Better explicating the strengths and shortcomings of these models will help refine projections and improve transparency in the years ahead.
Image credit: Witsawat.S.
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Contributed by Jean-Pierre Changeux, October 31, 2007 (received for review August 30, 2007)
Abstract
Nicotine acetylcholine receptors (nAChRs) comprise a family of ligand-gated channels widely expressed in the mammalian brain. The β2 subunit is an abundant protein subunit critically involved in the cognitive and behavioral properties of nicotine as well as in the mechanisms of nicotine addiction. In this work, we used matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS) to uncover protein interactions of the intracellular loop of the β2 subunit and components of immunoprecipitated β2–nAChR complexes from mouse brain. Using the β2-knockout mouse to exclude nonspecific binding to the β2 antibody, we identify 21 nAChR-interacting proteins (NIPs) expressed in brain. Western blot analysis confirmed the association between the β2 subunit and candidate NIPs. Based on their functional profiles, the hypothesis is suggested that the identified NIPs can regulate the trafficking and signaling of the β2–nAChR. Interactions of the β2 subunit with NIPs such as G protein α, G protein-regulated inducer of neurite outgrowth 1, and G protein-activated K+ channel 1 suggest a link between nAChRs and cellular G protein pathways. These findings reveal intracellular interactions of the β2 subunit and may contribute to the understanding of the mechanisms of nAChR signaling and trafficking in neurons.
Nicotine is a common drug of addiction and a leading cause of preventable deaths in developed countries (1, 2). The target of nicotine is a class of nicotinic acetylcholine receptors (nAChRs) existing as pentameric channels made up of various receptor subunits and distributed throughout the brain (3). To date, 11 neuronal subunits have been identified: α2–α9 and β2–β4. The specific combination of these subunits confers the pharmacological and physiological properties of the nAChR (4). Studies show that ≈90% of the high-affinity nicotine receptor sites within the brain consist of the α4β2–nAChR (5, 6). Topological analysis of nAChR subunits indicates the presence of a large, highly divergent intracellular loop (M3–M4 loop) that is implicated in trafficking and targeting properties of nAChRs (7, 8). Recent analysis of a prokaryotic homolog of the nAChR family reveals that the M3–M4 loop is unique to the evolution of the nAChR in eukaryotes (9).
The generation of subunit-specific knockout (KO) mice has proven valuable in defining the role of individual nAChR subunits in brain physiology and animal behavior (10). Experiments in mice lacking the β2 subunit (β2−/−) demonstrate an important role for this receptor subunit in neuronal development and plasticity (10), protection from excitotoxicity (11), and the mechanisms underlying cognitive and social behaviors (12). β2−/− mice also exhibit a loss of nicotine self-administration and nicotine-elicited firing and dopamine release from dopaminergic neurons of the ventral tegmental area (13, 14). Because reexpression of the β2 subunit in β2−/− mice was found sufficient to restore the electrophysiological and behavioral effects of nicotine (5), this subunit is likely a central mediator in the mechanism of nicotine addiction.
The major functional properties of receptor systems have been found to depend on their association with various intracellular molecules (15). Receptor proteomics has thus emerged as a primary approach in the molecular analysis of receptor signaling and regulation (16–18). Multiprotein complexes (termed signaling complexes or signalplexes) have been found to associate with receptors such as glutamate N-methyl-d-aspartate (NMDA) and mGluR5 (19, 20), glucocorticoid (21), serotonin 5-hydroxytryptamine type 2A and 2C (22), dopamine D1 and D2 (23), and the ATP-gated channel (P2X7) (24). Earlier studies based on yeast two-hybrid screens indicate that the α4-nAChR binds the scaffold molecule 14-3-3η (8) and the calcium sensor visinin-like protein 1 (25). In this work, we explore interactions of the β2 subunit by using matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS/MS). Defining the constituents of the β2–nAChR signalplex represents an essential step in understanding the mechanisms of nAChR-mediated signaling and regulation in brain.
Results
Proteins Interacting with the M3–M4 Loop of the β2 Subunit.
