Erbin Is a Protein Concentrated at Postsynaptic Membranes That Interacts with PSD-95

Neuregulin is a factor essential for synapse-specific transcription of acetylcholine receptor genes at the neuromuscular junction. Its receptors, ErbB receptor tyrosine kinases, are localized at the postjunctional membrane presumably to ensure localized signaling. However, the molecular mechanisms underlying synaptic localization of ErbBs are unknown. Our recent studies indicate that ErbB4 interacts with postsynaptic density (PSD)-95 (SAP90), a PDZ domain-containing protein that does not interact with ErbB2 or ErbB3. Using as bait the ErbB2 C terminus, we identified Erbin, another PDZ domain-containing protein that interacts specifically with ErbB2. Erbin is concentrated in postsynaptic membranes at the neuromuscular junction and in the central nervous system, where ErbB2 is concentrated. Expression of Erbin increases the amount of ErbB2 labeled by biotin in transfected cells, suggesting that Erbin is able to increase ErbB2 surface expression. Furthermore, we provide evidence that Erbin interacts with PSD-95 in both transfected cells and synaptosomes. Thus ErbB proteins can interact with a network of PDZ domain-containing proteins. This interaction may play an important role in regulation of neuregulin signaling and/or subcellular localization of ErbB proteins. Neuregulin is a factor essential for synapse-specific transcription of acetylcholine receptor genes at the neuromuscular junction. Its receptors, ErbB receptor tyrosine kinases, are localized at the postjunctional membrane presumably to ensure localized signaling. However, the molecular mechanisms underlying synaptic localization of ErbBs are unknown. Our recent studies indicate that ErbB4 interacts with postsynaptic density (PSD)-95 (SAP90), a PDZ domain-containing protein that does not interact with ErbB2 or ErbB3. Using as bait the ErbB2 C terminus, we identified Erbin, another PDZ domain-containing protein that interacts specifically with ErbB2. Erbin is concentrated in postsynaptic membranes at the neuromuscular junction and in the central nervous system, where ErbB2 is concentrated. Expression of Erbin increases the amount of ErbB2 labeled by biotin in transfected cells, suggesting that Erbin is able to increase ErbB2 surface expression. Furthermore, we provide evidence that Erbin interacts with PSD-95 in both transfected cells and synaptosomes. Thus ErbB proteins can interact with a network of PDZ domain-containing proteins. This interaction may play an important role in regulation of neuregulin signaling and/or subcellular localization of ErbB proteins. postsynaptic density ErbB2-binding protein (Erbin) leucine-rich repeat glutathione S-transferase human embryonic kidney phosphate-buffered saline synaptosomal plasma membrane polyacrylamide gel electrophoresis 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid The neuromuscular junction is a cholinergic synapse that conveys signals rapidly from motoneurons to muscle cells. The fast and accurate neurotransmission at this synapse is guaranteed by the high concentration of acetylcholine receptors in the postsynaptic membrane, which accounts for only 0.1% of total muscle surface (1Hall Z.W. Sanes J.R. Cell. 1993; 72: 99-121Abstract Full Text PDF PubMed Scopus (591) Google Scholar, 2Sanes J.R. Lichtman J.W. Annu. Rev. Neurosci. 1999; 22: 389-442Crossref PubMed Scopus (1239) Google Scholar). Muscle fibers are multinucleated cells. Remarkably, it is only the synaptic nuclei that actively transcribe genes encoding acetylcholine receptor subunits. Such synapse-specific transcription is mediated by neuregulin, a factor used by motoneurons to stimulate acetylcholine receptor synthesis at the neuromuscular junction. Neuregulin receptors are transmembrane tyrosine kinases of the ErbB family: ErbB2, ErbB3, and ErbB4. Stimulation by neuregulin of ErbB proteins leads to their tyrosine phosphorylation (3Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (131) Google Scholar, 4Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (554) Google Scholar, 5Jo S.A. Burden S.J. Development. 