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• W2070036656 abstract "The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between α4β2 AChRs and the presynaptic cell adhesion molecule, neurexin-1β. In non-neuronal tsA 201 cells, recombinant neurexin-1β and mature α4β2 AChRs form complexes. α4β2 AChRs and neurexin-1β also coimmunoprecipitate from rat brain lysates. When exogenous α4β2 AChRs and neurexin-1β are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1β. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1β lacking the extracellular domain. Additionally, when α4β2 AChRs, α7 AChRs, and neurexin-1β are coexpressed in the same neuron, only the α4β2 AChR colocalizes with neurexin-1β at presynaptic terminals. Collectively, these data suggest that neurexin-1β targets α4β2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on α4β2 AChRs, may contribute to the etiology of these neurological disorders. The mechanisms involved in the targeting of neuronal nicotinic acetylcholine receptors (AChRs), critical for their functional organization at neuronal synapses, are not well understood. We have identified a novel functional association between α4β2 AChRs and the presynaptic cell adhesion molecule, neurexin-1β. In non-neuronal tsA 201 cells, recombinant neurexin-1β and mature α4β2 AChRs form complexes. α4β2 AChRs and neurexin-1β also coimmunoprecipitate from rat brain lysates. When exogenous α4β2 AChRs and neurexin-1β are coexpressed in hippocampal neurons, they are robustly targeted to hemi-synapses formed between these neurons and cocultured tsA 201 cells expressing neuroligin-1, a postsynaptic binding partner of neurexin-1β. The extent of synaptic targeting is significantly reduced in similar experiments using a mutant neurexin-1β lacking the extracellular domain. Additionally, when α4β2 AChRs, α7 AChRs, and neurexin-1β are coexpressed in the same neuron, only the α4β2 AChR colocalizes with neurexin-1β at presynaptic terminals. Collectively, these data suggest that neurexin-1β targets α4β2 AChRs to presynaptic terminals, which mature by trans-synaptic interactions between neurexins and neuroligins. Interestingly, human neurexin-1 gene dysfunctions have been implicated in nicotine dependence and in autism spectrum disorders. Our results provide novel insights as to possible mechanisms by which dysfunctional neurexins, through downstream effects on α4β2 AChRs, may contribute to the etiology of these neurological disorders. The clustering of ion channels or receptors and precise targeting to pre- and postsynaptic specializations in neurons is critical to efficiently regulate synaptic transmission. Within the central nervous system, neuronal nicotinic acetylcholine receptors (AChRs) 5The abbreviations used are: AChRneuronal nicotinic acetylcholine receptorAbantibodyASDautism spectrum disordersBACbromoacetylcholineGABAγ-aminobutyric acidNLGneuroligin-1NRXneurexin-1β lacking the insert at splice site 4mAbmonoclonal antibodyHAhemagglutininBSAbovine serum albuminPBSphosphate-buffered salineDIVday(s) in vitromiRNAmicro-RNA interference-expressing constructVSV-Gvesicular stomatitis virus G.5The abbreviations used are: AChRneuronal nicotinic acetylcholine receptorAbantibodyASDautism spectrum disordersBACbromoacetylcholineGABAγ-aminobutyric acidNLGneuroligin-1NRXneurexin-1β lacking the insert at splice site 4mAbmonoclonal antibodyHAhemagglutininBSAbovine serum albuminPBSphosphate-buffered salineDIVday(s) in vitromiRNAmicro-RNA interference-expressing constructVSV-Gvesicular stomatitis virus G. regulate the release of neurotransmitters at presynaptic sites (1.Wonnacott S. Trends Neurosci. 