Interaction of the ERC Family of RIM-binding Proteins with the Liprin-α Family of Multidomain Proteins
2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês
10.1074/jbc.m307561200
ISSN1083-351X
AutoresJaewon Ko, Moonseok Na, Se-Ho Kim, Jae-Ran Lee, Eunjoon Kim,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoLiprin-α/SYD-2 is a family of multidomain proteins with four known isoforms. One of the reported functions of liprin-α is to regulate the development of presynaptic active zones, but the underlying mechanism is poorly understood. Here we report that liprin-α directly interacts with the ERC (ELKS-Rab6-interacting protein-CAST) family of proteins, members of which are known to bind RIMs, the active zone proteins that regulate neurotransmitter release. In vitro results indicate that ERC2/CAST, an active zone-specific isoform, interacts with all of the known isoforms of liprin-α and that liprin-α1 associates with both ERC2 and ERC1b, a splice variant of ERC1 that distributes to both cytosolic and active zone regions. ERC2 colocalizes with liprin-α1 in cultured neurons and forms a complex with liprin-α1 in brain. Liprin-α1, when expressed alone in cultured neurons, shows a partial synaptic localization. When coexpressed with ERC2, however, liprin-α1 is redistributed to synaptic sites. Moreover, roughly the first half of ERC2, which contains the liprin-α-binding region, is sufficient for the synaptic localization of liprin-α1 while the second half is not. These results suggest that the interaction between ERC2 and liprin-α may be involved in the presynaptic localization of liprin-α and the molecular organization of presynaptic active zones. Liprin-α/SYD-2 is a family of multidomain proteins with four known isoforms. One of the reported functions of liprin-α is to regulate the development of presynaptic active zones, but the underlying mechanism is poorly understood. Here we report that liprin-α directly interacts with the ERC (ELKS-Rab6-interacting protein-CAST) family of proteins, members of which are known to bind RIMs, the active zone proteins that regulate neurotransmitter release. In vitro results indicate that ERC2/CAST, an active zone-specific isoform, interacts with all of the known isoforms of liprin-α and that liprin-α1 associates with both ERC2 and ERC1b, a splice variant of ERC1 that distributes to both cytosolic and active zone regions. ERC2 colocalizes with liprin-α1 in cultured neurons and forms a complex with liprin-α1 in brain. Liprin-α1, when expressed alone in cultured neurons, shows a partial synaptic localization. When coexpressed with ERC2, however, liprin-α1 is redistributed to synaptic sites. Moreover, roughly the first half of ERC2, which contains the liprin-α-binding region, is sufficient for the synaptic localization of liprin-α1 while the second half is not. These results suggest that the interaction between ERC2 and liprin-α may be involved in the presynaptic localization of liprin-α and the molecular organization of presynaptic active zones. The active zone is a specialized presynaptic plasma membrane region where synaptic vesicles dock and fuse. The cytoskeletal matrix assembled at active zones (CAZ) 1The abbreviations used are: CAZ, cytoskeletal matrix assembled at active zones; aa, amino acid; EGFP, enhanced green fluorescent protein; HA, hemagglutinin; GST, glutathione S-transferase; HEK, human embryonic kidney; DIV, days in vitro. is a complex proteinaceous structure implicated in the organization of the site of neurotransmitter release (1Garner C.C. Kindler S. Gundelfinger E.D. Curr. Opin. Neurobiol. 2000; 10: 321-327Crossref PubMed Scopus (170) Google Scholar, 2Dresbach T. Qualmann B. Kessels M.M. Garner C.C. Gundelfinger E.D. Cell Mol. Life Sci. 2001; 58: 94-116Crossref PubMed Scopus (158) Google Scholar), but little is known regarding the molecular mechanisms by which the CAZ is formed and maintained. Liprin-α is a family of multidomain proteins with four known isoforms (3Serra-Pages C. Kedersha N.L. Fazikas L. Medley Q. Debant A. Streuli M. EMBO J. 1995; 14: 2827-2838Crossref PubMed Scopus (293) Google Scholar, 4Serra-Pages C. Medley Q.G. Tang M. Hart A. Streuli M. J. Biol. Chem. 1998; 273: 15611-15620Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). Liprin-α was originally isolated as a binding partner of the LAR receptor protein tyrosine phosphatase (3Serra-Pages C. Kedersha N.L. Fazikas L. Medley Q. Debant A. Streuli M. EMBO J. 1995; 14: 2827-2838Crossref PubMed Scopus (293) Google Scholar). The presynaptic function of liprin-α was first demonstrated by a study on syd-2 (for synapse-defective), a Caenorhabditis elegans homolog of liprin-α (5Zhen M. Jin Y. Nature. 1999; 401: 371-375Crossref PubMed Scopus (298) Google Scholar). Deletion of syd-2 was found to result in the lengthening of the presynaptic active zones and impaired synaptic transmission (5Zhen M. Jin Y. Nature. 