| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Biol Chem, Vol. 273, Issue 41, 26269-26272, October 9, 1998
,,§,,,,,¶, and
**
From the Takai Biotimer Project, ERATO, Japan Science
and Technology Corporation, c/o JCR Pharmaceuticals Co. Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-2241, Japan and the Department of
Molecular Biology and Biochemistry, Osaka University Medical School,
2-2 Yamada-Oka, Suita, Osaka 565, Japan
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
|
|---|
PSD-95/SAP90 is a synaptic membrane-associated guanylate kinase with three PDZ, one SH3, and one guanylate kinase (GK) domain. PSD-95/SAP90 binds various proteins through the PDZ domains and organizes synaptic junctions. PSD-95/SAP90 also interacts with the postsynaptic density (PSD) fraction-enriched protein, named SAPAP (also called GKAP and DAP), through the GK domain. SAPAP is Triton X-100-insoluble and recruits PSD-95/SAP90 into the Triton X-100-insoluble fraction in the transfected cells, suggesting that SAPAP may fix PSD-95/SAP90 to the PSD. Here we report a novel protein interacting with the GK domain of PSD-95/SAP90, BEGAIN. BEGAIN is specifically expressed in brain and enriched in the PSD fraction. BEGAIN is Triton X-100-soluble in the transfected cells but is recruited to the Triton X-100-insoluble fraction by SAPAP when coexpressed with PSD-95/SAP90. BEGAIN may be a novel PSD component associated with the core complex of PSD-95/SAP90 and SAPAP.
| |
INTRODUCTION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
|
|---|
Synaptic junctions accumulate molecules involved in neurotransmissions. The organization of components of synaptic junctions is orchestrated by several scaffolding proteins (reviewed in Refs. 1-5). PSD-95/SAP90 is a prototypic synaptic scaffolding protein and has three isoforms, SAP97, PSD-93/chapsyn, and SAP102 (6-12). They have three PDZ, one SH3, and one guanylate kinase (GK)1 domain. The PDZ domain is a protein-interacting module (reviewed in Refs. 13-16), and those of PSD-95/SAP90 bind to the C termini of NMDA receptors, K+ channels, neuroligins, synGAP, and CRIPT (8-12, 17-21). The SH3 domain is also a protein-interacting module (reviewed in Ref. 22), but the molecules interacting with the SH3 domain of PSD-95/SAP90 have not been identified. The GK domain shows a homology to the yeast guanylate kinase. Three groups including ours have recently identified a protein interacting with the GK domain of PSD-95/SAP90 and named it GKAP, SAPAP, and DAP, respectively (23-25). SAPAP is neuronal-specific and tightly attached to the synaptic plasma membranes, and in the transfected cells, SAPAP recruits PSD-95/SAP90 to the plasma membranes. We have subsequently identified a protein interacting with SAPAP and named it S-SCAM (26). S-SCAM has five PDZ domains and binds to NMDA receptors and neuroligins. PSD-95/SAP90, SAPAP, and S-SCAM may cooperate to assemble receptors and cell adhesion molecules at the synaptic junctions.
In the yeast two-hybrid screening using the GK domain of PSD-95/SAP90 as bait, we have identified two novel proteins in addition to SAPAP. One of them is ubiquitously expressed, and the other is specifically expressed in brain. In this study, we have characterized this brain-specific protein and named it BEGAIN (brain-enriched guanylate-kinase domain-associated protein). BEGAIN is expressed in neurons and rather enriched at synaptic junctions. BEGAIN may be involved also in the organization of the components of synaptic junctions.
| |
MATERIALS AND METHODS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
|
|---|
Yeast Two-hybrid Screening and cDNA Cloning--
Rat brain
yeast two-hybrid library was constructed using pVP16 vector and
screened (27). Rat brain cDNA libraries were screened with the
[
-32P]dCTP-labeled random-primed probes (27).