1997; 20: 92-98Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar) and mediate fast synaptic transmission at postsynaptic sites of neurons (2.Role L.W. Berg D.K. Neuron. 1996; 16: 1077-1085Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar). These receptors are part of a family of acetylcholine-gated ion channels that are assembled from various combinations of α2–α10 and β2–β4 subunits (3.Millar N.S. Biochem. Soc. Trans. 2003; 31: 869-874Crossref PubMed Scopus (180) Google Scholar). AChRs participate in the regulation of locomotion, affect, reward, analgesia, anxiety, learning, and attention (4.Dani J.A. Bertrand D. Annu. Rev. Pharmacol. Toxicol. 2007; 47: 699-729Crossref PubMed Scopus (932) Google Scholar, 5.Picciotto M.R. Caldarone B.J. King S.L. Zachariou V. Neuropsychopharmacology. 2000; 22: 451-465Crossref PubMed Scopus (299) Google Scholar). neuronal nicotinic acetylcholine receptor antibody autism spectrum disorders bromoacetylcholine γ-aminobutyric acid neuroligin-1 neurexin-1β lacking the insert at splice site 4 monoclonal antibody hemagglutinin bovine serum albumin phosphate-buffered saline day(s) in vitro micro-RNA interference-expressing construct vesicular stomatitis virus G. neuronal nicotinic acetylcholine receptor antibody autism spectrum disorders bromoacetylcholine γ-aminobutyric acid neuroligin-1 neurexin-1β lacking the insert at splice site 4 monoclonal antibody hemagglutinin bovine serum albumin phosphate-buffered saline day(s) in vitro micro-RNA interference-expressing construct vesicular stomatitis virus G. The α4β2 subtype is the most abundant AChR receptor expressed in the brain. Multiple lines of evidence support a major role for α4β2 AChRs in nicotine addiction. α4β2 AChRs show high affinity for nicotine (6.Whiting P.J. Lindstrom J.M. J. Neurosci. 1988; 8: 3395-3404Crossref PubMed Google Scholar) and are located on the dopaminergic projections of ventral tegmental area neurons to the medium spiny neurons of the nucleus accumbens (7.Charpantier E. Barnéoud P. Moser P. Besnard F. Sgard F. Neuroreport. 1998; 9: 3097-3101Crossref PubMed Scopus (141) Google Scholar, 8.Wada E. Wada K. Boulter J. Deneris E. Heinemann S. Patrick J. Swanson L.W. J. Comp. Neurol. 1989; 284: 314-335Crossref PubMed Scopus (925) Google Scholar). Furthermore, β2 AChR subunit knock-out mice lose their sensitivity to nicotine in passive avoidance tasks (9.Picciotto M.R. Zoli M. Léna C. Bessis A. Lallemand Y. Le Novère N. Vincent P. Pich E.M. Brûlet P. Changeux J.P. Nature. 1995; 374: 65-67Crossref PubMed Scopus (560) Google Scholar) and show attenuated self-administration of nicotine (10.Picciotto M.R. Zoli M. Rimondini R. Léna C. Marubio L.M. Pich E.M. Fuxe K. Changeux J.P. Nature. 1998; 391: 173-177Crossref PubMed Scopus (1116) Google Scholar). α4 AChR subunit knock-out mice also exhibit a loss of tonic control of striatal basal dopamine release (11.Marubio L.M. Gardier A.M. Durier S. David D. Klink R. Arroyo-Jimenez M.M. McIntosh J.M. Rossi F. Champtiaux N. Zoli M. Changeux J.P. Eur. J. Neurosci. 2003; 17: 1329-1337Crossref PubMed Scopus (209) Google Scholar). Finally, experiments with knock-in mice expressing α4β2 AChRs hypersensitive to nicotine demonstrate that α4β2 AChRs indeed mediate the essential features of nicotine addiction including reward, tolerance, and sensitization (12.Tapper A.R. McKinney S.L. Nashmi R. Schwarz J. Deshpande P. Labarca C. Whiteaker P. Marks M.J. Collins A.C. Lester H.A. Science. 2004; 306: 1029-1032Crossref PubMed Scopus (586) Google Scholar). High resolution ultrastructural studies show that α4 subunit-containing AChRs are clustered at dopaminergic axonal terminals (13.Arroyo-Jiménez M.M. Bourgeois J.P. Marubio L.M. Le Sourd A.M. Ottersen O.P. Rinvik E. Fairén A. Changeux J.P. J. Neurosci. 