1999; 401: 371-375Crossref PubMed Scopus (298) Google Scholar). In addition, mutations in the Dliprin-α gene, a Drosophila homolog of liprin-α, led to changes in the size and shape of active zones (6Kaufmann N. DeProto J. Ranjan R. Wan H. Van Vactor D. Neuron. 2002; 34: 27-38Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), suggesting that liprin-α regulates the formation and/or maintenance of presynaptic active zones. However, the question remains as to how liprin-α regulates presynaptic development. Previous results suggest that the three following mechanisms may be involved. First, liprin-α associates with active zone components. Liprin-α directly interacts with RIMs (7Schoch S. Castillo P.E. Jo T. Mukherjee K. Geppert M. Wang Y. Schmitz F. Malenka R.C. Sudhof T.C. Nature. 2002; 415: 321-326Crossref PubMed Scopus (481) Google Scholar), active zone proteins that regulate neurotransmitter release (8Wang Y. Okamoto M. Schmitz F. Hofmann K. Sudhof T.C. Nature. 1997; 388: 593-598Crossref PubMed Scopus (537) Google Scholar, 9Wang Y. Sugita S. Sudhof T.C. J. Biol. Chem. 2000; 275: 20033-20044Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar), and is indirectly linked to the active zone protein Piccolo/aczonin through GITs (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar, 11Fenster S.D. Chung W.J. Zhai R. Cases-Langhoff C. Voss B. Garner A.M. Kaempf U. Kindler S. Gundelfinger E.D. Garner C.C. Neuron. 2000; 25: 203-214Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 12Kim S. Ko J. Shin H. Lee J.R. Lim C. Han J.H. Altrock W.D. Garner C.C. Gundelfinger E.D. Premont R.T. Kaang B.K. Kim E. J. Biol. Chem. 2003; 278: 6291-6300Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 13Wang X. Kibschull M. Laue M.M. Lichte B. Petrasch-Parwez E. Kilimann M.W. J. Cell Biol. 1999; 147: 151-162Crossref PubMed Scopus (153) Google Scholar, 14Cases-Langhoff C. Voss B. Garner A.M. Appeltauer U. Takei K. Kindler S. Veh R.W. De Camilli P. Gundelfinger E.D. Garner C.C. Eur. J. Cell Biol. 1996; 69: 214-223PubMed Google Scholar), which are GTPase-activating proteins for ADP-ribosylation factor small GTPases (15Turner C.E. West K.A. Brown M.C. Curr. Opin. Cell Biol. 2001; 13: 593-599Crossref PubMed Scopus (114) Google Scholar). Second, liprin-α may regulate membrane traffic at the active zone. In support of this hypothesis, liprin-α distributes to both cytosolic and active zone regions (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar, 16Wyszynski M. Kim E. Dunah A.W. Passafaro M. Valtschanoff J.G. Serra-Pages C. Streuli M. Weinberg R.J. Sheng M. Neuron. 2002; 34: 39-52Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). ARFs that are negatively modulated by liprin-α-binding GITs are expressed in neurons (17Hernandez-Deviez D.J. Casanova J.E. Wilson J.M. Nat. Neurosci. 2002; 5: 623-624Crossref PubMed Scopus (95) Google Scholar) and are known to regulate membrane traffic and the actin cytoskeleton (18Chavrier P. Goud B. Curr. Opin. Cell Biol. 1999; 11: 466-475Crossref PubMed Scopus (422) Google Scholar). Third, we recently reported that liprin-α associates with KIF1A (19Shin H. Wyszynski M. Huh K.H. Valtschanoff J.G. Lee J.R. Ko J. Streuli M. Weinberg R.J. Sheng M. Kim E. J. Biol. Chem. 2003; 278: 11393-11401Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), a neuronal kinesin motor protein (20Okada Y. Yamazaki H. Sekine-Aizawa Y. Hirokawa N. Cell. 1995; 81: 769-780Abstract Full Text PDF PubMed Scopus (496) Google Scholar). This suggests the possibility that liprin-α may be involved in the KIF1A-mediated long range transport of active zone components in neurons. However, additional data may be needed for a more comprehensive understanding of the liprin-α-dependent regulation of presynaptic development. Recently, a novel family of active zone proteins termed ERC (ELKS-Rab6-interacting protein-CAST) with two known members (ERC1 and ERC2/CAST) was identified as a binding partner of RIMs (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar, 22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar, 23Monier S. Jollivet F. Janoueix-Lerosey I. Johannes L. Goud B. Traffic. 2002; 3: 289-297Crossref PubMed Scopus (130) Google Scholar, 24Nakata T. Kitamura Y. Shimizu K. Tanaka S. Fujimori M. Yokoyama S. Ito K. Emi M. Genes Chromosomes Cancer. 1999; 25: 97-103Crossref PubMed Scopus (112) Google Scholar). There are two known splice variants of ERC1 that differ at their C termini (ubiquitous ERC1a and brain-specific ERC1b), whereas no splice variants are known for the brain-specific ERC2 (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar, 22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar). Intriguingly, ERC1b and ERC2 show different subcellular distribution patterns in neurons. ERC1b is expressed as a cytosolic protein as well as an active zone component, whereas ERC2 is an active zone-specific protein (22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar). Despite this difference, both ERC1b and ERC2 share a common motif at their C termini (the class II PDZ domain binding motif) through which they interact with the PDZ domain of RIMs (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar, 22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar). Functionally, a RIM1 mutant lacking the PDZ domain showed a reduced presynaptic targeting in cultured neurons, suggesting that ERC2 may play a role in the presynaptic localization of RIM1 (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar). In addition, ERC1 interacts with Rab6 (22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar, 23Monier S. Jollivet F. Janoueix-Lerosey I. Johannes L. Goud B. Traffic. 