- Research article
- Open Access
The function of Shp2 tyrosine phosphatase in the dispersal of acetylcholine receptor clusters
© Qian et al; licensee BioMed Central Ltd. 2008
Received: 10 March 2008
Accepted: 23 July 2008
Published: 23 July 2008
A crucial event in the development of the vertebrate neuromuscular junction (NMJ) is the postsynaptic enrichment of muscle acetylcholine (ACh) receptors (AChRs). This process involves two distinct steps: the local clustering of AChRs at synapses, which depends on the activation of the muscle-specific receptor tyrosine kinase MuSK by neural agrin, and the global dispersal of aneural or "pre-patterned" AChR aggregates, which is triggered by ACh or by synaptogenic stimuli. We and others have previously shown that tyrosine phosphatases, such as the SH2 domain-containing phosphatase Shp2, regulate AChR cluster formation in muscle cells, and that tyrosine phosphatases also mediate the dispersal of pre-patterned AChR clusters by synaptogenic stimuli, although the specific phosphatases involved in this latter step remain unknown.
Using an assay system that allows AChR cluster assembly and disassembly to be studied separately and quantitatively, we describe a previously unrecognized role of the tyrosine phosphatase Shp2 in AChR cluster disassembly. Shp2 was robustly expressed in embryonic Xenopus muscle in vivo and in cultured myotomal muscle cells, and treatment of the muscle cultures with an inhibitor of Shp2 (NSC-87877) blocked the dispersal of pre-patterned AChR clusters by synaptogenic stimuli. In contrast, over-expression in muscle cells of either wild-type or constitutively active Shp2 accelerated cluster dispersal. Significantly, forced expression in muscle of the Shp2-activator SIRPα1 (signal regulatory protein α1) also enhanced the disassembly of AChR clusters, whereas the expression of a truncated SIRPα1 mutant that suppresses Shp2 signaling inhibited cluster disassembly.
Our results suggest that Shp2 activation by synaptogenic stimuli, through signaling intermediates such as SIRPα1, promotes the dispersal of pre-patterned AChR clusters to facilitate the selective accumulation of AChRs at developing NMJs.
Synapses facilitate efficient neuronal communication by bringing close together the organelles and molecules involved in the release and detection of neurotransmitters. Thus, to understand how synapses function, it is necessary to elucidate the signaling pathways that regulate the development of cellular specializations unique to synapses. For this purpose, one particular synaptic specialization – found at the vertebrate neuromuscular junction (NMJ) – has been examined extensively over the past several decades. Enriched in the postsynaptic domain of the NMJ are ion channels named acetylcholine (ACh) receptors (AChRs) that open upon binding to the neurotransmitter ACh; opening of AChRs depolarizes the synaptic region of muscle to trigger contraction. The synaptic aggregation of AChRs during NMJ assembly is therefore a process of paramount importance, one that is recognized to be tightly controlled by the combined actions of both muscle- and nerve-derived factors.
AChR clustering in muscle is initiated by the activation of the receptor tyrosine kinase MuSK (muscle-specific kinase) and it is mediated by the cytoplasmic protein rapsyn that binds to AChRs and tethers them to the cytoskeleton [1–3]. During development, rudimentary AChR clusters first appear before innervation in a MuSK-dependent manner and bestride the midline of embryonic muscle fibers [4, 5]; these clusters are referred to as "pre-patterned" clusters because they form in the absence of neural influence. Later, in innervated muscle, AChRs become concentrated at incipient synapses where MuSK is locally stimulated by the nerve-derived factor agrin [4–6]. This synaptic accumulation of AChRs is furthermore coupled with the disassembly of the pre-patterned AChR clusters [7–10], and these two distinct processes together help generate a 1000-fold higher density of AChRs at the NMJ compared to extra-synaptic muscle .