1992; 115: 673-680PubMed Google Scholar, 6Si J. Luo Z. Mei L. J. Biol. Chem. 1996; 271: 19752-19759Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and subsequent activation of multiple intracellular signaling cascades (6Si J. Luo Z. Mei L. J. Biol. Chem. 1996; 271: 19752-19759Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 7Gassmann M. Lemke G. Curr. Opin. Neurobiol. 1997; 7: 87-92Crossref PubMed Scopus (108) Google Scholar, 8Burden S. Yarden Y. Neuron. 1997; 18: 847-855Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar, 9Si J. Wang Q. Mei L. J. Neurosci. 1999; 19: 8489-8508Crossref Google Scholar), essential for compartmental synthesis of acetylcholine receptors. In the central nervous system, neuregulin regulates expression of neuronal nicotinic acetylcholine receptor (10Yang X. Kuo Y. Devay P., Yu, C. Role L. Neuron. 1998; 20: 255-270Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), N-methyl-d-aspartate receptor (11Ozaki M. Sasner M. Yano R. Lu H.S. Buonanno A. Nature. 1997; 390: 691-694Crossref PubMed Scopus (236) Google Scholar), and γ-aminobutyric acid receptor (12Rieff H.I. Raetzman L.T. Sapp D.W. Yeh H.H. Siegel R.E. Corfas G. J. Neurosci. 1999; 19: 10757-10766Crossref PubMed Google Scholar). Recent studies suggest that in addition to an essential role during development, neuregulin appears to regulate synaptic plasticity in the adult brain (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). ErbB proteins are not expressed evenly on the surface of cells. On the contrary, they are localized in subcellular compartments. In the nervous system, ErbB proteins are concentrated in postsynaptic membranes both at the neuromuscular junction (3Altiok N. Bessereau J.-L. Changeux J.-P. EMBO J. 1995; 14: 4258-4266Crossref PubMed Scopus (131) Google Scholar, 14Zhu X. Lai C. Thomas S. Burden S.J. EMBO J. 1995; 23: 5842-5848Crossref Scopus (152) Google Scholar, 15Moscoso L.M. Chu G.C. Gautam M. Noakes P.G. Merlie J.P. Sanes J.R. Dev. Biol. 1995; 172: 158-169Crossref PubMed Scopus (160) Google Scholar, 16Trinidad J.C. Fischbach G.D. Cohen J.B. J. Neurosci. 2000; 20: 8762-8770Crossref PubMed Google Scholar) and in the central nervous system (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 17Garcia R.A.G. Vasudevan K. Buonanno A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3596-3601Crossref PubMed Scopus (244) Google Scholar). In epithelial cells, ErbB2 appears to be enriched in basolateral membranes (18Borg J.-P. Marchetto S. Le Bivic A. Ollendorff V. Jaulin-Bastard F. Saito H. Fournier E. Adelaide J. Margolis B. Birnbaum D. Nature Cell Biol. 2000; 2: 407-413Crossref PubMed Scopus (258) Google Scholar). The mechanism by which ErbB proteins are localized in the subcellular compartments remains largely unknown. The intracellular portions of ErbB receptor tyrosine kinases contain large C termini in addition to kinase domains. Thus, it is conceivable that ErbBs may interact with proteins that regulate their localization, surface expression, or kinase activity. Indeed, recent studies demonstrated that ErbB4, via its C terminus, interacts with postsynaptic density (PSD)1-95 (or SAP90), a PDZ domain-containing protein (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 17Garcia R.A.G. Vasudevan K. Buonanno A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3596-3601Crossref PubMed Scopus (244) Google Scholar). PDZ domains are motifs of 80–90 amino acids which often bind to specific sequences at the extreme C termini of target proteins (19Bredt D.S. Cell. 1998; 94: 691-694Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 20Kennedy M.B. Trends Neurosci. 1997; 20: 264-268Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar, 21Sheng M. Lee S.H. Nat. Neurosci. 