2002; 3: 289-297Crossref PubMed Scopus (130) Google Scholar), a small GTPase that is implicated in the regulation of post-Golgi membrane traffic in neurons (25Tixier-Vidal A. Barret A. Picart R. Mayau V. Vogt D. Wiedenmann B. Goud B. J. Cell Sci. 1993; 105: 935-947PubMed Google Scholar), suggesting that ERCs may regulate membrane traffic at the active zone. However, other than their association with RIMs, little is known of the role of ERCs in the organization of the active zone. Here, we provide in vitro and in vivo evidence that ERCs associate with liprin-α and we demonstrate that active zone-specific ERC2 promotes the synaptic accumulation of liprin-α in cultured neurons. These results suggest that the ERC-liprin-α interaction is involved in the presynaptic localization of liprin-α, active zone assembly, and perhaps in the regulation of membrane traffic at the active zone. Yeast Two-hybrid Screen and Assays—Two-hybrid screen and assays were performed as described previously (26Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (897) Google Scholar). Bait liprin-α4 (the region corresponding to aa 351-1202 of liprin-α1) and full-length GIT1 (aa 1-770) in pBHA have been described previously (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar). Regions of ERC2 (aa 1-701 and 833-957) and PDZ4-6 (aa 447-749) of GRIP2 were subcloned into the EcoRI-BamHI site of pBHA (a bait vector containing LexA DNA-binding domain). The following regions of ERC2 and liprin-α were subcloned into the indicated restriction sites of pGAD10, a prey vector (Clontech): ERC2 (aa 1-701, 1-463, 1-314, 1-183, 1-142, 118-383, 118-463, 118-535, 136-535, 136-463, 136-383, 136-309, and 136-183, BamHI-EcoRI); liprin-α1 (aa 1-848, 217-350, 351-512, 390-512, 351-486, 351-602, and 603-673, BamHI-EcoRI); liprin-α2 (aa 369-696. XhoI-EcoRI); and liprin-α3 (aa 333-645 from KIAA0654, EcoRI). Liprin-α1 (aa 1-221, 1-673, 217-553, 217-673, 351-553, 351-673, 513-673, and 674-1202) and liprin-α4 (the region corresponding to aa 351-1202 of liprin-α1) in pGAD10 have been described previously (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar). All of the constructs were verified by nucleotide sequencing. Expression Constructs—Full-length ERC2/KIAA0378 (aa 1-957) was subcloned into the KpnI-EcoRI site of GW1 vector (British Biotechnology). For EGFP-tagged ERC2 and liprin-α1 constructs, the following regions were subcloned into pEGFP-C1: ERC2 (aa 1-957, 1-954, 1-693, and 773-957, EcoRI-BamHI) and ERC1b/KIAA1081 (aa 1-992, full-length, EcoRI). The following expression constructs have been described previously: pMT2 HA-tagged liprin-α1 and liprin-α2 (3Serra-Pages C. Kedersha N.L. Fazikas L. Medley Q. Debant A. Streuli M. EMBO J. 1995; 14: 2827-2838Crossref PubMed Scopus (293) Google Scholar) and GW1 Myc-tagged GRIP1 and GRIP2 (27Mok H. Shin H. Kim S. Lee J.R. Yoon J. Kim E. J. Neurosci. 2002; 22: 5253-5258Crossref PubMed Google Scholar). Antibodies—The following fusion proteins were used for the generation of the following polyclonal antibodies: GST-ERC2 (aa 427-698; 1292 rabbit and 1296 guinea pig); H6-ERC2 (aa 725-957; 1284 rabbit); GST-liprin-α1 (aa 513-673; 1288 rabbit and 1290 guinea pig); and H6-GKAP (clone 2.1 region in GKAP; 1243 guinea pig) (28Kim E. Naisbitt S. Hsueh Y.P. Rao A. Rothschild A. Craig A.M. Sheng M. J. Cell Biol. 1997; 136: 669-678Crossref PubMed Scopus (433) Google Scholar). Specific antibodies were affinity-purified using immunogens immobilized in polyvinylidene difluoride membranes. The following antibodies have been described: EGFP 1173 guinea pig (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar); EGFP 1167 rabbit (29Choi J. Ko J. Park E. Lee J.R. Yoon J. Lim S. Kim E. J. Biol. Chem. 2002; 277: 12359-12363Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar); GIT1 1177 (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar); Piccolo 1203 (12Kim S. Ko J. Shin H. Lee J.R. Lim C. Han J.H. Altrock W.D. Garner C.C. Gundelfinger E.D. Premont R.T. Kaang B.K. Kim E. J. Biol. Chem. 2003; 278: 6291-6300Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar); Shank 3856 (30Naisbitt S. Kim E. Tu J.C. Xiao B. Sala C. Valtschanoff J. Weinberg R.J. Worley P.F. Sheng M. Neuron. 1999; 23: 569-582Abstract Full Text Full Text PDF PubMed Scopus (804) Google Scholar); GRIP2 1757 (31Wyszynski M. Kim E. Yang F.C. Sheng M. Neuropharmacology. 