The disassembly of pre-patterned AChR clusters, which is our focus here, was first shown to occur independently of AChR activity [7–10]; however, it was also reported that in rat and chick myotubes AChR agonists trigger the loss of AChR clusters [12, 13], an issue that has recently attracted renewed attention. It has been observed that pre-patterned AChR aggregates, which are eliminated by embryonic day 17 or 18 (E17-18) in innervated muscle fibers of normal mice, are retained in the muscles of mutant mice with defective motor innervation [4, 5] and of mice lacking the gene encoding the ACh biosynthetic enzyme choline acetyltransferase (CHAT) [14, 15]. These findings have engendered the view that ACh directs the disassembly of AChR aggregates, and it has been proposed that neural agrin protects synaptic AChRs against ACh-induced dispersal . The dispersal of AChR clusters by ACh is further thought to involve the ser/thr kinase Cdk5 [17, 18] whose activity, in turn, appears to depend on the regulation of the protease calpain by rapsyn .
AChR activity-independent AChR dispersal has been well documented in frog and fish muscle where synaptogenic stimuli trigger both the assembly and disassembly of AChR aggregates [7, 9, 10, 20–22]. In our studies we have used embryonic Xenopus muscle cultures to focus on the regulation of the AChR dispersal process by tyrosine phosphatases [23–25] because it is known that tyrosine phosphorylation stabilizes AChR clusters by strengthening links between AChRs and the actin cytoskeleton [26–31]. We showed that tyrosine phosphatases mediate the dispersal of pre-patterned AChR clusters by synaptogenic stimuli and that tyrosine dephosphorylation of pre-patterned clusters precedes the loss of AChRs from these sites in situ [2, 23–25, 32]. To date, however, the specific phosphatases involved in dispersing AChR clusters and the signaling pathways that stimulate them have remained unknown. These issues were addressed here using pharmacological and molecular methods and we describe for the first time a role of the tyrosine phosphatase Shp2, and its activator signal regulatory protein α1 (SIRPα1), in the disassembly of pre-patterned AChR clusters.
Recombinant heparan-binding growth associated molecule (HB-GAM) was generously provided by Dr. Heikki Rauvala (University of Helsinki). Neural agrin was obtained as described . The following reagents were purchased: NSC-87877 (Acros Organics; Fairlawn, NJ); tetramethylrhodamine-conjugated α-bungarotoxin (R-BTX) (Molecular Probes; Eugene, OR); anti-phosphotyrosine (mAb4G10) and anti-STEP tyrosine phosphatase monoclonal antibodies (Upstate Biotechnology; Lake Placid, NY); anti-Shp2 monoclonal antibody and protein tyrosine phosphatase antibody kit (BD Biosciences; San Jose, CA); secondary antibodies conjugated to FITC (Zymed; South San Francisco, CA) and horseradish-peroxidase (HRP) (Jackson Immuno Research Laboratories, Inc; West Grove, PA); Triton X-100 (TX-100) (Pierce; Rockford, IL).
Whole-mount in situ hybridization
Xenopus embryos collected at different developmental stages were fixed in MEMFA solution (0.1 M MOPS, pH 7.4, with 2 mM EGTA, 1 mM MgSO4, and 3.7% formaldehyde) for 2 h at room temperature and then stored in absolute methanol at -20°C. Embryos were re-hydrated through a graded methanol series before carrying out in situ hybridization as described . The cDNA encoding Xenopus SH2 domain-containing tyrosine phosphatase Shp2 in pCS2+ vector was kindly provided by Dr. Benjamin Neel (Beth Israel Deaconess Medical Center). This was used to synthesize digoxigenin-labeled cRNA probes in the antisense and sense directions with SP6 and T3 RNA polymerases with the Riboprobe In Vitro Transcription kit (Promega; Madison, WI). Hybridization reactions were carried out in parallel with these probes at a concentration of 0.1 μg/ml (in the presence of 1 mg/ml Torula RNA) overnight at ~60°C. Embryos were washed extensively with several buffers, including those containing RNaseA (20 μg/ml) and RNase T1 (10 U/ml), and then stained overnight at 4°C with alkaline phosphatase-linked anti-digoxigenin antibody. Chromogenic reaction was carried out by using NBT/BCIP as substrate in the alkaline phosphatase buffer (100 mM Tris HCL, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween-20, 5 mM levamisol). Embryos were made transparent with a 2:1 mixture of benzyl benxoate:benzyl alcohol and imaged using a color CCD camera.