Construction of Expression Vectors-- Various expression vectors were constructed by conventional molecular biology techniques and a polymerase chain reaction method using pBTM116, pBTM116-2, pClneo (Promega), pClneo Myc, pGex5X-3 (Amersham Pharmacia Biotech), pGex4T-1 (Amersham Pharmacia Biotech), and pGexKG. pBTM116-2 and pClneo Myc were constructed from pBTM116 and pClneo, respectively (26). pBTM116 PSD-95-3, -9, -11, SAP97-5, p55-1, CASK-10, -11, ZO-1-1, dlg-2, pGexKG PSD-95-1, -2, and -4 were described previously (19, 24). The following constructs contained the following amino acid (aa) residues of BEGAIN-2; pGex5X-3 BEGAIN-2-4, 407-611; pClneo Myc BEGAIN-2-1, 1-611; pClneo Myc BEGAIN-2-2, 1-226; pClneo Myc BEGAIN-2-3, 216-415; pClneo Myc BEGAIN-2-4, 407-611; pClneo BEGAIN-2, 1-611; and pBTM116 BEGAIN-2-8, 501-611. pGex4T-1 PSD-95-17 and -18 contained the residues 435-495 and 534-724 of PSD-95/SAP90, respectively. pClneo BEGAIN-1 and pClneo PSD-95 were constructed by ligating the NotI fragment from p1314-196 at NotI site and the EcoRI/XbaI fragment from pCMV PSD-95-1 at EcoRI/XbaI sites of pClneo, respectively.
Antibodies-- A rabbit antibody was raised against the product of pGex5X-3 BEGAIN-2-4 and purified (24). Monoclonal anti-PSD-95/SAP90 and anti-Myc-tag and polyclonal anti-SAPAP1 and anti-PSD-95/SAP90 antibodies were described previously (24). The anti-GABAA receptor antibody was purchased from Chemicon.
Preparation of COS Cell Extracts-- COS cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum under 10% CO2 at 37 °C and transfected with DEAE-dextran (24). COS cells of one 10-cm plate were homogenized in 0.5 ml of 20 mM Hepes/NaOH, pH 8.0, containing 100 mM NaCl and 1% (w/v) Triton X-100, and centrifuged at 100,000 × g for 15 min. The supernatant was diluted with 2 volumes of 20 mM Hepes/NaOH, pH 8.0, and used as COS cell extracts.
In Vitro Binding Experiment Using GST-fusion Proteins and COS Cell Extracts-- 1.5-ml aliquots of the COS cell extracts were incubated with 200 pmol of various glutathione S-transferase (GST) constructs fixed on 20 µl of glutathione-Sepharose 4B beads. After the beads were washed with Buffer A (20 mM Hepes/NaOH, pH 8.0, containing 33 mM NaCl and 0.33% (w/v) Triton X-100), proteins on the beads were detected with the immunoblottings.
Stable Transformants-- CHO cells were transfected with pClneo PSD-95 by a calcium phosphate method using Mammalian transfection kit (Stratagene) and cultured in DMEM with 10% (w/v) fetal bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin, 40 µg/ml L-proline, and 700 µg/ml Geneticin (Life Technologies, Inc.). After the three cycles of the subcloning, the cells expressing PSD-95/SAP90 were selected.
Coimmunoprecipitation-- Crude synaptosomes were prepared from four rat brains (26). Synaptosomes were homogenized in 16 ml of 20 mM Hepes/NaOH, pH 8.0, containing 6 M urea and 1% (w/v) Triton X-100, and centrifuged at 100,000 × g for 30 min. The supernatant was dialyzed against 5 liters of 20 mM Hepes/NaOH, pH 8.0, containing 100 mM NaCl with one exchange and centrifuged at 100,000 × g for 30 min to remove the precipitate. 4-ml aliquots of the supernatant were incubated with various antibodies fixed on 20 µl of protein G-Sepharose Fast Flow beads. After the beads were washed with Buffer A, proteins on the beads were detected with the immunoblottings.
Subcellular Fractionation of CHO Cells-- CHO cells stably expressing PSD-95/SAP90 were transfected with various eukaryote expression vectors using TransFast Transfection Reagent (Promega). After 48 h, the cells were collected and homogenized in 300 µl of 20 mM Hepes/NaOH, pH 7.4, by sonication. 80 µl of the homogenates were kept for the analysis, and the remaining samples were centrifuged at 100,000 × g for 30 min to separate the supernatant and the pellet. The pellet was homogenized in 220 µl of 20 mM Hepes/NaOH, pH 7.4, containing 1% (w/v) Triton X-100, and centrifuged at 10,000 × g for 10 min to separate the supernatant and the pellet.
Miscellaneous Procedures-- Other procedures including subcellular fractionation of rat brain, primary cultures of rat hippocampal neurons, immunocytostaining, SDS-polyacrylamide gel electrophoresis, and protein determination were performed as described (24). Northern and Western blottings were performed using multiple tissue Northern blots (CLONTECH) and ECL reagents (Amersham Pharmacia Biotech), respectively.
| |
RESULTS AND DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
|
|---|
Identification of BEGAIN--
We performed the yeast two-hybrid
screening using the SH3 and GK domains (the residues 430-724) of
PSD-95/SAP90 as bait. We obtained six positive clones from 1.4 × 106 clones of a rat brain yeast two-hybrid library. Four
clones were SAPAPs. One clone, pPrey1294, was ubiquitously expressed.