The M3–M4 loop of nAChRs mediates important receptor properties such as export from the endoplasmic reticulum (ER) (25, 26), targeting to axonal or dendritic compartments (27), and interactions with scaffold and signaling molecules (8, 28). To identify proteins that associate with the β2–M3–M4 loop, we generated a fusion protein encoding the M3–M4 loop (amino acid residues 326–454 of mouse β2) in association with GST [GST-β2 (M3–M4)], and we used a pulldown strategy to isolate the interactions of GST-β2 (M3–M4) from mouse brain [supporting information (SI) Fig. 4A ]. We used a Sepharose matrix to purify GST-β2 (M3–M4), and the purified product was verified by Coomassie staining and Western blotting with an anti-GST antibody (SI Fig. 4 B and C ). Western blot analysis of purified GST-β2 (M3–M4) indicates expression of a prominent band at 42 kDa, the expected molecular mass of GST-β2 (M3–M4) on an SDS/polyacrylamide gel (SI Fig. 4C ). We also detected a minor band with reactivity to anti-GST at ≈38 kDa (SI Fig. 4C ). Because this band did not stain with an antibody directed against the β2 subunit (data not shown), we conclude that it represents a nonspecific interaction of the GST antibody on the blot.
For pulldown experiments we immobilized GST-β2 (M3–M4) or GST alone (as a control) onto the Sepharose matrix. Beads were then incubated with solubilized preparations of mouse brain to isolate proteins that associate with GST-β2 (M3–M4) or GST alone. Results of the pulldown assay are presented in Fig. 1. Coomassie blue staining of the gel shows a difference in the protein interaction profile of GST-β2 (M3–M4) and GST alone (Fig. 1 B). In total, seven unique bands were visualized in the GST-β2 (M3–M4) pulldown lane (annotated bands in Fig. 1 B), and these bands were excised for MALDI-TOF-TOF MS/MS analysis [the pulldown experiment was performed four times (n = 4), and the results were similar in each case (data not shown)]. A sample MS spectrum (for synaptotagmin 1) is shown in Fig. 1 C. The mass of the parent peptides used to identify the interacting proteins is listed in SI Table 3). A complete list of nAChR-interacting proteins (NIPs) derived from the GST-β2 (M3–M4) pulldown experiment is presented in Table 1.
Fig. 1.
Identification of NIPs from GST-β2 (M3–M4) pulldown experiments. (A) Western blots showing immunoreactivity for anti-dynamin 1, anti-clathrin HC, anti-synatotagmin 1, and anti-NSF in the pulldown assay of GST-β2 (M3–M4). (B) Coomassie blue-stained gel showing a spectrum of protein interactions for GST-β2 (M3–M4) and GST alone (control) in pulldown experiments. Arrowheads indicate the positions of bands (listed in Table 1) analyzed by MALDI-TOF-TOF MS/MS. The asterisk band shows the position of GST-β2 (M3–M4) on the gel. (C) MS spectrum for synaptotagmin 1 (T = trypsin autolysis product) isolated by the pulldown experiment.
View this table:
Table 1.
Intracellular proteins that associate with GST-β2 (M3–M4)
We tested interaction between GST-β2 (M3–M4) and NIPs by Western blotting. As shown in Fig. 1 A, immunoblot analysis of protein complexes derived from the pulldown assay confirmed the interaction of dynamin 1, clathrin heavy chain (HC), synaptotagmin 1, and N-ethylmaleimide-sensitive factor (NSF) with GST-β2 (M3–M4).
Interactions of the β2–nAChR in the Brain Defined by the Immunoprecipitation Method.
We used a monoclonal antibody (mAb 270) directed against the N terminus of the β2 subunit (29) to isolate β2 subunit-containing nAChRs (β2–nAChRs) from the brain of mouse (SI Fig. 5). Previous studies indicate that mAb 270 immunoprecipitates the β2 subunit from membrane preparations of cultured cells and native brain tissue (29, 30). We assessed the ability of mAb 270 to immunoprecipitate the β2 subunit from the mouse brain. Western blot experiments, with polyclonal anti-β2 (H-92) as an immunoprobe, indicate that mAb 270 successfully immunoprecipitated the β2 subunit from transfected human embryonic kidney (HEK) 293 cells as well as from brain tissue of wild type (WT) mice (Fig. 2 A and B). In samples of β2−/− mice, subject to immunoprecipitation with mAb 270 under equivalent conditions, anti-β2 reactivity was not detected on the Western blot (Fig. 2 A). Probing the blot with a polyclonal antibody directed against the α4 subunit (H-133), however, revealed the presence of α4 within the immunoprecipitated complex of WT and β2−/− mouse brain (Fig. 2 A). These findings indicate reactivity between mAb 270 and the mouse α4 subunit within the immunoprecipitation complex derived from brain, and they suggest reactivity between mAb 270 and non-β2–nAChR subunits under these conditions (31).