Protein extract preparation and immuno-blotting
C2 mouse myoblasts (American Type Culture Collection) were differentiated into myotubes (4–5 d) and proteins were extracted from them using a TX-100 buffer (100 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% TX-100, and 1 mM Na-pervanadate; 1 ml buffer/10 cm dish) as described . To extract Xenopus embryonic muscle proteins, dissected tadpole tails were homogenized in the above Triton buffer and the homogenates were incubated for 30 min on ice (with frequent mixing) before centrifuging them to obtain clarified extracts. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes, which were blocked in Tris-buffered saline containing 0.1% Tween-20 and 5% milk and immuno-blotted with antibodies diluted in the same blocking buffer. Primary antibodies were detected using appropriate HRP-linked secondary antibodies and enhanced chemiluminescence substrate (West Pico, Pierce).
Shp2 and SIRPα1 cDNA constructs and mRNA synthesis
Human wild-type and mutant Shp2 cDNA constructs were from Dr. Benjamin Neel (Beth Israel Deaconess Medical Center). The mutant Shp2s were Shp2 E76A, which has a point mutation in the N-SH2 domain and produces a constitutively active Shp2 , and Shp2 deltaP, which has a deletion in the catalytic domain and, lacking both catalytic activity and substrate-binding ability, acts as a dominant-negative mutant . Wild-type Shp2 (Shp2 WT), Shp2 E76A and Shp2 deltaP cDNAs were cloned into pSP64R1 vector . Human full-length SIRPα1 (SIRP-FL) and truncated SIRPα1 (SIRP-TR) constructs were from Dr. David Clemmons (University of North Carolina at Chapel Hill). In SIRP-TR, the Y475 site of the tandem phosphorylation domain of SIRPα1 that binds to Shp2 is eliminated, generating a SIRPα1 mutant that does not activate Shp2 and functions as a dominant-negative suppressor of Shp2 signaling . SIRPα1 sequences were in pcDNA3.1 vector  as were those encoding the reporter green fluorescent protein (GFP). Shp2 mRNAs were prepared with the SP6 in vitro transcription kit purchased from Ambion (Austin, TX) and the SIRPα1 and GFP mRNAs were prepared with the T7 ULTRA kit from the same company. Shp2 and SIRPα1 mRNAs were mixed with GFP mRNA before microinjection.
Xenopus embryo microinjection and preparation of primary muscle cultures
Mixtures of Shp2 or SIRPα1 and GFP mRNAs (total 10 ng or less, in a volume of 4.6–9.2 nl) were injected into one cell of 2–4 cell stage Xenopus embryos with a Drummond Nanoject oocyte injector (Drummond Scientific Co., Broomall, PA). Injected embryos were maintained in Holfreter's solution containing 4% Ficoll and those expressing exogenous mRNAs were identified by green fluorescence and used for preparing muscle cultures . Myotomal muscle cells were cultured from stage 20–22 embryos as described  and plated on glass coverslips coated with ECL (entactin-collagen IV-laminin) substrate (Upstate Biotechnology). AChR redistribution experiments used muscle cells maintained in culture for 2–6 days.