1999; 19: 6475-6487Crossref PubMed Google Scholar), and a sequence motif has been identified within the α4 AChR subunit cytoplasmic domain that is essential for receptor trafficking to axons (14.Xu J. Zhu Y. Heinemann S.F. J. Neurosci. 2006; 26: 9780-9793Crossref PubMed Scopus (80) Google Scholar). However, the mechanisms underlying the targeting and clustering of α4β2 AChRs to presynaptic sites in neurons remain elusive. Recently, bi-directional interactions between neurexins and neuroligins have been shown to promote synapse assembly and maturation by fostering pre- and postsynaptic differentiation (reviewed in Refs. 15.Dean C. Dresbach T. Trends Neurosci. 2006; 29: 21-29Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 16.Craig A.M. Kang Y. Curr. Opin Neurobiol. 2007; 17: 43-52Crossref PubMed Scopus (448) Google Scholar, 17.Südhof T.C. Nature. 2008; 455: 903-911Crossref PubMed Scopus (1265) Google Scholar). The neurexins are encoded by three genes corresponding to neurexins I–III (18.Missler M. Südhof T.C. Trends Genet. 1998; 14: 20-26Abstract Full Text PDF PubMed Scopus (291) Google Scholar, 19.Lisé M.F. El-Husseini A. Cell Mol. Life Sci. 2006; 63: 1833-1849Crossref PubMed Scopus (133) Google Scholar), each encoding longer α-neurexins and shorter β-neurexins, because of differential promoter use. Neurexins recruit N- and P/Q-type calcium channels via scaffolding proteins, including calmodulin-associated serine/threonine kinase (20.Hata Y. Butz S. Südhof T.C. J. Neurosci. 1996; 16: 2488-2494Crossref PubMed Google Scholar), to active zones of presynaptic terminals (21.Missler M. Zhang W. Rohlmann A. Kattenstroth G. Hammer R.E. Gottmann K. Südhof T.C. Nature. 2003; 423: 939-948Crossref PubMed Scopus (516) Google Scholar, 22.Zhang W. Rohlmann A. Sargsyan V. Aramuni G. Hammer R.E. Südhof T.C. Missler M. J. Neurosci. 2005; 25: 4330-4342Crossref PubMed Scopus (124) Google Scholar). Recently, α-neurexins were shown to specifically induce GABAergic postsynaptic differentiation (23.Kang Y. Zhang X. Dobie F. Wu H. Craig A.M. J. Biol. Chem. 2008; 283: 2323-2334Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Neuroligins, postsynaptic binding partners of neurexins, cluster N-methyl-d-aspartate receptors and GABAA receptors by recruiting the scaffolding proteins PSD-95 (post-synaptic density 95) and gephyrin, respectively (24.Nam C.I. Chen L. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 6137-6142Crossref PubMed Scopus (244) Google Scholar, 25.Chih B. Gollan L. Scheiffele P. Neuron. 2006; 51: 171-178Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Interestingly, neurexins and neuroligins also modulate the postsynaptic clustering of α3-containing AChRs in chick ciliary ganglia (26.Conroy W.G. Nai Q. Ross B. Naughton G. Berg D.K. Dev. Biol. 2007; 307: 79-91Crossref PubMed Scopus (32) Google Scholar, 27.Ross B.S. Conroy W.G. Dev. Neurobiol. 2008; 68: 409-419Crossref PubMed Scopus (11) Google Scholar). In this study, using multiple experimental strategies, we provide evidence for the formation of complexes between neurexin-1β and α4β2 AChRs and a role for neurexin in the targeting of α4β2 AChRs to presynaptic terminals of neurons. All of the constructs were made by PCR using appropriate pairs of forward and reverse synthetic oligonucleotide primers (Invitrogen) and Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). Rat α4, rat β2, and chicken α7 AChR subunit cDNAs were cloned into the mammalian cell expression vector pEF6/Myc-His A as described previously (28.Jeanclos E.M. Lin L. Treuil M.W. Rao J. DeCoster M.A. Anand R. J. Biol. Chem. 2001; 276: 28281-28290Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Mouse neurexin-1β lacking the insert at splice site 4 with an extracellular VSV-G epitope tag at the mature N terminus of the protein (NRX) and mouse neuroligin-1 with an extracellular HA epitope tag at the mature N terminus of the protein (NLG) were kind gifts from Dr. Peter Scheiffele (29.Dean C. Scholl F.G. Choih J. DeMaria S. Berger J. Isacoff E. Scheiffele P. Nat. Neurosci. 