The full-length clone of this gene encoded a protein composed of 1,822 aa, which was homologous to SPA-1, a GTPase-activating protein for Ran
(28). The sequence of this gene was submitted to GenBankTM
with an accession number of AF026504. The remaining clone, pPrey1314,
was brain-specific. To obtain a full-length clone, we screened two
ZAP (Stratagene) and one gt11 (CLONTECH) rat brain cDNA libraries and obtained 200 positive clones. Two clones, p1314-113 and p1314-196, contained a putative initiation codon and a
termination codon. p1314-196 encoded a protein composed of 605 aa (Fig.
1A). p1314-113 encoded a
protein composed of 611 aa. We named these proteins BEGAIN-1 and -2, respectively. The residues 1-8 of BEGAIN-1 and the residues 1-14 of
BEGAIN-2 were different, whereas the remaining residues were the same
(Fig. 1B). The sequences around the initiation codons of
BEGAIN-1 and -2 were confirmed by the sequencing of three and two
independent clones, respectively. To further confirm whether these
clones were the full-length ones, COS cell extracts transfected with pClneo BEGAIN-1 and -2 were immunoblotted with the antibody against residues 407-611 of BEGAIN-2. A protein with a
Mr of 83,000 was detected in the COS cells
transfected with pClneo BEGAIN-1, whereas a protein with a
Mr of 74,000 was detected in the COS cells
transfected with pClneo BEGAIN-2 (Fig.
2A). The reason BEGAIN-1 with
605 aa migrated slower than BEGAIN-2 with 611 aa on SDS-polyacrylamide gel electrophoresis is unknown. In the synaptic plasma membrane (SPM)
fraction, proteins with Mr of 83,000 and 74,000 were detected (Fig. 2A). Therefore, pClneo BEGAIN-1 and -2 encoded proteins with the same sizes as the native proteins. The
software COILS version 2.1 predicted a coiled-coil structure in the
N-terminal region of BEGAIN-1. This region showed a weak homology to
the repeats of Drosophila CLIP-190, which were also
predicted to be coiled-coil regions (29).
|
|
Tissue and Subcellular Distribution of BEGAIN-- The Northern blot analysis revealed a 3.4-kilobase pair message only in brain (Fig. 2B). No message was detected in heart, spleen, lung, liver, kidney, skeletal muscles, or testis. In the subcellular fractionation of rat brain, BEGAIN was detected mainly in the SPM and PSD fractions (Fig. 2C).
Interaction of BEGAIN with PSD-95/SAP90-- To confirm the interaction of BEGAIN with PSD-95/SAP90, BEGAIN was immunoprecipitated from the crude synaptosome fraction. PSD-95/SAP90 was coprecipitated with BEGAIN as well as with SAPAP (Fig. 3A). To determine the BEGAIN-interacting region of PSD-95/SAP90, the extracts of COS cell expressing Myc-tagged BEGAIN-2 were incubated with GST constructs containing various regions of PSD-95/SAP90. The GK domain interacted with BEGAIN, whereas the PDZ and SH3 domains did not (Fig. 3B). The same results were obtained using BEGAIN-1 (data not shown). The GK domain of PSD-95/SAP90 is necessary and sufficient for the interaction with BEGAIN. In rat hippocampal neurons, BEGAIN showed a somatodendritic pattern and was colocalized with PSD-95/SAP90 (Fig. 3C), whereas the staining of GABAA receptor did not overlap with that of BEGAIN (data not shown). These findings suggest that BEGAIN is localized at the excitatory synapses and interacts with PSD-95/SAP90 in vivo.
|
Interaction of BEGAIN with Other MAGUKs--
To test the
interactions of BEGAIN with other MAGUKs, 
-galactosidase assay was
performed using the prey clone of BEGAIN and various baits.