2000; 3: 633-635Crossref PubMed Scopus (44) Google Scholar, 22Garner C.C. Nash J. Huganir R.L. Trends Cell Biol. 2000; 10: 274-280Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). They were originally identified in PSD-95, the Drosophila septate junction protein discs large, and the epithelial tight-junction protein zona occludens 1 (23Cho K.O. Hunt C.A. Kennedy M.B. Neuron. 1992; 9: 929-942Abstract Full Text PDF PubMed Scopus (1008) Google Scholar, 24Woods D.F. Bryant P.J. Cell. 1991; 66: 451-464Abstract Full Text PDF PubMed Scopus (773) Google Scholar, 25Kistner U. Wenzel B.M. Veh R.W. Cases-Langhoff C. Garner A.M. Appeltauer U. Voss B. Gundelfinger E.D. Garner C.C. J. Biol. Chem. 1993; 268: 4580-4583Abstract Full Text PDF PubMed Google Scholar, 26Willott E. Balda M.S. Fanning A.S. Jameson B. Itallie C.V. Anderson J.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7834-7838Crossref PubMed Scopus (425) Google Scholar). PDZ domain-containing proteins appear to coordinate the assembly of functional subcellular domains. PSD-95 uses multiple PDZ domains to cluster ion channels, receptors, and cytosolic signaling proteins in subcellular domains including synapses and cellular junctions (27Fanning A.S. Anderson J.M. J. Clin. Invest. 1999; 103: 767-772Crossref PubMed Scopus (401) Google Scholar). The interaction of PSD-95 with ErbB4 potentially may allow for a localized signaling complex at synapses while minimizing unwanted cross-talk. Moreover, PSD-95 could enhance neuregulin signaling probably by promoting dimerization of ErbB4 receptor tyrosine kinases (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). However, PSD-95 interacts with ErbB2 poorly and does not interact with ErbB3 (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 17Garcia R.A.G. Vasudevan K. Buonanno A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3596-3601Crossref PubMed Scopus (244) Google Scholar), which raises the possibility that other PDZ domain-containing protein may exist. Using a yeast two-hybrid strategy, we identified a novel PDZ domain-containing protein that interacts specifically with ErbB2, but not ErbB3 or ErbB4. This protein was named B2BP for ErbB2-binding protein. B2BP was a polypeptide of 180 kDa. It had 16 leucine-rich repeats (LRRs) in the N terminus and a PDZ domain in the C terminus. While the study was in progress, Borg et al. reported the cloning of Erbin as an ErbB2-interacting protein (18Borg J.-P. Marchetto S. Le Bivic A. Ollendorff V. Jaulin-Bastard F. Saito H. Fournier E. Adelaide J. Margolis B. Birnbaum D. Nature Cell Biol. 2000; 2: 407-413Crossref PubMed Scopus (258) Google Scholar, 28Jaulin-Bastard F. Saito H. Le Bivic A. Ollendorff V. Marchetto S. Birnbaum D. Borg J.-P. J. Biol. Chem. 2001; 276: 15256-15263Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Sequence analysis suggests that B2BP is the mouse homolog of Erbin. We will refer B2BP as Erbin in the manuscript. We demonstrate that Erbin is concentrated at the neuromuscular junction and a component of the PSD in the central nervous system. Erbin interacts with ErbB2 in synaptosomes. Moreover, Erbin increases surface expression of ErbB2 in transfected cells. Furthermore, Erbin interacts with PSD-95 in synaptosomes and in mammalian cells. These results suggest that ErbB receptor tyrosine kinases interact with a network of PDZ domain-containing proteins that may regulate neuregulin signaling and localization. To identify ErbB2-interacting proteins, the ErbB2 carboxyl terminus (amino acids 1251–1260) was generated by two complimentary oligonucleotides and subcloned into the pGBT9 yeast vector containing the Gal4 DNA binding domain (CLONTECH). The bait plasmid was then transformed into the yeast strain Y190 and used to screen mouse cDNA libraries in λACT2 or in pACT2 that contained the Gal4 transcription activation domain. Positive clones were selected on plates lacking leucine, tryptophan, and histidine and were confirmed further by a filter assay for β-galactosidase activity as described previously (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Constructs containing PDZ domains of PSD-95, nNOS, α1-syntrophin, β1-syntrophin, or