1998; 37: 1335-1344Crossref PubMed Scopus (65) Google Scholar); and PSD-95 SM55 (29Choi J. Ko J. Park E. Lee J.R. Yoon J. Lim S. Kim E. J. Biol. Chem. 2002; 277: 12359-12363Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Other antibodies were obtained from the following sources: HA (Roche Applied Science), RIM (Transduction Laboratories), Myc 9E10 (Santa Cruz Biotechnology), FLAG (Sigma), and His-probe (Santa Cruz Biotechnology). GST Pull-down, Immunoprecipitation, and Coclustering Assays in Heterologous Cells—For GST fusion proteins, the following regions of liprin-α were subcloned into pGEX-4T-1 (Amersham Biosciences): liprin-α1 (aa 351-512, 513-673, and 351-673, BamHI and EcoRI); liprin-α2 (aa 369-696, XhoI-EcoRI); liprin-α3 (aa 333-645, EcoRI); and liprin-α4 (the region corresponding to aa 351-673 of liprin-α1, BamHI-EcoRI). For hexahistidine fusion proteins containing ERC2, aa 1-957 and aa 725-957 of ERC2 were subcloned into the BamHI-EcoRI site of pET32a (Novagen) and pRSETB (Invitrogen), respectively. GST pull-down assays were performed as described previously (12Kim S. Ko J. Shin H. Lee J.R. Lim C. Han J.H. Altrock W.D. Garner C.C. Gundelfinger E.D. Premont R.T. Kaang B.K. Kim E. J. Biol. Chem. 2003; 278: 6291-6300Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). For immunoprecipitation, lysates of HEK293T cells transfected with EGFP-ERC2 and/or HA-liprin-α1 constructs were extracted in phosphate-buffered saline containing 1% Triton X-100, immunoprecipitated with HA (3 μg/ml) or EGFP (1173, 3 μg/ml) antibodies, and immunoblotted with EGFP (1167, 1 μg/ml) and HA (1 μg/ml) antibodies. Coclustering assay was performed as described previously (26Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Nature. 1995; 378: 85-88Crossref PubMed Scopus (897) Google Scholar). Immunoprecipitation in Rat Brain—In vivo coimmunoprecipitations were performed as described previously (32Wyszynski M. Valtschanoff J.G. Naisbitt S. Dunah A.W. Kim E. Standaert D.G. Weinberg R. Sheng M. J. Neurosci. 1999; 19: 6528-6537Crossref PubMed Google Scholar). Deoxycholate extracts of the crude synaptosomal fraction of adult rat brain were immunoprecipitated with liprin-α1 (1288, 5 μg/ml), ERC2 (1292, 5 μg/ml) or ERC2 (1296, 5 μg/ml), and rabbit or guinea pig IgG (5 μg/ml) antibodies. Immunoblotting of the immunoprecipitates was performed using the following antibodies: liprin-α1 (1288, 1 μg/ml); ERC2 (1292, 1 μg/ml); GIT1 (1:2000); GRIP2 (1 μg/ml); RIM (1 μg/ml); GKAP (1 μg/ml); and PSD-95 (1:1000). Neuron Culture, Transfection, and Immunocytochemistry—Cultured hippocampal neurons were prepared from embryonic (E18) rat brain as described previously (33Goslin K. Banker G. Banker G. Goslin K. Culturing Nerve Cells. The MIT Press, Cambridge, MA1991: 339-370Google Scholar). For double immunofluorescence staining, cultured neurons were incubated with combinations of ERC2 (1292; 1 μg/ml), liprin-α1 (1288) and liprin-α1 (1290, 2 μg/ml), Shank (3856, 1:300), and Piccolo (1203, 1 μg/ml) antibodies followed by Cy3- or fluorescein isothiocyanate-conjugated secondary antibodies (Jackson Immunoresearch). For targeting experiments, neurons were transfected at 5 days in vitro (DIV) using mammalian transfection kit (Stratagene). Two days after transfection (DIV 7), neurons were fixed with cold 100% methanol for 15 min and incubated with primary and secondary antibodies in phosphate-buffered saline containing 3% horse serum, 0.1% crystalline grade BSA, and 0.5% Triton X-100. The following antibodies were used for the immunocytochemistry of transfected neurons: EGFP (1173, 1 μg/ml), HA (1 μg/ml), Piccolo (1203, 1 μg/ml), and ERC2 (1292, 1 μg/ml). Image Acquisition and Quantitative Analysis—Fluorescent images were acquired using confocal laser-scanning microscope (LSM510, Zeiss) and analyzed using MetaMorph software (Universal Imaging). The image acquisition settings were kept constant during scanning. Images of distal thin neurites of cultured neurons from 3 to 10 independent experiments were captured for analysis. In image analysis, clusters were defined as discrete regions of immunoreactivity with an average fluorescence intensity at least 10-fold higher than that in background regions. Non-discrete regions were excluded from quantitative analysis. Colocalization between two puncta was defined as an overlap of >50% of each region, and colocalization analyses were performed blind. Approximately, 30-50 clusters were analyzed per cell and average values from each of the cells were used to obtain final mean ± S.E. Statistical significance was assessed using Student's t test. Characterization of the Interaction between ERC and Liprin-α by a Yeast Two-hybrid Assay—In a yeast two-hybrid screen (one million colonies) of a human brain cDNA library using liprin-α4 as bait, we obtained a fragment of ERC2/CAST containing roughly the first half of the protein (aa 34-535; full-length is 957 aa). The minimal liprin-α-binding region in ERC2 was narrowed down to aa 118-535 (Fig. 1A). Conversely, the minimal ERC-binding region in liprin-α1 was aa 351-602 (Fig. 1B), which is distinct from the minimal GIT1-binding region (aa 603-673; Fig. 1B) and the reported RIM-binding region (aa 200-350) in liprin-α1 (7Schoch S. Castillo P.E. Jo T. Mukherjee K. Geppert M. Wang Y. Schmitz F. Malenka R.C. Sudhof T.C. Nature. 