PSD-95/SAP90, SAP97, and Drosophila dlg-A interacted with
BEGAIN, whereas p55, ZO-1, or CASK did not (Table
I). Although the interaction of BEGAIN
with PSD-95/SAP90 was much weaker than that of SAPAP with PSD-95/SAP90,
these results suggest that BEGAIN specifically interacts with
PSD-95/SAP90 and its isoforms.
|
Binding of BEGAIN to SAPAP via PSD-95/SAP90-- Because BEGAIN and SAPAP interact with the same region of PSD-95/SAP90, BEGAIN and SAPAP may compete for the binding to PSD-95/SAP90 and cannot interact with PSD-95/SAP90 simultaneously. If this is the case, BEGAIN cannot participate in the core complex formation of PSD-95/SAP90 and SAPAP in the PSD. PSD-95/SAP90 is multimerized through the disulfide-linkage at the N terminus (30). Therefore, PSD-95/SAP90 binding BEGAIN may interact with another PSD-95/SAP90 binding SAPAP. If such interactions take place in vivo, BEGAIN can be a component of the core complex of PSD-95/SAP90 and SAPAP. In the transfected HEK293 cells, PSD-95/SAP90 is translocated from the cytosol to the membrane in the presence of SAPAP (24). We have confirmed this observation using the CHO cells stably expressing PSD-95/SAP90 (CHO/PSD-95 cells). In CHO/PSD-95 cells, PSD-95/SAP90 was distributed in the cytosol and membrane fractions and was solubilized in the Triton X-100 extraction (Fig. 4A). When SAPAP was transfected in these cells, SAPAP was distributed in the Triton X-100-insoluble fraction, and PSD-95/SAP90 was recruited into the Triton X-100-insoluble fraction (Fig. 4B). BEGAIN was distributed in the Triton X-100-soluble fraction and did not affect the distribution of PSD-95/SAP90 (Fig. 4C). When BEGAIN and SAPAP were coexpressed in CHO/PSD-95 cells, BEGAIN was recruited into the Triton X-100-insoluble fraction with PSD-95/SAP90 (Fig. 4D). When BEGAIN and SAPAP were coexpressed in the wild CHO cells, BEGAIN remained in the Triton X-100-soluble fraction (Fig. 4E). These findings suggest that BEGAIN binds to SAPAP via PSD-95/SAP90 and forms a ternary complex in vivo.
|
| |
FOOTNOTES |
|---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF064869 (BEGAIN-1) and AF064868 (BEGAIN-2).
§ Present address: Shionogi Pharmaceuticals Co. Ltd., 5-12-4 Sagizu, Fukushima-ku, Osaka 553-0002, Japan.
¶ Present address: Dept. of Agriculture, Ibaragi University, 3-21-1 Chuoh, Ami-machi, Inashiki, Ibaragi 300-0332, Japan.
** To whom correspondence should be addressed. Tel.: 81-06-879-3410; Fax: 81-06-879-3419; E-mail: ytakai{at}molbio.med.osak-u.ac.jp.
The abbreviations used are:
GK, guanylate
kinase; NMDA, N-methyl-D-aspartateBEGAIN, brain-enriched guanylate kinase-associated proteinGST, glutathione
S-transferaseDMEM, Dulbecco's modified Eagle's mediumMAGUK, membrane-associated guanylate kinasePDZ, PSD-95/Dlg/ZO-1PSD, postsynaptic densityaa, amino acidSPM, synaptic plasma membraneGABA, 
-aminobutyric acid.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results & Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Richter, K. K. Murai, C. Bourgin, D. T. Pak, and E. B. Pasquale The EphA4 Receptor Regulates Neuronal Morphology through SPAR-Mediated Inactivation of Rap GTPases J. Neurosci., December 19, 2007; 27(51): 14205 - 14215. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Asaba, T. Hanada, A. Takeuchi, and A. H. Chishti Direct Interaction with a Kinesin-related Motor Mediates Transport of Mammalian Discs Large Tumor Suppressor Homologue in Epithelial Cells J. Biol. Chem., February 28, 2003; 278(10): 8395 - 8400. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Olsen and D. S. Bredt Functional Analysis of the Nucleotide Binding Domain of Membrane-associated Guanylate Kinases J. Biol. Chem., February 21, 2003; 278(9): 6873 - 6878. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Paarmann, O. Spangenberg, A. Lavie, and M. Konrad Formation of Complexes between Ca2+{middle dot}Calmodulin and the Synapse-associated Protein SAP97 Requires the SH3 Domain-Guanylate Kinase Domain-connecting HOOK Region J. Biol. Chem., October 18, 2002; 277(43): 40832 - 40838. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Yao, J. Iida, W. Nishimura, and Y. Hata Synaptic and Nuclear Localization of Brain-Enriched Guanylate Kinase-Associated Protein J. Neurosci., July 1, 2002; 22(13): 5354 - 5364. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. McMahon, R. Legouis, J.-L. Vonesch, and M. Labouesse Assembly of C. elegans apical junctions involves positioning and compaction by LET-413 and protein aggregation by the MAGUK protein DLG-1 J. Cell Sci., March 8, 2002; 114(12): 2265 - 2277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Li, O. Spangenberg, I. Paarmann, M. Konrad, and A. Lavie Structural Basis for Nucleotide-dependent Regulation of Membrane-associated Guanylate Kinase-like Domains J. Biol. Chem., February 1, 2002; 277(6): 4159 - 4165. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Imamura, S. Maeda, T. Doi, and Y. Fujiyoshi Ligand Binding of the Second PDZ Domain Regulates Clustering of PSD-95 with the Kv1.4 Potassium Channel J. Biol. Chem., January 25, 2002; 277(5): 3640 - 3646. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kawabe, H. Nakanishi, M. Asada, A. Fukuhara, K. Morimoto, M. Takeuchi, and Y. Takai Pilt, a Novel Peripheral Membrane Protein at Tight Junctions in Epithelial Cells J. Biol. Chem., December 14, 2001; 276(51): 48350 - 48355. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Shin, Y.-P. Hsueh, F.-C. Yang, E. Kim, and M. Sheng An Intramolecular Interaction between Src Homology 3 Domain and Guanylate Kinase-Like Domain Required for Channel Clustering by Postsynaptic Density-95/SAP90 J. Neurosci., May 15, 2000; 20(10): 3580 - 3587. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hirao, Y. Hata, I. Yao, M. Deguchi, H. Kawabe, A. Mizoguchi, and Y. Takai Three Isoforms of Synaptic Scaffolding Molecule and Their Characterization. MULTIMERIZATION BETWEEN THE ISOFORMS AND THEIR INTERACTION WITH N-METHYL-D-ASPARTATE RECEPTORS AND SAP90/PSD-95-ASSOCIATED PROTEIN J. Biol. Chem., January 28, 2000; 275(4): 2966 - 2972. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Kawabe, Y. Hata, M. Takeuchi, N. Ide, A. Mizoguchi, and Y. Takai nArgBP2, a Novel Neural Member of Ponsin/ArgBP2/Vinexin Family That Interacts with Synapse-associated Protein 90/Postsynaptic Density-95-associated Protein (SAPAP) J. Biol. Chem., October 22, 1999; 274(43): 30914 - 30918. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Yao, Y. Hata, K. Hirao, M. Deguchi, N. Ide, M. Takeuchi, and Y. Takai Synamon, a Novel Neuronal Protein Interacting with Synapse-associated Protein 90/Postsynaptic Density-95-associated Protein J. Biol. Chem., September 24, 1999; 274(39): 27463 - 27466. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Yao, Y. Hata, N. Ide, K. Hirao, M. Deguchi, H. Nishioka, A. Mizoguchi, and Y. Takai MAGUIN, a Novel Neuronal Membrane-associated Guanylate Kinase-interacting Protein J. Biol. Chem., April 23, 1999; 274(17): 11889 - 11896. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hanada, L. Lin, E. V. Tibaldi, E. L. Reinherz, and A. H. Chishti GAKIN, a Novel Kinesin-like Protein Associates with the Human Homologue of the Drosophila Discs Large Tumor Suppressor in T Lymphocytes J. Biol. Chem., September 8, 2000; 275(37): 28774 - 28784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. L. Nix, A. H. Chishti, J. M. Anderson, and Z. Walther hCASK and hDlg Associate in Epithelia, and Their Src Homology 3 and Guanylate Kinase Domains Participate in Both Intramolecular and Intermolecular Interactions J. Biol. Chem., December 22, 2000; 275(52): 41192 - 41200. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
80 °C for 9 days. Lane 1,
heart; lane 2, brain; lane 3, spleen; lane
4, lung; lane 5, liver; lane 6, skeletal
muscle; lane 7, kidney; lane 8, testis.
C, Western blot analysis of the subcellular fractions of rat
brain. Equal aliquots of the subcellular fractions of rat brain (25 µg of protein each) were immunoblotted with the anti-BEGAIN antibody.
Lane 1, the homogenate; lane 2, the
nuclear pellet; lane 3, the crude synaptosome; lane
4, the cytosolic synaptosome; lane 5, the crude
synaptosomal pellet; lane 6, the crude synaptic vesicle;
lane 7, the lysed synaptosomal membrane; lane 8,
the SPM; lane 9, the 0.5% (w/v) Triton X-100-soluble
fraction of the SPM; lane 10, the 0.5% (w/v) Triton
X-100-insoluble fraction of the SPM; lane 11, the 1% (w/v)
Triton X-100-soluble fraction of the SPM; lane 12, the 1%
(w/v) Triton X-100-insoluble fraction of the SPM.