β2-syntrophin, or C termini of ErbB3 or ErbB4 have been described previously (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). C termini of ErbB2 mutants and NR2A (amino acids 1416–1464) were generated by polymerase chain reaction and subcloned in pGBT9. Sequences of all constructs were confirmed by DNA sequencing. The yeast vectors are transformed into HF7c and Y190. Interactions were characterized by growth without leucine, tryptophan, and histidine and by a filter assay for β-galactosidase activity. The GST fusion protein containing the PDZ of Erbin (amino acid residues 1241–1371) was produced, affinity purified, and concentrated as described previously (29Mei L. Doherty C.A. Huganir R.L. J. Biol. Chem. 1994; 269: 12254-12262Abstract Full Text PDF PubMed Google Scholar). Antiserum against the GST-Erbin/PDZ fusion protein was raised in a New Zealand White rabbit by standard procedures (30Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993Google Scholar). To purify the antibodies, two affinity columns were prepared by coupling GST protein and GST- Erbin/PDZ fusion protein to Affi-Gel 15 (Bio-Rad), respectively, according to the manufacturer's instruction. The columns, 2 ml each, were washed sequentially with 10 ml of 100 mm glycine, pH 2.5, 10 ml of 10 mm Tris/HCl, pH 8.8, 10 ml of 100 mm triethylamine, pH 11.5, and equilibrated with 10 ml of 10 mm Tris/HCl, pH 7.5. 2 ml of antiserum was diluted in 18 ml of 10 mm Tris/HCl, pH 7.5, and passed through the GST-coupled Affi-Gel 15 column three times. The flow-through was then loaded on the GST-Erbin/PDZ-coupled Affi-Gel 10 column three times. After washing the column with 20 ml of 10 mm Tris/HCl, pH 7.5, and 20 ml of 500 mm NaCl in 10 mm Tris/HCl, pH 7.5, antibodies were eluted with aliquots of 100 mm glycine, pH 2.5, and collected in vials containing 500 μl of 1 m Tris/HCl, pH 8.0. The antibodies were dialyzed overnight against 10 mm Tris/HCl, pH 7.5, 0.9% saline and stored in 0.02% sodium azide. Affinity-purified antibodies were used in all experiments unless otherwise specified. Commercially available antibodies used were from Upstate Technology (PSD-95), Santa Cruz (ErbB2, ErbB3, ErbB4, and Myc), and Sigma (anti-FLAG antibodies). HEK 293T cells (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), C2C12 muscle cells (6Si J. Luo Z. Mei L. J. Biol. Chem. 1996; 271: 19752-19759Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 9Si J. Wang Q. Mei L. J. Neurosci. 1999; 19: 8489-8508Crossref Google Scholar, 31Tanowitz M. Si J., Yu, D.-H. Feng G.-S. Mei L. J. Neuorsci. 1999; 19: 9426-9435Crossref PubMed Google Scholar), and hippocampal neurons (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar) were cultured as described previously. Primary rat muscle cell cultures were prepared as described previously (32Gilmour B.P. Goldman D. Chahine K.G. Gardner P.D. Dev. Biol. 1995; 168: 416-428Crossref PubMed Scopus (20) Google Scholar) with minor modifications. Muscles were isolated from hind legs of day 19 rat embryos. Muscles were tweezed apart in PBS and incubated in PBS containing 0.25% trypsin at 37 °C for 50 min with frequent trituration. Dissociated cells were filtered through a 20-mesh screen and pelleted twice. They were resuspended in the growth medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 10% horse serum, 2% chick embryo extract, and 25 μg/ml gentamicin). The cells were plated on regular culture dishes for 30 min to get rid of fibroblasts prior to being plated on collagen-coated culture dishes at a density of 7.5 × 106 cells/10-cm dish. By the 4th day in vitro, myotubes were formed, and the cultures were treated with 3 μg/ml cytosine arabinoside for 24 h to inhibit fibroblast proliferation. Cells were transfected using the standard calcium phosphate technique. 