2002; 415: 321-326Crossref PubMed Scopus (481) Google Scholar). It should be noted that the minimal GIT1-binding region in liprin-α1 (aa 603-673) was further narrowed down in this study from the one that we previously reported (aa 513-673) (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar). ERC2 interacted with all of the known liprin-α family members (liprin-α1, liprin-α2, liprin-α3, and liprin-α4) (Fig. 1C). ERC and Liprin-α Colocalize in Cultured Neurons and Heterologous Cells—To study ERC and liprin-α proteins in vivo, we generated ERC and liprin-α polyclonal antibodies against regions of ERC2 (aa 427-698, 1292 rabbit and 1296 guinea pig; aa 725-957, 1284 rabbit) and liprin-α1 (aa 513-673, 1288 rabbit and 1290 guinea pig) (Fig. 1, A and B). The 1292, 1296, and 1284 ERC2 antibodies reacted much more strongly with ERC2 than ERC1, whereas the 1288 and 1290 liprin-α1 antibodies reacted specifically with liprin-α1 rather than liprin-α2 in immunoblot analysis (Fig. 2, A and B). In brain, these antibodies recognized a single band of ERC2 and liprin-α1 whose apparent molecular weight matched that of these proteins expressed in heterologous cells (Fig. 2, C and D). In immunofluorescence staining of cultured hippocampal neurons at 21 DIV, ERC2 showed a punctate distribution pattern and the majority of the ERC2 puncta colocalized with those of Piccolo, a presynaptic marker protein (Fig. 2E). This finding suggests that ERC2 mainly distributes to synaptic sites and is consistent with the reported ultrastructural localization of ERC2 at active zones and the colocalization of ERC2 with other presynaptic marker proteins including Bassoon, RIM, and synapsin (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar, 22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar). In parallel immunohistochemical analyses, liprin-α1 exhibited a similar punctate distribution pattern, but only a portion of the liprin-α1 puncta colocalized with those of Piccolo (Fig. 2F). This finding suggests that liprin-α1 is partially synaptic, a result that is consistent with previous reports (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar, 16Wyszynski M. Kim E. Dunah A.W. Passafaro M. Valtschanoff J.G. Serra-Pages C. Streuli M. Weinberg R.J. Sheng M. Neuron. 2002; 34: 39-52Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). In double-labeled immunofluorescence staining assays of cultured neurons, ERC2 often colocalized with liprin-α1 at various subcellular sites (probably presynaptic sites) of mature neurons (DIV 21, Fig. 3A). Previously, ERC2 and liprin-α (liprin-α1 and liprin-α2) have been shown to distribute to small puncta in the growth cones of young cultured neurons (DIV 2-4) (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar, 16Wyszynski M. Kim E. Dunah A.W. Passafaro M. Valtschanoff J.G. Serra-Pages C. Streuli M. Weinberg R.J. Sheng M. Neuron. 2002; 34: 39-52Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar). When tested for their colocalization in young cultured neurons (DIV 2) in our experiments, both proteins were detected in a large number of puncta within the growth cones and some of the ERC2 puncta clearly colocalized with liprin-α1 puncta (Fig. 3B). These results suggest that ERC2 and liprin-α1 colocalize in vivo. In heterologous cells, singly expressed liprin-α1 was diffusely distributed throughout the cells (Fig. 3C), whereas ERC2 formed intracellular aggregates (Fig. 3D), which extensively colocalized with BiP, a marker protein for the endoplasmic reticulum (data not shown). When both proteins were coexpressed, a significant portion of liprin-α1 was redistributed to and colocalized with ERC2 aggregates (Fig. 3E). These results further suggest that ERC associates with liprin-α. ERC Forms a Complex with Liprin-α in Vitro and in Vivo—We tested for the interaction between ERC and liprin-α in a GST pull-down assay (Fig. 4A-D). GST fusion proteins containing liprin-α isoforms (liprin-α1, liprin-α2, liprin-α3, and liprin-α4) pulled down ERC2 expressed in heterologous cells (Fig. 4A). GST-liprin-α1 (aa 351-673) containing the minimal ERC-binding region but not the control GST fusion proteins brought down ERC2 (Fig. 4B). Conversely, GST-liprin-α1 pulled down all of the deletion variants of ERC2 that contain the minimal liprin-α-binding region (Fig. 4C), consistent with the yeast two-hybrid results (Fig. 1, A and B). Moreover, GST-liprin-α1 (aa 351-673) brought down hexahistidine (H6)-tagged ERC2 full-length (aa 1-957) but not H6-ERC2 (aa 725-957) lacking the liprin-α-binding region (Fig. 4D), suggesting that ERC directly interacts with liprin-α. In doubly transfected