Fig. 2.
Immunoprecipitation of the β2 subunit from brain. (A) Western blots showing the expression of β2 subunits within immunoprecipitated complexes from WT (+/+) mouse, using mAb 270. Expression of α4 was also detected within immunoprecipitated complexes of WT (+/+) and β2−/− mouse brain. (B) Immunoblot confirming comigration of the β2 subunit from the WT (+/+) mouse brain with the β2 subunit expressed in HEK 293 cells. (C) Coomassie blue-stained gel showing proteins that coimmunoprecipitate with the β2 subunit in WT (+/+) and β2−/− brain preparations. Arrowheads point to the position of bands analyzed by MALDI-TOF-TOF MS/MS (listed in Table 2). Arrowheads without a number indicate the positions of bands that correspond to unknown proteins within NCBI. Asterisks point to the light- and heavy-chain Ig subunits of the monoclonal antibody.
We compared the constituents of the immunoprecipitated β2 subunit complex between WT and β2−/− mice to eliminate the nonspecific binding of mAb 270 in brain tissue (32). A representative Coomassie blue-stained gel shows the results of an immunoprecipitation experiment conducted in WT and β2−/− mice (Fig. 2 C). Protein bands that appeared common to the two sample lanes were omitted from the MALDI-TOF-TOF MS/MS analysis, and only the bands unique to the WT (annotated bands in Fig. 2 C) were analyzed. A total of 23 unique bands were found in the WT sample lane, and proteomic analysis of these bands revealed the identity of 16 NIPs from NCBI (Fig. 2 C and Table 2). In addition, 6 other putative NIPs matched “hypothetical” protein products of mouse genomic DNA sequences listed in the National Center for Biotechnology Information (NCBI) (annotated bands lacking numbers in Fig. 2 C).
View this table:
Table 2.
Intracellular proteins that coimmunoprecipitate with the β2 subunit in brain
The MS spectrum for a representative NIP [G protein regulated inducer of neurite outgrowth (GPRIN) 1] is shown in Fig. 3 B, and the mass of the parent peptides used to identify NIPs is presented in SI Table 4. A complete list of NIPs identified by immunoprecipitation is presented in Table 2.
Fig. 3.
Confirmation of NIP–β2 subunit interactions. (A) Representative blots showing immunoreactivity to dynamin 1, GIRK1, and Goα subunits within the immunoprecipitated complexes from the brain of WT (+/+) and β2−/− mice. (B) MS spectrum for GPRIN1 (T = trypsin autolysis product). (C) Western blots confirming the presence of synatotagmin 1 and NSF within immunoprecipitated complexes of the β2 subunit from mouse brain. Note that a weak reactivity to both antibodies was also detected in lanes of the β2−/−.
Western blot analysis confirmed the presence of dynamin 1, G protein-coupled inwardly rectifying potassium channel (GIRK) 1, and guanine nucleotide-binding protein O (Goα) within complexes of the β2 subunit (Fig. 3 A). These NIPs were not detected within immunoprecipitated complexes of the β2−/− brain under the same experimental conditions (Fig. 3 A). To test whether NIPs identified by pulldown were present within the immunoprecipitated β2 subunit complex, we probed the blot with antibodies against NSF and synaptotagmin 1 (Fig. 3 C). Antibodies directed against these two proteins also reacted weakly with the immunoprecipitated complexes from β2−/− mice (Fig. 3 C). Multiple repeats (n = 3) of this experiment confirmed the difference in band intensity between the WT and β2−/− sample lane for these proteins (data not shown), suggesting that they have been excluded from the proteomic analysis because of their presence in the β2−/− lane. The presence of NIPs within the immunoprecipitated complex of the β2−/− suggests that they may interact with additional nAChR subunits, such as α4, immunoprecipitated by the antibody.