2003; 6: 708-716Crossref PubMed Scopus (469) Google Scholar). The reading frame of full-length mouse NRX (NRX1–447) was amplified by PCR and subcloned between the EcoRI and XbaI sites of pEF6A vector. Truncation mutants were also made by PCR to create NRXΔC (NRX1–389) lacking the C-terminal cytoplasmic domain and NRXΔEC (Δ47–360) lacking the entire extracellular domain. Numbering includes the VSV-G tag. The following antibodies (Abs) were used: rat mAbs to the α4 AChR subunit (mAb 299) and to the β2 AChR subunit (mAbs 295 and 270); a mouse mAb to the α7 AChR subunit (mAb 306); a goat polyclonal Ab against the β2 AChR subunit (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) that binds to denatured β2 subunits on immunoblots; rabbit polyclonal Abs against VSV-G (Sigma for immunoblots and Clontech for immunostaining); a mouse monoclonal Ab against HA (AbCam, Cambridge, MA); a goat polyclonal Ab against neurexin I (P-15, sc-14334; Santa Cruz Biotechnology); a mouse monoclonal Ab to neuroligin-1 (Synaptic Systems; Gottingen, Germany); and a mouse monoclonal Ab to synapsin-1 (Synaptic Systems). The bovine anti-goat, goat anti-rat, anti-mouse, and anti-rabbit horseradish peroxidase-conjugated secondary Abs were obtained from Pierce. The Alexa Fluor 488-, Alexa Fluor 594-, and Alexa Fluor 647-conjugated goat anti-rat, goat anti-rabbit, and goat anti-mouse secondary Abs were obtained from Molecular Probes (Eugene, OR). Human tsA 201 cells, a derivative of the human embryonic kidney cell line 293 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) as previously described (30.Ren X.Q. Cheng S.B. Treuil M.W. Mukherjee J. Rao J. Braunewell K.H. Lindstrom J.M. Anand R. J. Neurosci. 2005; 25: 6676-6686Crossref PubMed Scopus (44) Google Scholar). tsA 201 cells were transfected using FuGENE 6 (Roche Applied Science). The cultures were prepared essentially as previously described (31.Banker G. Goslin K. Culturing Nerve Cells. 2nd Ed. The MIT Press, Cambridge, MA1998: 339-370Google Scholar). Briefly, the hippocampi were isolated from embryonic day 18 rat embryos and dissociated by trituration after incubation in 0.25% trypsin/Hanks' balanced salt solution for 15 min at 37 °C (Invitrogen). The cells were plated on poly-l-lysine (Sigma)-coated glass coverslips at 100,000 cells/well and maintained in 500 μl of neurobasal media supplemented with B27 and 0.5 mm l-glutamine (Invitrogen). The cells were washed and refed with fresh medium after 16 h. 200 μl of medium was exchanged on DIV 3 and 6–7. The neurons were transfected at DIV 7–10 using the Clontech CalPhos mammalian transfection kit (BD Bioscience, Palo Alto, CA) as described (32.Jiang M. Chen G. Nat. Protoc. 2006; 1: 695-700Crossref PubMed Scopus (228) Google Scholar). The experiments were performed essentially as previously described (33.Biederer T. Scheiffele P. Nat. Protoc. 2007; 2: 670-676Crossref PubMed Scopus (116) Google Scholar). After 24 h, transfected tsA 201 cells were coplated at 10,000 cells/well with the neurons (at DIV 12–13), transferred to 30 °C to up-regulate α4β2 AChR expression, and processed for immunocytochemistry at DIV 14–15. Affi-Gel 401 was prepared from Affi-Gel 102 (Bio-Rad). 