Shp2-inhibitor, R-BTX/antibody labeling and microscopy
The Shp2-inhibitor NSC-87877  was dissolved in water and then diluted into culture medium as required. R-BTX-labeled muscle cells were incubated in culture medium without or with NSC-87877, in the absence or presence of agrin or HB-GAM beads. Polystyrene beads (10 μm; Polysciences, Warrington, PA) were coated with HB-GAM as described . In cases where muscle cells expressed exogenous mRNAs, live cultures were used to examine cells of comparable GFP fluorescence intensity and AChR cluster redistribution data were collected from a minimum of five separate mRNA injections and culture preparations. In experiments using wild-type cultures, muscle cells were fixed with cold 95% ethanol and stained with primary and fluorescent secondary antibodies. Labeled cells were imaged using an Olympus IX70 microscope equipped with a Hamamatsu ORCAII cooled-CCD or ORCA-ER camera controlled by MetaMorph software (Universal Imaging, West Chester, PA).
Shp2 expression in Xenopus embryonic muscle
Shp2 inhibition and AChR cluster dispersal
The above results and those showing enhanced AChR clustering in C2 mouse myotubes following the depletion or inhibition of Shp2 [25, 43] support the conclusion that in Xenopus muscle cells, as in mouse myotubes, Shp2 limits the formation of AChR clusters. But does Shp2 also mediate the dispersal of pre-patterned AChR clusters? To answer this we again used NSC-treatment and examined the AChR clusters that form spontaneously in Xenopus muscle cells that have been in culture for 2 or more days. These large AChR clusters, which are often more than 10–15 μm across and were previously referred as "hot spots", were the ones considered here to be "pre-patterned" because they develop without synaptogenic stimulation. These pre-patterned clusters are stable for several days and do not disperse on their own, but they are rapidly disassembled when muscle cells are innervated by co-cultured nerves or when the cells are exposed to agrin or growth factor-coated beads [23–25]. We have previously demonstrated that the rate of dispersal of a pre-patterned cluster is directly related to its distance from a synaptogenic stimulation site as well as to the strength of the external stimulus [10, 23]. In all experiments described below, muscle cells were labeled with R-BTX before synaptogenic stimulation so that the true disassembly of pre-patterned clusters could be followed, and, in all cases, untreated (control) cells from the same culture preparation were examined in parallel to confirm that pre-patterned clusters had developed normally. These control cells additionally showed that during the course of the assays (none of which lasted longer than one day) there was no significant increase in spontaneous AChR clustering, which is in accord with our previous work . This point is important to consider because the rapid, net decrease in the number of pre-patterned AChR clusters that follows synaptogenic stimulation demonstrates that external stimuli actively disperse old AChRs clusters rather than merely preventing (if this occurs at all) the assembly of additional, new pre-patterned clusters (leaving the original pre-patterned clusters intact). Indeed, we have previously used growth factor-coated beads and time-lapse imaging of identified pre-patterned clusters to show their dispersal by applied stimuli in live cells . Lastly, in this study pure muscle cultures were used throughout to avoid any (currently unknown) presynaptic effect of NSC-87877 that could potentially influence AChR redistribution in nerve-muscle co-cultures.
The above NSC-87877 data suggest that Shp2 signaling promotes the disassembly of pre-patterned AChR clusters, which agrees with and extends our earlier finding that tyrosine phosphatase activity mediates AChR cluster dispersal [2, 23, 25].
Exogenous Shp2 expression and AChR cluster dispersal
Here we also tested whether ectopic expression of the inactive Shp2 in Xenopus muscle cells would significantly hinder AChR cluster dispersal and found that not to be the case (images not shown; see quantified data in Figures 6, 7). This is possibly because we obtained only low-level expression of inactive Shp2 in cells (as indicated by GFP fluorescence) and attempts to enhance this mutant's expression by injecting more of its mRNA into embryos disrupted development as previously noted [35, 36]. That Shp2 signaling plays a role in mediating the dispersal of AChR clusters, however, is suggested by experiments described below using the Shp2-activator SIRPα1 and a mutant form of it.