Discussion
We have examined constituents of the β2 subunit complex from brain by using MALDI-TOF-TOF MS/MS analysis of multiprotein complexes isolated by affinity purification with pulldown and immunoprecipitation methods. The isolation of protein complexes from native tissue provides a significant advantage over traditional protein interaction screens, such as the yeast two-hybrid, because it allows for the analysis of interactions within the normal cellular milieu (33). We conclude that the β2 subunit associates with well >20 cellular proteins termed NIPs. NIPs represent components of diverse β2–nAChR complexes existing within various brain regions, cell types, and subcellular compartments. At present, we do not know the specific profile and distribution of the various β2–nAChR complexes that exist in the brain. Moreover, because many protein interactions are transient, our proteomic analysis more likely identifies stable interactions of the β2 subunit. This is underscored by the discovery of cytoskeletal and scaffold molecules, such as clathrin and tubulin, and a dearth in the identification of signaling molecules such as kinases among the list of NIPs. Our findings are consistent with those of others on protein interactions for receptor and channel molecules and highlight a limitation of the current high-throughput proteomic approach (33).
The discovery of NIPs was achieved by pulldown and immunoprecipitation assays, and several NIPs (such as clathrin, dynamin, and tubulin) were found in both approaches. Some of the identified proteins (such as lysozyme, creatine kinase, and GAPDH) may represent interaction artifacts that arise from nonspecific binding to the antibody or the fusion protein or even possibly contaminants within the sample preparation. A protein such as myelin basic protein, for example, may associate with the receptor complex by means of (nonspecific) hydrophobic interactions. However, it is important to consider that myelin basic protein is known to bind various channel and receptor molecules (34) and that nAChRs are expressed within glial cells such as oligodendrocytes (35), suggesting that interaction between these two proteins cannot be excluded. Because the results of the pulldown study are based on interactions of the M3–M4 loop fusion protein with proteins expressed in brain, we propose that the NIPs identified by the pulldown strategy (such as synaptotagmin 1 and NSF) associate with the β2 subunit by coupling to the intracellular region of the receptor either directly or indirectly (as by an intermediate protein). Moreover, because interactions between proteins depend on conditions defined by the experimental paradigm, such as those imposing constraints on the stability of protein bonds as well as the proximity of interaction motifs (36), it is expected that NIPs identified by pulldown will differ from NIPs identified by immunoprecipitation (33, 37). Clearly, the combined use of multiple techniques provides the best strategy for identifying the full spectrum of interactions for any given protein (38). This fact is underscored by a report on the proteomic analysis of the NMDA receptor from brain, showing >77 binding partners for this receptor by use of immunoprecipitation, pulldown, and yeast two-hybrid procedures (19).
Antibody cross-reactivity can lead to the identification of false-positive interactions, thereby confounding the proteomic results. To overcome problems associated with cross-reactivity of the β2 antibody, we have conducted control experiments using β2−/− mice. This strategy aims to ensure selection of interactions that are specific to the β2 subunit expressed in the WT brain. It was somewhat perplexing that we did not detect association between β2 and other nAChRs (such as α4, α6, α5, and β3) as expected for mature β2–nAChRs (39). Because Western blotting experiments revealed that nAChRs, such as α4, are present in immunoprecipitated complexes of the β2−/−, we conclude that additional nAChR subunits have been excluded from the MS analysis because of their presence in the β2−/− control lane. These findings indicate a secondary reactivity of the anti-β2 antibody (mAb 270) to non-β2–nAChRs during the immunoprecipitation procedure. This reactivity is consistent with previous observations on cross-reactivity among nAChR antibodies in tissue (31) and may underlie the presence of some of the polypeptides within the immunoprecipitated complexes of the β2−/− mouse.