HEK293T cells, liprin-α1 formed a complex with the deletion variants of ERC2 that contain the minimal liprin-α-binding region (full-length, aa 1-954, and aa 1-693) but not with ERC2 (aa 773-957) (Fig. 4, E-I). In addition to liprin-α1, liprin-α2 formed a complex with ERC2 (Fig. 4J) and liprin-α1 formed a complex with ERC1 in addition to ERC2 (Fig. 4K). These results along with the yeast two-hybrid and GST pull-down results (Figs. 1C and 4A) suggest that the ERC family proteins (ERC1 and ERC2) interact with all of the known members of the liprin-α family. From the lysates of the crude synaptosomal fraction of adult rat brain, liprin-α1 (1288) antibodies brought down liprin-α1 and coprecipitated ERC2 and other liprin-α1-associated proteins including GIT1 and GRIP2 but not GKAP and PSD-95 (Fig. 4L). In addition, ERC2 antibodies coimmunoprecipitated liprin-α1, GIT1, GRIP2, and RIM but not PSD-95 and GKAP (Fig. 4, L and M). These results suggest that ERC and liprin-α form a complex in vivo. Overexpression of ERC2 Increases the Synaptic Levels of Liprin-α1 in Cultured Neurons—It has been reported that ERC2 plays a role in the presynaptic localization of RIM1 (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar). Thus, we tested whether ERC2 is also involved in the presynaptic localization of liprin-α. To this end, we first determined the subcellular distribution of ERC2 and liprin-α1 in cultured neurons. When expressed alone in cultured hippocampal neurons (DIV 7), EGFP-tagged ERC2 (EGFP-ERC2) showed a punctate distribution pattern along the length of neurites and EGFP-ERC2 clusters colocalized well with endogenous Piccolo (Piccolo-positive EGFP-ERC2 clusters = 97.5 ± 1.4%, n = 10 cells; EGFP-ERC2-positive Piccolo clusters = 69.4 ± 3.1%, n = 23, 1271 clusters, Fig. 5A). This finding suggests that despite the ∼5.5-fold higher expression level of EGFP-ERC2 compared with that of endogenous ERC2 (determined by comparison of immunofluorescence intensity, data not shown), EGFP-ERC2 mainly distributes to synaptic sites, similar to the distribution pattern of endogenous ERC2 (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar, 22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar). HA-tagged liprin-α1 (HA-liprin-α1) expressed alone in cultured neurons also showed a punctate distribution pattern, but each liprin-α1 cluster was often indiscrete and had an elongated shape. In addition, liprin-α1 clusters only partially colocalized with endogenous ERC2 or Piccolo clusters (Fig. 5B, an example of double staining for HA-liprin-α1 and ERC2). In quantitative analysis, only 20.7 ± 2.4% (n = 17) of ERC2 clusters and 10.1 ± 2.0% Piccolo clusters (n = 15) were HA-liprin-α1-positive. Measurement of the percentage of ERC2- or Piccolo-positive HA-liprin-α1 clusters was not attempted because liprin-α1 often formed indiscrete clusters along the length of neurites. These results suggest that HA-liprin-α1 is partially synaptic, similar to the distribution pattern of endogenous liprin-α1 (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar, 16Wyszynski M. Kim E. Dunah A.W. Passafaro M. Valtschanoff J.G. Serra-Pages C. Streuli M. Weinberg R.J. Sheng M. Neuron. 2002; 34: 39-52Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). We then determined the subcellular distribution of EGFP-ERC2 and HA-liprin-α1 coexpressed in cultured neurons. Intriguingly, HA-liprin-α1 showed a prominent colocalization with EGFP-ERC2 clusters (liprin-α1-positive ERC2 clusters = 90.8 ± 2.6%, n = 17, Fig. 5C; quantitation summarized in Fig. 5J). These results indicate that the synaptic localization of liprin-α1 is increased by ERC2 coexpression. To determine the regions of ERC2 that promote synaptic localization of liprin-α1, we employed deletion variants of EGFP-ERC2 (aa 1-954, 1-693, and 773-957; schematic diagrams shown in Fig. 4C). We first tested whether these variants are localized to synaptic sites by themselves. When compared with the full-length ERC2 (97.5% synaptic localization), ERC2 aa 1-954 and 1-693 showed a slightly reduced but still significant synaptic localization (83.2 ± 3.1% (n = 16) and 79.4 ± 4.2% (n = 8) of their clusters, respectively, were Piccolo-positive), whereas aa 773-957 of ERC2 showed a mainly diffuse distribution, similar to the previous results (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar) (Fig. 5, D-F; see quantitation in Fig. 5K). When tested for their ability to enhance liprin-α1 targeting, ERC2 (aa 1-954 and 1-693 but not aa 773-957) promoted the synaptic localization of liprin-α1 (91.0 ± 2.4% (n = 10) and 89.8 ± 4.3% (n = 7) of ERC clusters, respectively, were HA-liprin-α1-positive in Fig. 5, G-I; see quantitation in Fig. 5L), similar to that induced by full-length ERC2 (90.8 ± 2.6%). These results suggest that roughly the N-terminal half of ERC2 (aa 1-693) plays a major role in promoting the synaptic localization of liprin-α1. Functions of the Interaction between ERC and Liprin-α—Our in vitro data indicate that both ERC isoforms (ERC1b and ERC2) associate with liprin-α1 and that ERC2 associates with all of the known isoforms of liprin-α (Figs. 1 and 4). Different ERC isoforms show different subcellular distribution patterns. ERC1b distributes to both cytosolic and active zone regions while ERC2 localizes to active zones (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar, 22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar). Similar to ERC1b, liprin-α distributes to both synaptic and nonsynaptic sites (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar, 16Wyszynski M. Kim E. Dunah A.W. Passafaro M. Valtschanoff J.G. Serra-Pages C. Streuli M. Weinberg R.J. Sheng M. Neuron. 