10 ml of Affi-Gel 102 was washed with 0.5 m NaHCO3, followed by incubation with 10% N-acetyl-dl-homocysteine thiolactone, 0.5 m NaHCO3, pH 8.5, overnight at 4 °C with stirring. The following day, the gel was washed with 0.1 m NaCl and then 0.2 m NaOAc, 0.1 m 2-mercaptoethanol. The gel was slurried with deionized H2O, and the pH was adjusted to between 6 and 7. One ml of acetic anhydride was added in five 200-μl aliquots at 10-min intervals, adjusting the pH to between 6 and 7 after each addition with 10 m NaOH. Ten min after the last aliquot was added; the pH was adjusted to 9.5 and incubated for 30 min. The gel was rinsed with deionized H2O until the pH was under 9.0 and then slurried with 20 ml deionized H2O. While stirring, 0.63 g of NaCl, 0.104 g of EDTA, 0.014 g of sodium azide, 0.42 g of Tris, and 55 μl of 2-mercaptoethanol were added. The pH was adjusted to 7.75, and the gel was stored at 4 °C until use. BAC was coupled to Affi-Gel-401 as described (34.Ellena J.F. Blazing M.A. McNamee M.G. Biochemistry. 1983; 22: 5523-5535Crossref PubMed Scopus (162) Google Scholar). tsA 201 cells were homogenized and incubated in 2% Nonidet P-40 lysis buffer for 2 h at 4 °C and then centrifuged at 12,000 × g for 15 min at 4 °C. The cleared lysates were incubated with 50 μl of BAC-Affi-Gel 401 overnight at 4 °C. For the negative control, the lysate was incubated with 10 μm nicotine for 15 min prior to the addition of BAC-Affi-Gel 401. The gel was then washed three times with lysis buffer and eluted in sample buffer at 60 °C for 30 min, and then β-mercaptoethanol was added to the eluted samples prior to analysis by SDS-PAGE. Transfected tsA 201 cells were solubilized in 1% Nonidet P-40 and subject to pulldowns using the FLAG M2 beads (Sigma) as previously described (30.Ren X.Q. Cheng S.B. Treuil M.W. Mukherjee J. Rao J. Braunewell K.H. Lindstrom J.M. Anand R. J. Neurosci. 2005; 25: 6676-6686Crossref PubMed Scopus (44) Google Scholar). For native AChR pulldowns, the Abs were covalently coupled to protein G-Sepharose beads. Briefly, ∼5 μg of affinity purified anti-β2 (mAb 295) or rat or mouse IgG were incubated with 50 μl of a 1:1 slurry of Sepharose beads for 2 h at room temperature in PBS containing 0.1% azide with gentle rotation. After washing with 200 mm borate buffer + 3 m NaCl, pH 9.0, the beads were incubated with 20 μm dimethylpimelimidate in the 200 mm borate buffer + 3 m NaCl for 30 min at room temperature. After several washes with 200 mm borate buffer + 3 m NaCl, the unreacted sites on the beads were then blocked using 200 mm ethanolamine, pH 8.0, for 2 h at room temperature. The beads were washed with PBS several times and finally with 200 mm glycine once and then stored at 4 °C in PBS containing 0.1% sodium azide. Frozen rat brains were homogenized and solubilized in 1% Nonidet P-40 buffer as previously described (28.Jeanclos E.M. Lin L. Treuil M.W. Rao J. DeCoster M.A. Anand R. J. Biol. Chem. 2001; 276: 28281-28290Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Detergent-solubilized brain extracts (typically 1–3 ml) were precleared with 50 μl of protein G-Sepharose bead slurry and then incubated with 50 μl of mAb covalently cross-linked protein G-Sepharose beads (∼5–10 μg of antibody/50 μl of beads) for 3–4 h at 4 °C. The beads were washed and eluted with sample buffer (lacking β-mercaptoethanol to avoid reduction of the disulfide linkage of the IgG chains) at 60 °C for 30 min, and then β-mercaptoethanol was added to the eluted samples prior to analysis by SDS-PAGE. Following separation using SDS-PAGE, the proteins were transferred onto polyvinylidene difluoride membrane and incubated with diluted Abs in PBS containing 5% nonfat milk powder. The binding of the primary Abs to proteins was detected using appropriate secondary Abs as previously described (30.Ren X.Q. Cheng S.B. Treuil M.W. Mukherjee J. Rao J. Braunewell K.H. Lindstrom J.M. Anand R. J. Neurosci. 