2 days after transfection, cells were washed with PBS and lysed in the modified RIPA buffer (1 ml/100-mm plate), containing 20 mm sodium phosphate, pH 7.4, 50 mm sodium fluoride, 40 mm sodium pyrophosphate, 1% Triton X-100, 2 mm sodium vanadate, 50 μmphenylarsine, 10 mm p-nitrophenyl phosphate, including protease inhibitors (6Si J. Luo Z. Mei L. J. Biol. Chem. 1996; 271: 19752-19759Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Lysed cells were incubated on ice for 30 min and centrifuged at 13,000 ×g for 10 min at 4 °C. The supernatant was designated as cell lysate. Northern blot was done using a membrane containing mRNAs from multiple tissues (CLONTECH). An Erbin cDNA fragment (nucleotides 4044–4651) was labeled with [α-32P]dCTP by a random prime method. The membrane was hybridized in a buffer (total 5 ml) containing 32P-labeled probe (∼4 × 108cpm/μg of cDNA), 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 100 μg/ml salmon sperm DNA at 42 °C overnight. It was then washed with 0.5 × SSC, 0.5% SDS at 55 °C three times each for 30 min and exposed to Kodak X-Omat AR film at −70 °C with an intensifying screen. Adult brains were homogenized in a homogenizing buffer containing 0.32 m sucrose, 4 mm HEPES/NaOH, pH 7.4, 5 mm EDTA, 5 mm EGTA, 20 units/ml Trasylol, and 0.1 mmphenylmethylsulfonyl fluoride with a glass-Teflon homogenizer as described previously (33Blackstone C.D. Moss S.J. Martin L.J. Levey A.I. Price D.L. Huganir R.L. J. Neurochem. 1992; 58: 1118-1126Crossref PubMed Scopus (217) Google Scholar). Briefly, the ground tissue was centrifuged at 800 × g for 10 min, and the supernatant was designated the homogenate (S1). The homogenate was centrifuged at 9,000 × g for 15 min, yielding P2 (the crude synaptosomal fraction) and S2. The P2 fraction was resuspended in the homogenizing buffer and used for coimmunoprecipitation studies. To purify PSDs, the resuspended P2 pellet was subjected to another centrifugation at 10,000 × g for 15 min, and the pellet was lysed by hypoosmotic shock in water, rapidly adjusted to 1 mm HEPES/NaOH, pH 7.4, and stirred on ice for 30 min. The lysate was then centrifuged at 25,000 × g for 20 min, yielding P3 and S3. The P3 pellet was resuspended in 0.25 mbuffered sucrose, layered onto a discontinuous sucrose gradient containing 0.8 m/1.0 m/1.2 msucrose, and centrifuged for 2 h at 65,000 × g in a Beckman SW-28 rotor. The gradient yielded a synaptosomal plasma membrane (SPM) fraction at the 1.0 m/1.2 msucrose interface. The SPM fraction was solubilized with 0.4% Triton X-100 in 0.5 mm HEPES/NaOH, pH 7.4, yielding an insoluble PSD fraction and a soluble SPM extract after a centrifugation at 65,000 × g for 20 min. Synaptophysin, a synaptic vesicle protein, was fractionated with the SPM fraction but was not present in the PSD fraction (see Fig. 8). Cell lysates (∼400 μg of protein) were incubated directly without or with indicated antibodies for 1 h at 4 °C. They were then incubated with protein A-agarose beads overnight at 4 °C on a rotating platform. After centrifugation, beads were washed four or five times with the modified RIPA buffer. Bound proteins were eluted with SDS sample buffer and subjected to SDS-PAGE. Immunoprecipitation of Erbin from rat brain P2 fraction was done as described previously (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). The GST fusion protein containing the PDZ of Erbin (amino acids 1241–1371) was induced in BL21 cells with 1 mm isopropyl β-d-thiogalactopyranoside and purified using glutathione-agarose beads (Roche Molecular Biochemicals, Indianapolis). Equal amounts of GST fusion protein beads (∼50 μg of protein) were incubated with cell lysates overnight at 4 °C on a rotating platform. After centrifugation, beads were washed four or five times with wash buffer (150 mm sodium chloride, 10 mmsodium phosphate, 1% Triton X-100, pH 7.4). Bound proteins were eluted with SDS sample buffer and subjected to SDS-PAGE and immunoblotting. Proteins resolved on SDS-PAGE were transferred to nitrocellulose membranes (Schleicher & Schuell). Nitrocellulose blots were incubated at room temperature for 1 h in Tris-buffered saline with 0.1% Tween (TBS-T) containing 5% milk followed by an