2002; 34: 39-52Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 19Shin H. Wyszynski M. Huh K.H. Valtschanoff J.G. Lee J.R. Ko J. Streuli M. Weinberg R.J. Sheng M. Kim E. J. Biol. Chem. 2003; 278: 11393-11401Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar), although the detailed subcellular distribution patterns of the four known liprin-α isoforms remain largely unknown. In characterizing the in vivo association of ERC and liprin-α in this study, we used antibodies against a subset of all of the known isoforms, namely ERC2 and liprin-α1. Therefore, the in vivo colocalization and coimmunoprecipitation of ERC2 and liprin-α1 revealed in this study (Figs. 3 and 4) may represent only a small fraction of the in vivo associations that may occur in various other subcellular compartments. The generation of additional isoform-specific antibodies will allow a more systematic analysis of their in vivo association in future studies. However, we may speculate on the functions of the ERC-liprin-α interaction observed in this study. Our data indicate that the synaptic levels of liprin-α1 are markedly increased by coexpression of ERC2 in cultured neurons (Fig. 5) and that this enhancement is mediated by the N-terminal half of ERC2 (Fig. 5), which contains the minimal liprin-α-binding region (Fig. 1). A simple interpretation of these results is that ERC2, through its interaction with liprin-α1, may recruit liprin-α1 to presynaptic active zones. However, this may not represent a physiological situation because it is not known whether the presynaptic levels of ERC2 are dynamically regulated. A more plausible hypothesis is that ERC2 may be involved in the stabilization of liprin-α at the presynaptic active zone, although further details remain to be determined. In the case of ERC1b, its association with liprin-α is likely to occur both in the cytosol and the active zone. At the active zone, ERC1b may be involved in the presynaptic stabilization of liprin-α in a manner similar to that hypothesized for ERC2. It is also possible that the ERC-liprin-α interaction may be involved in the regulation of membrane traffic at the active zone. Recent results have indicated that presynaptic active zones are formed by the insertion of preassembled active zone precursor vesicles into the presynaptic plasma membrane (34Shapira M. Zhai R.G. Dresbach T. Bresler T. Torres V.I. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2003; 38: 237-252Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 35Zhai R.G. Vardinon-Friedman H. Cases-Langhoff C. Becker B. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2001; 29: 131-143Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 36Ahmari S.E. Buchanan J. Smith S.J. Nat. Neurosci. 2000; 3: 445-451Crossref PubMed Scopus (487) Google Scholar). Thus, it is conceivable that ERC may associate with liprin-α on the surface of cytosolic precursor vesicles and that the ERC-liprin-α interaction may play a role in the surface delivery of these vesicles. This is supported by the observation that ERC1 binds to Rab6 (23Monier S. Jollivet F. Janoueix-Lerosey I. Johannes L. Goud B. Traffic. 2002; 3: 289-297Crossref PubMed Scopus (130) Google Scholar), a small GTPase implicated in the regulation of post-Golgi traffic in neurons (25Tixier-Vidal A. Barret A. Picart R. Mayau V. Vogt D. Wiedenmann B. Goud B. J. Cell Sci. 1993; 105: 935-947PubMed Google Scholar). Similarly, liprin-α is linked to ARFs, small GTPases known to regulate membrane traffic (18Chavrier P. Goud B. Curr. Opin. Cell Biol. 1999; 11: 466-475Crossref PubMed Scopus (422) Google Scholar) through GITs (10Ko J. Kim S. Valtschanoff J.G. Shin H. Lee J.R. Sheng M. Premont R.T. Weinberg R.J. Kim E. J. Neurosci. 2003; 23: 1667-1677Crossref PubMed Google Scholar). In this context, an interesting possibility is that the interaction between ERC and liprin-α may mediate the integration of the Rab6 and ARF signaling pathways for the regulation of membrane traffic. And finally, the ERC-liprin-α interaction may assist in a kinesin-mediated neuronal transport. We recently reported that the KIF1A kinesin motor associates with liprin-α and liprin-α-interacting RIMs, which suggests the possibility that liprin-α links KIF1A to cargo vesicles containing various liprin-α-binding proteins including RIMs and ERCs (19Shin H. Wyszynski M. Huh K.H. Valtschanoff J.G. Lee J.R. Ko J. Streuli M. Weinberg R.J. Sheng M. Kim E. J. Biol. Chem. 2003; 278: 11393-11401Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). This hypothesis is supported by immunohistochemical studies on cultured neurons that have indicated that both ERC1b and liprin-α are detected in neuronal cell bodies in addition to synaptic sites (16Wyszynski M. Kim E. Dunah A.W. Passafaro M. Valtschanoff J.G. Serra-Pages C. Streuli M. Weinberg R.J. Sheng M. Neuron. 