syt I, pEF-T7/Syt I-(∆C2AB), pEF-T7/Syt I-(∆C2A), pEF-T7/Syt I-(∆C2B), pGEX-2T/Syt I-(C2A), and pGEX-2T/Syt I-(C2B) were constructed as described (Fukuda et al. 2001; Fukuda2002). pMAL-c2/Syt I-(C2A), pMAL-c2/Syt I-(C2B), pMAL-c2/Syt I-(C2AB), pGEX-2T/syntaxin-1A, pGEX-4T/SNAP-25,pGEX-4T-3/synaptobrevin, pOZ/Dvl-1 (wildtype), pCGN/Dvl-2, and pVIKS/Dvl-1-(∆141–227) were constructed usingstandard techniques.

RNAi

The nucleotide sequences of rat Dvl-2 (accession number;XM_239254) and Dvl-3 (accession number; XM_221304) werepredicted by automated computational analysis from an annotatedgenomic sequence (accession number; NW_047334). ThesiRNA for rat Dvl was derived from the sequence for mRNAnucleotides 970–990 of rat Dvl-2. The siRNA for rat Dvl-2 hasone and two nucleotides differences from the siRNA for ratDvl-1 and Dvl-3, respectively. To generate retroviruses, pSUPER-retro.neo+gfp/Dvl-2 was transfected into ampho-pack 293 cells(BD Biosciences Clontech, Palo Alto, CA). These cells werecultured for 2 days, and the conditioned medium containingretroviruses were collected and filtrated. Retrovirus-containingmedia were incubated with PC12 cells in the presence of 4 µg/mL polybrene for 48 h. Then, the cells were selected in thepresence of 400 µg/mL G418 to obtain PC12 cells stably expressingsiRNA for Dvl. Stably expressing cells were pooled from eachtransfection, rather than individual clones, to avoid the effects ofclonal variations.

Dopamine release assay

PC12 cells were plated onto 35-mm-diameter dishes at a densityof 1.5×106 cells per dish and cultured for 48 h. The cells were



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washed once with a low K+ solution (Buffer A (20 mm Hepes–NaOH, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2, 1.2 mm MgSO4,1.2 mm KH2PO4, and 1.1 mm glucose) containing 4.7 mm KCl),and incubated in the low K+ solution for 10 min. After thesolutions were subsequently replaced with 1 mL of the low K+

solution or a high K+ solution (Buffer A containing 60 mm KCl)for the indicated periods of times, the solutions were collected andthe cells were extracted with 0.5 mL of 0.4 % (w/v) perchloricacid. Contents of dopamine were determined by high pressureliquid chromatography using a reverse-phase column and anelectrochemical detector system (Eicom, Kyoto, Japan)(Nagano et al. 2003). The rate of dopamine release was expressedas a percentage of secreted dopamine relative to the sum ofdopamine secreted from cells and dopamine remaining in the cells.

Imaging of vesicle endocytosis using antibody against lumenal domain of Syt I

PC12 cells plated onto 18-mm glass cover slips coated withpoly-d-lysine (Sigma, St Louis, MO) were cultured with 100 ng/mL NGF for 48 h. Syt I exposed to the cell surface and internalizedas a result of exocytotic stimulation was labelled as described withmodifications (Matteoli et al. 1992). Briefly, the cells were washedwith the low K+ solution and incubated with the low K+ solutionfor 10 min. After stimulation with high K+ solution containingantibody against lumenal domain of Syt I conjugated withOyster®550 (Synaptic Systems GmbH, Germany) for theindicated periods of time at 37°C, the cells were rinsed with icecold 0.5% acetic acid (w/v) and 0.5 M NaCl for 10 min to removethe antibody which was not internalized and then were fixed with4% paraformaldehyde in PBS for 10 min. The cells were viewedwith the LSM510 confocal microscope system (Carl Zeiss,Germany).

To quantitate the internalization of Syt I, 50 cells from threeindependent experiments were selected randomly and all imageswere acquired under identical conditions. The integrated fluorescenceof each cell was determined by taking the mean fluorescence ofthe whole cell (fluorescence of neurites and cell body/area) usingMetamorph software (Molecular devices Corporation, SunnyvaleCA). As the total expression level of Syt I was not changed duringthe treatment with the high K+ or low K+ solution up to 20 min(by Western blot and immunostaining, data not shown), the meanfluorescence per cell (nurites and cell body) represented theendocytosis of the Syt I containing vesicles.

Binding of Dvl, Syt I, and µµµµ2

To examine the direct interaction of Dvl and Syt I in vitro, GST-Dvl immobilized on glutathione-Sepharose 4B was incubatedwith MBP, MBP-Syt I-(C2A) or MBP-Syt I-(C2B) in 50 uL ofreaction mixture (20 mm Hepes–NaOH, pH 7.4, 150 mm NaCl,0.5% (w/v) Triton X-100, and 1 mm EGTA) for 1 h at 4°C. Afterthe GST-fusion proteins were precipitated by centrifugation, theprecipitates were resolved by electrophoresis and stained withCoomassie Brilliant Blue (CBB) or probed with anti-MBP anti-body. In some experiments, Syt I-(C2AB), which was obtained by

digestion of GST-fusion protein, bound to anti-Syt I antibody andprotein A complex was incubated with GST-Dvl-1 (WT) or GST-Dvl-1-(∆141–227) under the same conditions as above. Theamounts of each protein were quantified with densitometry usingNIH image software.