2005; 25: 6676-6686Crossref PubMed Scopus (44) Google Scholar). Cell surface α4β2 AChRs and NRX were measured using an enzyme-linked immunoassay previously described (28.Jeanclos E.M. Lin L. Treuil M.W. Rao J. DeCoster M.A. Anand R. J. Biol. Chem. 2001; 276: 28281-28290Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Briefly, transfected, tsA 201 cells (0.5 × 106 cells/well) were blocked with 3% BSA/PBS and incubated for 1 h with anti-β2 subunit (mAb 295) or anti-VSV-G antibodies in 3% BSA/PBS, washed, fixed with formaldehyde (3%), washed, and blocked again. The cells were incubated with horseradish peroxidase-conjugated goat anti-rat secondary Ab for 1 h in the presence of 3% BSA, washed, and incubated with 300 μl of the horseradish peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (Sigma) for 1 h. The absorbance of the supernatant was then measured at 655 nm in a Beckman spectrophotometer. The values obtained using this assay are the mean ± S.E. and were statistically analyzed using an analysis of variance test. The significance level was set at p < 0.05. The nonspecific background to nontransfected cells was typically <0.5% of the total binding observed for transfected cells. For the mixed neuron/tsA 201 cell assays, the cultures were fixed in 4% paraformaldehyde, 4% sucrose, Hanks' balanced salt solution (with Ca2+ and Mg2+), pH 7.3 (15 min at room temperature), blocked with 3% normal goat serum, 3% BSA, Hanks' balanced salt solution with 0.2% Triton X-100 (30 min at room temperature), and incubated with the appropriate primary (overnight at 4 °C) and secondary (90 min at room temperature) antibodies. Coverslips were mounted onto slides with ProLong Gold antifade reagent (Invitrogen). The cells were visualized using an Olympus IX81 spinning disc confocal microscope (Tokyo, Japan) with a xenon arc illumination source through a 60× (numerical aperture, 1.42) or 40× (numerical aperture, 1.35) Olympus oil immersion objective. Single-plane fluorescence images were captured using a Hamamatsu EM camera, and the images were processed using the Slide Book version 4.2 software. When the observed fluorescence intensity of antibody staining observed was weak, post acquisition intensities of images were adjusted in the different channels using the gamma function of the slide book software to enhance visibility of axons and terminal in the figures shown. In all cases, the essential features of the original images were not altered. The figures were then processed with Adobe Photoshop CS. Targeting quantification was determined from 29–71 cells/condition from three independent experiments. Random neuroligin-1-expressing cells were imaged, and the targeting of the constructs was quantified as the number of neurons with targeting/number of neuroligin-1 cells contacted. The values obtained are the means ± S.E. and were statistically analyzed by a Student's t test. α4β2 AChRs in the central nervous system are targeted to presynaptic terminals, but the mechanisms underlying their recruitment remain unclear. We investigated the possibility that β-neurexins, which are also highly enriched at axon terminals, have a functional role in the synaptic targeting of α4β2 AChRs. A neurexin-1β isoform was tested in subsequent functional studies with recombinant α4β2 AChRs. To determine whether NRX forms complexes with recombinant α4β2 AChRs, we coexpressed VSV-G-tagged neurexin-1β (NRX) with the α4 and the N-terminal FLAG-tagged β2 AChR subunits (labeled as α4β2FLAG AChRs) by transfecting tsA 201 cells with their respective