2002; 34: 39-52Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar). ERC2 colocalizes with liprin-α1 in fine puncta in growth cones of young neurons (Fig. 3B), which are thought to represent active zone precursor vesicles (34Shapira M. Zhai R.G. Dresbach T. Bresler T. Torres V.I. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2003; 38: 237-252Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 35Zhai R.G. Vardinon-Friedman H. Cases-Langhoff C. Becker B. Gundelfinger E.D. Ziv N.E. Garner C.C. Neuron. 2001; 29: 131-143Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). In addition, ERC2 and RIM1 are detected in vesicles immunoisolated with antibodies against Bassoon (a good marker of active zone precursor vesicles) (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar). ERC-Liprin-α Interaction and Organization of the CAZ—The interaction of liprin-α with RIMs is mediated by a region of liprin-α (aa 200-350 in liprin-α1) that is distinct from the ERC-binding region (aa 351-602, Fig. 1) and that associates with the C2B domain of RIMs (7Schoch S. Castillo P.E. Jo T. Mukherjee K. Geppert M. Wang Y. Schmitz F. Malenka R.C. Sudhof T.C. Nature. 2002; 415: 321-326Crossref PubMed Scopus (481) Google Scholar). This finding suggests that ERC2, in addition to its direct interaction with the PDZ domain of RIMs through its C terminus, is indirectly linked to RIMs through liprin-α. Although the function of this tripartite interaction remains to be determined, one possibility is that ERC may employ two distinct molecular mechanisms, direct and indirect, to ensure the synaptic accumulation of RIMs, which are important regulators of neurotransmitter release and presynaptic long-term potentiation (7Schoch S. Castillo P.E. Jo T. Mukherjee K. Geppert M. Wang Y. Schmitz F. Malenka R.C. Sudhof T.C. Nature. 2002; 415: 321-326Crossref PubMed Scopus (481) Google Scholar, 37Castillo P.E. Schoch S. Schmitz F. Sudhof T.C. Malenka R.C. Nature. 2002; 415: 327-330Crossref PubMed Scopus (328) Google Scholar, 38Koushika S.P. Richmond J.E. Hadwiger G. Weimer R.M. Jorgensen E.M. Nonet M.L. Nat. Neurosci. 2001; 4: 997-1005Crossref PubMed Scopus (254) Google Scholar). In support of the role of liprin-α in the ERC-dependent synaptic localization of RIMs, we note that the RIM1 mutant lacking the PDZ domain shows some (although mainly diffuse) synaptic localization in cultured neurons (21Ohtsuka T. Takao-Rikitsu E. Inoue E. Inoue M. Takeuchi M. Matsubara K. Deguchi-Tawarada M. Satoh K. Morimoto K. Nakanishi H. Takai Y. J. Cell Biol. 2002; 158: 577-590Crossref PubMed Scopus (237) Google Scholar), suggesting that regions of RIMs other than the PDZ domain, such as their Zn2+-fingers and C2 domains, may assist its synaptic localization. In addition, RIM/UNC-10 is mislocalized in C. elegans liprin-α/SYD-2 mutants (7Schoch S. Castillo P.E. Jo T. Mukherjee K. Geppert M. Wang Y. Schmitz F. Malenka R.C. Sudhof T.C. Nature. 2002; 415: 321-326Crossref PubMed Scopus (481) Google Scholar). Conversely, the abundance and solubility of ERC and liprin-α proteins are not changed in RIM1 knock-out mice (7Schoch S. Castillo P.E. Jo T. Mukherjee K. Geppert M. Wang Y. Schmitz F. Malenka R.C. Sudhof T.C. Nature. 2002; 415: 321-326Crossref PubMed Scopus (481) Google Scholar, 22Wang Y. Liu X. Biederer T. Sudhof T.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14464-14469Crossref PubMed Scopus (181) Google Scholar). There are only a few known active zone scaffold (or CAZ) proteins including Piccolo, Bassoon, ERC, RIM, Munc13, and liprin-α (1Garner C.C. Kindler S. Gundelfinger E.D. Curr. Opin. Neurobiol. 2000; 10: 321-327Crossref PubMed Scopus (170) Google Scholar, 2Dresbach T. Qualmann B. Kessels M.M. Garner C.C. Gundelfinger E.D. Cell Mol. Life Sci. 2001; 58: 94-116Crossref PubMed Scopus (158) Google Scholar), but the molecular mechanisms that link them together remain largely unknown. It is interesting to note that our finding of the ERC-liprin-α interaction provides a molecular link to bring some of the CAZ components together: (RIM or ERC)-liprin-α-GIT-Piccolo. Although this may not be a complete picture, our work may provide a useful first step toward a more comprehensive understanding of the molecular organization of the active zone. We thank the Kazusa DNA Research Institute for their generous gift of the KIAA clones (KIAA0378, KIAA0654, and KIAA1081).
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