To examine the formation of a complex between Dvl and Sytsin intact cells, COS cells (60-mm-diameter dishes) expressingepitope-tagged Dvls and Syts were lysed in 300 µL of NP-40buffer (20 mm Tris–HCl (pH 7.5), 135 mm NaCl, 1% (w/v)Nonidet-P40, 20 µg/mL aprotinin, 20 µg/mL leupeptin, and 1 mmphenylmethylsulfonyl fluoride). After the proteins were immuno-precipitated with anti-FLAG, anti-Myc, or anti-T7 antibody, theimmunoprecipitates were probed with anti-Dvl, anti-FLAG, anti-Myc, anti-T7, or anti-HA antibody. To examine the formation ofa complex between Dvl, Syt, and µ2-adaptin, COS cells (60-mm-diameter dishes) expressing various combinations of Myc-Dvl-1,T7-Syt I, and HA-µ2 were lysed in 300 µL of NP-40 buffer. Afterthe proteins were immunoprecipitated with anti-HA antibody,the immunoprecipitates were probed with anti-HA, anti-T7, oranti-Dvl antibody.

Subcellular fractionation

PC12 cells (four 100-mm-diameter dishes) cultured in thepresence or absence of 100 ng/mL NGF for 48 h were harvestedand washed with PBS once. The cells were homogenized in 1 mLof homogenization buffer (10 mm Hepes–NaOH, pH 7.4, 0.32 Msucrose, 1 mm phenylmethylsulfonyl fluoride, 20 µg/mLaprotinin, and 20 µg/mL leupeptin) with ten strokes in a Teflon/glass Potter homogenizer and the homogenates were centrifuged at10 000 g for 10 min at 4°C. The supernatant was loaded on a 9-mLlinear 0.6–1.6 M sucrose gradient and centrifuged at 150 000 gfor 6 h in a P40ST rotor (Hitachi Koki, Co., Ltd., Hitachi, Japan).Twenty fractions of 0.5 mL each were collected from top of thegradient and the aliquots were analyzed by immunoblotting.

Binding of Syt I to syntaxin-1A and SNARE complex

The binding of Syt I to syntaxin-1A and SNARE complex wasmeasured as described (Gerona et al. 2000). Syntaxin-1A wasobtained by digestion of GST-fusion protein with thrombin.Syt I-(C2AB) (4 µg) was immunoprecipitated with the anti-Syt Iantibody and protein A Sepharose for 1 h at 4°C and washed withPBS containing 0.5% Triton X-100 3 times. Syntaxin-1A (3 µM)was incubated with Syt I-(C2AB) immobilized on protein ASepharose in the presence GST-Dvl-1 or GST-Dvl-1-(∆141–227)in 100 µL of reaction mixture (20 mm Hepes–NaOH, pH 7.4,150 mm NaCl, 0.5% (w/v) Triton X-100, 1 mm EGTA, and 1.1mm CaCl2) for 1.5 h at 4°C. After Syt I-(C2AB) was precipitatedby centrifugation, the precipitates were probed with anti-syntaxinantibody. In some experiments, binary SNARE complex includ-ing syntaxin-1A and GST-SNAP-25 was incubated withMBP-Syt I-(C2AB) immobilized to amylose resin in the presenceor absence of GST-Dvl-1 (WT) in the same reaction mixture asabove.

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Others

Yeast two-hybrid screening was carried out using Dvl-1 as a bait(Kishida et al. 1999). Immunoblotting, immunoprecipitation, andimmunohistochemical analyses were performed as described(Hino et al. 2003; Kishida et al. 2004). Retrovirus carryingFlag-HA-Dvl-1 was produced as described (Ikura et al. 2000).Sendai virus carrying the Myc-Dvl-1 and HA-Dvl-1-(∆141–227)gene were produced as described (Li et al. 2000). SPR spectrometryfor the binding of MBP-Syt I-(C2B) to GST-Dvl-1 wasperformed by the methods as described (Oshiro et al. 2002).

AcknowledgementsWe are grateful to Dr A. Mizoguchi (Mie University, Tsu, Japan)for helpful discussion, to Drs K. Yano-Aoki, A. Oshita, and A.Inoue (Hiroshima University, Hiroshima, Japan) for theirtechnical assistances, and to T. Yamashita (DNAVEC Corporation,Tsukuba, Japan) for Sendai virus construction. We also thank DrV. Haucke (George-August-University, Göttingen, Germany) fora plasmid. This work was supported by Grants-in-aid for ScienceResearch from Ministry of Education, Culture, Sports, Scienceand Technology of Japan (2004, 2005, 2006).

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Received: 23 August 2006 Accepted: 26 September 2006

Dvl regulates endo- and exocytotic processes through binding to synaptotagmin

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