cDNAs. Forty-eight hours post-transfection, 1% Nonidet P-40-solubilized cell lysates were incubated with FLAG M2 antibody covalently attached to agarose beads. Proteins eluted from these beads were then fractionated by SDS-PAGE and subjected to immunoblot analyses using Abs recognizing the α4 AChR subunit, the β2 AChR subunit, and the VSV-G tag. NRX was found in complexes with Nonidet P-40-solubilized α4β2FLAG AChRs (Fig. 1A). To confirm that the complex formation between NRX and α4β2 AChRs is not an artifact of detergent solubilization, we mixed Nonidet P-40-solubilized extracts from cells expressing NRX alone and cells expressing α4β2FLAG AChRs alone in a pulldown experiment using FLAG M2 beads (Fig. 1B). No NRX was coimmunoprecipitated with α4β2FLAG AChRs, indicating that the complex formation between NRX and α4β2 AChRs was not induced by detergent solubilization but instead was due to complex formation within the cell membrane. To further verify that NRX forms complexes with assembled α4β2 AChRs, tsA 201 cells were cotransfected with untagged α4β2 AChRs and NRX and processed as in Fig. 1A. However, in this case (Fig. 1C), the α4β2 AChRs and their associated proteins were captured with a ligand that has a high affinity for α4β2 AChRs (BAC-conjugated to Affi-Gel 401 resin) (35.Anand R. Conroy W.G. Schoepfer R. Whiting P. Lindstrom J. J. Biol. Chem. 1991; 266: 11192-11198Abstract Full Text PDF PubMed Google Scholar). When α4β2 AChRs and NRX were coexpressed, BAC-conjugated beads captured both α4 and β2 AChR subunits, as well as NRX (Fig. 1C, BAC affinity capture, first lane). As a control for BAC capture specificity, lysates were incubated with 10 μm nicotine for 15 min prior to the addition of the BAC-coupled beads. Pretreatment with nicotine blocked the binding of BAC to the receptor complex (Fig. 1C, BAC, second lane). Additionally, BAC failed to capture the α4 AChR subunit if it was not coexpressed with the β2 AChR subunit (data not shown), indicating that only fully formed pentamers are affinity-purified. When the N-terminal HA-tagged neuroligin-1 (NLG), a trans-synaptic binding partner of NRX, was coexpressed with α4β2 AChRs and incubated with BAC, the anti-HA antibody did not detect NLG in the pulldown (Fig. 1C, BAC, third lane), suggesting that the complex formation between NRX and α4β2 AChRs is specific. To determine whether neurexin-1β forms complexes with native α4β2 AChRs, as it does with recombinant α4β2 AChRs, 1% Nonidet P-40-solubilized rat brain membrane extracts were incubated with a β2 AChR subunit-specific mAb (mAb 295 or 270) or nonspecific rat IgGs (as a control), and the eluates were fractionated by SDS-PAGE and immunoblotted using Abs to the α4 AChR subunit, the β2 AChR subunit, and neurexin-1 (that was reported to cross-react with both the 1α and 1β isoforms). Both α4 and β2 AChR subunits are captured by the anti-β2 antibody. Note that the endogeneous levels of α4β2 AChRs in the lysates lanes of the blot are below the threshold for detection by the anti-α4 and anti-β2 AChR antibodies (Fig. 2A). The anti-neurexin antibody (P-15) detects a ∼66-kDa band in both the lysate lane and the β2 immunoprecipitation lane (Fig. 2A, IP, NRX). No band of this size was observed in the pulldown using the nonspecific IgG as a control. Furthermore, no bands corresponding to neurexin-1α isoforms expected at ∼165 kDa were observed in either the lysates or the pulldowns (Fig. 2A). Hence, we were unable to experimentally determine whether other neurexin-1α isoforms also form complexes with the α4β2 AChRs. When the immunoblots were probed with an anti-neuroligin-1 Ab, a strong band of the expected size (∼110 kDa) was detected in the lysate but was