incubation with 1% milk with the indicated antibodies except the anti-phosphotyrosine antibody, which required 3% bovine serum albumin in the blocking buffer and 1% bovine serum albumin in the blotting buffer. After washing three times for 15 min each with TBS-T, the blots were incubated with horseradish peroxidase-conjugated donkey anti-mouse or anti-rabbit IgG (Amersham Pharmacia Biotech) followed by washing. Immunoreactive bands were visualized with enhanced chemiluminescence substrate (Pierce). In some experiments, after visualizing an immunoactive protein, the nitrocellulose filter was incubated in a buffer containing 625 mm Tris/HCl, pH 6.7, 100 mm β-mercaptoenthanol, and 2% SDS at 50 °C for 30 min, washed with 0.1% Tween 20 in 50 mm TBS at room temperature for 1 h, and reblotted with different antibodies. Normal or denervated (5 days postdenervation) muscles were rapidly dissected, stretched on a board, and frozen in isopentane cooled with dry ice. 10-μm sections were prepared using a cryostat, thaw mounted on gelatin-coated slides, and stored at −80 °C. Sections of adult rat muscles were incubated with 2% normal goat serum (Vector Laboratories, Burlingame, CA) in PBS for 1 h at room temperature to reduce background staining and then incubated with the affinity-purified antibodies against Erbin or preimmune serum in 2% normal goat serum in PBS overnight at 4 °C. In some experiments, affinity-purified antibodies were preincubated with 10 nm GST-Erbin/PDZ overnight at 4 °C prior to immunohistochemical studies. After washing the sections five times with PBS, each for 30 min, the sections were incubated with a fluorescein isothiocyanate-conjugated anti-rabbit antibody (Zymed Laboratories Inc., San Francisco) and rhodamine-conjugated α-bungarotoxin (Molecular Probes, Eugene, OR). Fluorescent images of cells were captured on a Sony CCD camera mounted on a Nikon E600 microscope using Photoshop imaging software. To label surface proteins, cells were washed with cold PBS containing 1 mmMgCl2 and 0.1 mm CaCl2 and incubated with 0.5 mg/ml sulfo-NHS-LC-biotin in the same buffer at room temperature for 30 min. The labeling reaction was quenched by incubation with 100 mm glycine for 10 min at room temperature. Cells were then lysated in the modified RIPA buffer. Lysates were incubated with streptavidin-agarose beads (Molecular Probes) overnight at 4 °C. Bound proteins were subjected to SDS-PAGE. The protein was assayed according to the method of Bradford (34Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using bovine serum albumin as a standard. The C terminus (-DVPV*) of ErbB2 fits the consensus site for PDZ binding. However, whereas ErbB4 binds strongly to PSD-95, ErbB2 has little or no affinity for PSD-95 (13Huang Y.Z. Won S. Ali D.W. Wang Q. Tanowitz M. Du Q.S. Pelkey K.A. Yang D.J. Xiong W.C. Salter M.W. Mei L. Neuron. 2000; 26: 443-455Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). To identify proteins that bind to ErbB2, we generated several bait constructs composed of the ErbB2 C terminus in various lengths. Most of the baits showed autonomous transactivation activity in various yeast strains except the one with the last 10 amino acid residues (amino acids 1251–1260). Screens using this bait of mouse muscle, mouse brain, and human heart cDNA libraries led to isolation of cDNAs all of which encoded partial sequences of an apparently same protein with a PDZ domain in the C terminus. This protein was initially named as B2BP for ErbB2-binding protein because it only interacted with ErbB2 and not ErbB3 or ErbB4 (see below). While this study was in progress, Borget al. reported Erbin (18Borg J.-P. Marchetto S. Le Bivic A. Ollendorff V. Jaulin-Bastard F. Saito H. Fournier E. Adelaide J. Margolis B. Birnbaum D. Nature Cell Biol. 2000; 2: 407-413Crossref PubMed Scopus (258) Google Scholar). Sequence analysis indicated that B2BP was the mouse homolog of Erbin. Thus, this protein will be referred as Erbin in the rest of this manuscript. Erbin showed high homology to Densin-180, a protein identified previously as a postsynaptic component (35Apperson M.L. Moon I.S. Kennedy M.B. J. Neurosci. 