The presence of chaperone and transport proteins, such as eukaryotic elongation factor (eEF) and heat shock protein 90, suggests that our samples contain a fraction of unassembled β2 subunits from cells. Studies in cultured cells demonstrate the presence of large pools of partially assembled β2 subunits that are in association with Golgi and ER membranes. These subunits were found to assemble into mature β2–nAChRs in response to nicotine (40). Ultrastructural findings in rodent brain also support the notion that the majority of β2–nAChRs reside within the cell and in association with organelles such as the ER as well as with transport vesicles (41). Because studies show that chronic nicotine enhances the maturation of unassembled β2 subunits without altering β2 expression (30), it is tempting to speculate that molecules such as NIPs can contribute to the maturation and trafficking of β2–nAChRs in response to nicotine treatment. Interestingly, alterations in the expression of NSF and dynamin 1 have recently been documented in the brains of rodents subjected to chronic nicotine (42).
Protein Complexes for Trafficking the β2 Subunit in Cells.
The movement of receptors within cells requires the activity of molecules such as dynamin, a GTPase that mediates vesicle budding, and clathrin, a major constituent of the vesicle coat (8, 25). Several ligand-gated ion channels, such as GABAA, are found to traffic in a dynamin-dependent fashion (43). Our experiments indicate association of the β2 subunit with the neuron-specific dynamin 1 isoform and with the HC of the clathrin complex. Although it is not clear whether these interactions represent direct contacts between the nAChR and the NIP, our work supports the idea that β2–nAChRs are trafficked in a dynamin- and clathrin-dependent manner within neurons. Such a model of nAChR trafficking is in agreement with ultrastructural evidence of β2 subunit localization within clathrin-coated vesicles in neurons of the cortex (41).
The family of eEF proteins mediate protein synthesis and trafficking in cells and recently have been shown to contribute to plasticity within the brain (44). Studies indicate that eEF family proteins also bind to neurotransmitter receptors, such as the D3 dopamine and M4 muscarinic receptors, and mediate their trafficking to the plasma membrane (45, 46). We have uncovered an association between the β2 subunit and eEF2. It is possible that eEF2 interacts with β2–nAChRs within the ER, and this interaction contributes to subunit maturation and folding. Alternatively, it is possible that eEF2–β2 subunit associations mediate the assembly and targeting of β2–nAChRs to the plasma membrane.
NSF is an ATPase involved in the movement of transport vesicles within cells (47). NSF has been found to interact with neurotransmitter receptors such as the glutamate AMPA receptor and the β2-adrenergic receptor and to regulate their transport to synapses (48, 49). NSF has also been found to mediate the trafficking of the nAChR α7 subunit within postsynaptic regions of ciliary ganglion neuron (50). We find that the M3–M4 loop of the β2 subunit binds to NSF, and we confirm this interaction by using coimmunoprecipitation and Western blotting methods. Based on these findings, we hypothesize that interaction with NSF directs the trafficking of β2–nAChRs in neurons.
Potential Signaling Properties of the β2 Subunit.
nAChRs have been detected within the cell body, dendrites, and at synapses of neurons (39, 51). Presynaptically localized nAChRs, such as the α4β2, are known to play an important role in regulating neurotransmitter release in the brain (3). To date, however, little is known about the mechanisms by which nAChRs regulate neurotransmitter release. Our data indicate an interaction between the β2 subunit and synaptotagmin 1, a calcium sensor involved in vesicle docking and fusion with the synaptic plasma membrane (52). In previous studies, synaptotagmin 1 has also been found to interact with voltage-gated calcium channels (53) and with muscarinic receptors (54). Our work suggests that synaptotagmin 1 associates with the β2–nAChR within the M3–M4 loop and that this interaction may represent a possible mechanism whereby β2–nAChRs can regulate neurotransmitter release from synapses.