absent in the precipitate captured with the anti-β2 AChR antibody (Fig. 2B). Additionally, an antibody against N-cadherin, a protein expressed in both pre- and post-synaptic membranes, did not detect this protein in the pulldown. These data suggest that neurexin-1β and the α4β2 AChRs are present in specific complexes in vivo. Because the evidence that neurexins form complexes with native α4β2 AChRs relied on the use of a commercially generated anti-neurexin goat polyclonal antiserum (P-15, sc-1334; Santa Cruz) that has not been extensively characterized by other investigators, we additionally verified that this antiserum could recognize recombinant NRX expressed in tsA 201 cells (Fig. 2C). Equal amounts of samples from two eluates of pulldown experiments, one from cells coexpressing α4β2FLAG AChRs and VSV-G-tagged NRX and another from cells coexpressing α4β2FLAG AChRs and VSV-G-tagged NRX lacking its C terminus (NRXΔC), were loaded in parallel, and two sets of blots were probed with neurexin I or VSV-G antiserum. The results show that the neurexin I antiserum recognized both NRX and NRXΔC and that this antiserum weakly binds full-length NRX but is specific in its binding. Stronger reactivity of the Abs with the NRXΔC compared with the full-length NRX is observed possibly because truncation of the C terminus in the NRXΔC construct facilitates increased Ab access to highly antigenic terminal residues of the peptide epitope originally used to raise this antiserum. It is possible that our inability to detect the neurexin-1α isoforms may also be due to conformational masking of this epitope by the extracellular domains of the neurexin-1α isoforms. To investigate the functional significance of the interaction of NRX with the α4β2 AChRs, we first determined whether it affected the steady state levels of recombinant α4 or β2 AChR subunits. Either the pEF6A vector (as a control) or NRX was coexpressed in tsA 201 cells with α4β2 AChRs, and 48 h after transfection, the cells were lysed, separated by SDS-PAGE, and subjected to immunoblot analyses using Abs to the α4 and β2 AChR subunits. No significant change in the steady state levels of the α4 or β2 AChR subunits was observed, suggesting that NRX does not play a role in the early events that regulate AChR subunit stability (Fig. 3A). Next, we assessed whether coexpression of NRX with α4β2 AChRs altered the steady state levels of either the α4β2 AChRs or NRX itself on cell surface membranes. Surface expression of the α4β2 AChRs was measured using an Ab to the extracellular domain of the β2 AChR subunit (mAb 295) in conjunction with a previously described enzyme-linked immunoassay (28.Jeanclos E.M. Lin L. Treuil M.W. Rao J. DeCoster M.A. Anand R. J. Biol. Chem. 2001; 276: 28281-28290Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Similarly, the surface expression level of the NRX was measured with this same assay but using an Ab to the VSV-G tag. The coexpression of α4β2 AChRs with NRX did not significantly change their surface expression levels compared with when they were expressed alone (Fig. 3, B and C). The results suggest that NRX does not affect the trafficking of α4β2 AChRs to the cell surface membrane and vice versa and thus their rate" @default.
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• W2070036656 date "2009-08-01" @default.
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• W2070036656 title "Presynaptic Targeting of α4β2 Nicotinic Acetylcholine Receptors Is Regulated by Neurexin-1β" @default.
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• W2070036656 doi "https://doi.org/10.1074/jbc.m109.017384" @default.
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