1996; 16: 6839-6852Crossref PubMed Google Scholar, 36Walikonis R.S. Oguni A. Khorosheva E.M. Jeng C.-J. Asuncion F.J. Kennedy M.B. J. Neurosci. 2001; 21: 423-433Crossref PubMed Google Scholar). Like Densin-180, Erbin had 16 LRR domains in the N terminus. In the C terminus, there is a PDZ domain of group I which is characterized by a conserved histidine residue (37Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Science. 1997; 275: 73-77Crossref PubMed Scopus (1224) Google Scholar). The homology between Erbin and Densin-180 was 73% in LRR domains, 71% in the PDZ domain, and 39% in the middle region. Nice clones were isolated from the libraries that encoded two fragments of Erbin: Erbin965 and Erbin1254 (Fig.1 A). The binding of Erbin to ErbB2 was dependent on the PDZ domain of Erbin because deletion of the PDZ domain prevented the interaction. Furthermore, the PDZ alone was sufficient to bind to ErbB2. Although Erbin showed high homology with Densin-180, ErbB2 did not interact with the PDZ domain of Densin-180 (Fig. 1 A), nor did it interact with the PDZ domains of PSD-95 (Fig. 1 A) and of Scribble, nNOS, or α1-, β1-, and β2-syntrophin (data not shown). The binding of Erbin to ErbB2 was dependent on the ErbB2 C terminus. Mutation of the valine residues at the −1 and −3 positions to alanine prevented ErbB2 from interacting with Erbin (Fig. 1 B). On the other hand, Erbin interacted specifically with the C terminus of ErbB2 and did not interact with C termini of the ErbB3, ErbB4, or NR2A subunit of theN-methyl-d-aspartate receptor (Fig.1 C). To characterize further the interaction between Erbin and ErbB proteins, we examined the ability of Erbin's PDZ domain to bind to ErbBs inin vitro pull-down assays. Lysates from HEK 293T cells transfected with ErbB2, ErbB3, or ErbB4 were incubated with GST-Erbin fusion protein immobilized on agarose beads. Bound proteins were resolved on SDS-PAGE and immunoblotted with individual anti-ErbB antibodies. Consistent with the results from yeast two-hybrid assays, GST-Erbin was only able to pull down ErbB2 (Fig.2 A). In contrast, ErbB3 or ErbB4 was undetectable in the Erbin complex. To determine whether ErbB2 interacts with Erbin in mammalian cells, we expressed ErbB proteins with or without Myc-tagged Erbin in HEK 293T cells. Lysates of transfected cells were incubated with individual anti-ErbB antibodies, and the resulting immunocomplex was blotted with anti-Myc antibodies. Erbin was detected in the immunoprecipitates from cells that had been cotransfected with ErbB2 and Erbin (Fig. 2 B), suggesting that ErbB2 associates with Erbin in vivo. In contrast, Erbin was not detected in the ErbB3 or ErbB4 immunoprecipitates (Fig.2 C). Northern blot analysis was used to study mRNA expression of Erbin. The membrane loaded with mRNAs from multiple tissues was probed with a32P-labeled Erbin DNA fragment (encoding amino acids 1241–1371 plus the 3′-noncoding region). A major transcript at 7.5 kilobases was detected in various tissues (Fig.3, top panel). The expression was high in the lung, heart, and kidney, moderate in the brain, skeletal muscle, and testis, and little, if any, in the spleen and liver. In contrast, expression of the Densin-180 mRNA was brain-specific as reported previously (35Apperson M.L. Moon I.S. Kennedy M.B. J. Neurosci. 1996; 16: 6839-6852Crossref PubMed Google Scholar). The 7.4-kilobase transcript of Densin-180 was detected only in the brain, but not in any of tested periphery tissues (Fig. 3, middle panel). Note the exposure time of blots for Densin-180 (10 days) and Erbin (1 day), whereas both used a similar amount of probes (5 ng/ml, 5 ml) with same specific activity (4 × 108 cpm/μg of DNA). These results suggest that the expression level of Erbin may be at least five times higher than that of Densin-180 in the brain. To study Erbin expression at the protein level, an