Our results indicate an interaction between the β2 subunit and cellular signaling proteins such as Goα, GIRK1, and GPRIN1. These findings suggest an unexpected association between β2–nAChRs and cellular G protein signaling pathways. Although this idea is unique for the nAChR, it is supported by reports showing an effect of G protein inhibitors on the gating and functional response of nAChRs in various types of cells (55, 56). Recently, an interaction between β2–nAChRs and the G protein-coupled dopamine D2 receptor in rat striatum has been reported (57). Our immunoprecipitation experiments confirm interaction between β2–nAChRs, GIRK1 channels, and Goα subunits in the brain. However, the MASCOT score for GIRK1 appears low based on immunoprecipitation studies, suggesting that GIRK1 interacts with the β2 subunit indirectly. In one model, GIRK1 may interact with the β2–nAChR through a mutual association of the Goα subunit (58). This interaction is supported by Western blot findings that suggest that Goα is more abundant then GIRK1 within the immunoprecipitated β2–nAChR complex (compare band intensity in Fig. 3 A). This model of indirect coupling between β2–nAChRs and GIRK1 channels is consistent with other reports on the nature of associations between receptors and channels in cells (59). Lastly, because GPRIN1 is reported to bind activated Goα subunits (60), GPRIN1 may also play a role in the coupling among β2–nAChRs, Goα subunits, and GIRK channel molecules. These proteomic findings represent a model for the signaling and regulation of the β2–nAChR in the brain by their association to the cellular G protein pathway.
Methods
Dissection and Protein Preparation.
Brain tissue was pooled from adult C57BL/6 WT or β2−/− mice (61). The dissection was conducted in a cold buffer solution (4 mM Hepes, 1 mM EDTA, and 0.32 M sucrose, pH 7.4), and then the tissue was transferred to a cold cutting buffer [10 mM Tris (pH 7.4), 320 mM sucrose, 1 mM PMSF, and 1× a protease inhibitor mixture (Roche)] for homogenization at a medium speed. The homogenate was centrifuged at 700 × g for 10 min at 4°C, and the supernatant fraction was collected while the pellet was homogenized and recentrifuged. Supernatants were subject to ultracentrifugation at 40,000 × g for 60 min at 4°C, and the pellet was solubilized overnight at 4°C in a solution of 100 mM Tris (pH 7.4), 1% Triton X-100, and 1× the protease inhibitor mixture. Protein concentrations were determined with the Bradford reagent kit (Bio-Rad).
Preparation of the GST Fusion Protein and Pulldown Procedures.
GST fusion protein constructs encoding the M3–M4 loop of the β2 subunit [GST-β2 (M3–M4)] were generated corresponding to amino acid residues 326–454 of the mouse β2 subunit. Bacterial transformations and protein inductions were performed as described in ref. 62. Bacterial BL21 (DE3) cells were grown overnight in 2× YTA medium using ampicillin (Sigma) selection. Protein production was induced with 5 mM isopropyl-β-d-thiogalactopyranoside (Sigma) for 4 h. The bacteria were harvested by centrifugation at 7,500 × g for 15 min at 4°C, and the pellet was suspended in cold HNG buffer [20 mM Hepes (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM MgCl2, 150 mM NaCl, 10% (vol/vol) glycerol, 1% Triton X-100, and 1× the protease inhibitor mixture]. The cells were lysed on ice by ultrasonication, and the lysate was centrifuged at 16,000 × g for 10 min at 4°C.
Purification of fusion proteins was performed by using a Sepharose bead matrix (Amersham Pharmacia), and protein bound matrix was washed three times with HNG buffer before pulldown experiments. Pulldown experiments were performed by incubating the Sepharose matrix-immobilized GST/GST-β2 (M3–M4) with the solubilized protein preparations overnight at 4°C [50 μg of immobilized GST/GST-β2 (M3–M4) per 1,500 μg of solubilized protein preparations]. The beads were then washed three times with a cold wash buffer (1× PBS, 0.1% Triton X-100, and 1× protease inhibitor mixture) before proteins were eluted by boiling in 1% SDS for 5 min. Proteins were separated by an SDS/polyacrylamide (4–20%) gradient gel (Invitrogen), and the gel was stained with 0.1% (wt/vol) Coomassie blue R350 solution (Sigma) [20% (vol/vol) methanol, and 10% (vol/vol) acetic acid] for protein visualization or transferred onto a nitrocellulose membrane for Western blot analysis.
Immunoprecipitation and Western Blot Analysis.
HEK 293 cells were maintained in Dulbecco's modified Eagle's medium and 10% FBS (Gibco). Cellular transfection of the nAChR was done by using plasmids encoding human β2 and α4 subunits as described in ref. 63. Immunoprecipitation experiments were performed by using monoclonal antibody mAb 270, which has been characterized (29). Solubilized proteins (1,500 μg) were mixed with 2 μg of mAb 270 overnight at 4°C. Immunocomplexes were then captured with protein A/G Dynabeads (Invitrogen) for 1 h at 4°C before being washed three times with the wash buffer. Proteins were eluted with LDS buffer (Invitrogen) at 70°C for 15 min and then separated by SDS/PAGE. Gels were stained with Coomassie or transferred onto a nitrocellulose membrane.
Western blot analysis was performed with the following primary antibodies: anti-β2 (H-92) (Santa Cruz Biotechnology); anti-α4 (H-133) (Santa Cruz Biotechnology); anti-clathrin HC (Santa Cruz Biotechnology); anti-NSF (Upstate Biotechnology); anti-dynamin 1 (PharMingen–Becton Dickinson); anti-synaptotagmin 1 (Chemicon); anti-GTP-binding protein (G protein) Goα (Santa Cruz Biotechnology), anti-GIRK1 (Alomone Laboratories); and anti-GST (Amersham Pharmacia). Primary antibodies were complexed with species-specific horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) and then visualized by enhanced chemiluminescence (ECL) using the ECL plus kit (Amersham Pharmacia).
Protein Identification with MS and MS/MS Analysis.
Selected bands were excised into 96-well plates. Destaining, reduction, alkylation, and trypsin digestion of the proteins were followed by peptide extraction and carried out by using the Progest Investigator (Genomic Solutions). After desalting (C18-μZipTip; Millipore), peptides were eluted directly by using ProMS Investigator (Genomic Solutions) onto a 96-well stainless steel MALDI target plate (Applied Biosystems) by a 0.5-μl CHCA matrix (10 mg/ml in 70% CAN, 30% H2O, 0.1% trifluoroacetic acid).
MS and MS/MS data for protein identification were obtained by using a MALDI-TOF-TOF instrument (4800 proteomics analyzer; Applied Biosystems). For positive-ion reflector mode spectra, 3,000 laser shots were averaged. For MS calibration, autolysis peaks of trypsin ([M+H]+ = 842.5100 and 2,211.1046) were used as internal calibrates. Monoisotopic peak masses were determined within the mass range of 800–4,000 Da, and up to 12 of the most intense ion signals were selected as precursors for MS/MS acquisition, excluding the trypsin autolysis peaks and the matrix ion signals.
In MS/MS positive ion mode, 4,000 spectra were averaged, collision energy was 2 kV, collision gas was air, and default calibration was set by using the Glu1-Fibrino-peptide B ([M+H]+ = 1,570.6696) spotted onto 14 positions of the MALDI target. Combined peptide mass fingerprinting PMF and MS/MS queries were performed by using the MASCOT search engine 2.1 (Matrix Science, Ltd.) embedded into GPS-Explorer Software 3.5 (Applied Biosystems) on the NCBI database with the following parameter settings: 50 ppm mass accuracy, trypsin cleavage, one missed cleavage allowed, carbamidomethylation set as fixed modification, oxidation of methionine was allowed as variable modification, MS/MS fragment tolerance was set to 0.3 Da. Protein hits with MASCOT protein score ≥70 and a GPS Explorer protein confidence index ≥95% were used for further manual validation.
Acknowledgments
We thank Drs. Pierre-Jean Corringer, Thomas Grutter, and Abdelkader Namane for helpful discussions. This work was supported by the Institut Pasteur and the Centre National de la Recherche Scientifique (J.-P.C. and N.K.) and in part by an award from Philip Morris, Inc., and Philip Morris International (to N.K.) and National Institutes of Health Grants MH 068789 (to R.L.) and NS11323 (to J.M.L.).
Footnotes
- †To whom correspondence may be addressed. E-mail: kabbani.nadine{at}ijm.jussieu.fr or changeux{at}pasteur.fr
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Author contributions: N.K. and J.-P.C. designed research; N.K., M.P.W., and R.L. performed research; J.M.L. contributed new reagents/analytic tools; N.K. analyzed data; and N.K. wrote the paper.
-
The authors declare no conflict of interest.
-
This article contains supporting information online at www.pnas.org/cgi/content/full/0710314104/DC1.
- © 2007 by The National Academy of Sciences of the USA
References
Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics
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