SIRPα1 over-expression and AChR cluster dispersal
The disassembly of pre-patterned AChR clusters that occurs in muscle following motor innervation facilitates selective AChR concentration at the NMJ. Here we studied the process by which AChR clusters are disassembled by synaptogenic stimuli using primary cultures of Xenopus muscle cells. We found that AChR cluster dispersal was expedited when active or activatable Shp2, or the Shp2-activator SIRPα1, was introduced into muscle cells, but that cluster dispersal was blocked when cells were exposed to an inhibitor of Shp2 or when they over-expressed a mutant form of SIRPα1 that curbs Shp2 signaling. These results suggest that Shp2 and its regulators such as SIRPα1 promote the disassembly of pre-patterned AChR clusters by synaptogenic stimuli during NMJ development.
Innervation of muscle triggers AChR concentration at the NMJ. For this to occur, three proteins are indispensable: rapsyn, for clustering AChRs , MuSK, for initiating the AChR aggregation process , and agrin, for enabling synaptic AChR accumulation [16, 48]. AChR clusters are further stabilized by tyrosine kinases [29, 30, 49] and the dystrophin complex proteins  that directly or indirectly strengthen AChR-cytoskeleton linkage. Moreover, AChR clustering requires dynamic actin polymerization , which is regulated by small GTPases, their effectors and other modulators [51–54], and clustering is also promoted by a transmembrane protein, LRP4 , and the MuSK-binder, dok-7 .
In addition to AChR aggregation, innervation triggers the dispersal of pre-patterned AChR clusters. In Xenopus primary muscle cultures these two processes can be readily distinguished from one another and the redistribution of AChRs in response to various stimuli can be accurately quantified [23, 25, 32, 41]. We have previously shown that tyrosine phosphatases mediate AChR cluster dispersal [23, 25] and here we identified Shp2 as one of the phosphatases that facilitates dispersal. Treatment of muscle cells with NSC-87877, a selective Shp2 antagonist , hindered the dispersal of pre-patterned AChR clusters without preventing the induction of new AChR clusters, as also seen with the general phosphatase inhibitor pervanadate but not other drugs [23, 25, 32, 45].
NSC-treatment enhanced AChR clustering in Xenopus muscle cells whereas the expression of active Shp2 had the opposite effect, consistent with our results in C2 myotubes . In C2 myotubes, however, the expression of dominant-negative Shp2 promoted AChR cluster formation  but here no significant effect of this mutant on cluster induction was detected. Additionally, because the inactive Shp2 mutant was only poorly expressed in Xenopus muscle cells, we could not test whether "dominant-negative" inhibition of Shp2 would block AChR cluster dispersal, as suggested by our pharmacological assays. But because Shp2-dependent suppression of AChR clustering in C2 myotubes could be stimulated by the phosphoprotein SIRPα1 , we examined if exogenous SIRPα1 proteins can affect the dispersal of pre-patterned AChR clusters in Xenopus muscle cells. Expression of full-length and truncated SIRPα1, which enhance and inhibit Shp2 signaling , promoted and blocked AChR cluster dispersal, respectively. These results suggest that SIRPα1 (or a protein related to it) spreads the signal that globally activates Shp2 to facilitate AChR cluster disassembly.
Several studies (in addition to ours) have demonstrated that pre-patterned AChR cluster disassembly occurs in amphibian and fish muscle independently of AChR activity. For example, in Xenopus muscle cells, pre-patterned clusters are dispersed by innervation when all AChR channel activity is blocked using bungarotoxin or curare and no electrical activity or twitching is detected in muscle [7, 9, 20]. Moreover, during NMJ formation in vivo in zebrafish, pre-patterned AChR clusters are redistributed when saturating concentrations of bungarotoxin are used or even when the bungarotoxin is added together with inhibitors of sodium channels to further ensure blockade of all neuronal activity . Thus, in these muscle cells, ACh-dependent AChR channel opening appears to be unnecessary for redistributing pre-patterned AChR aggregates during NMJ formation. Whether ACh can subtly influence the effectiveness with which other synaptogenic stimuli disperse pre-patterned clusters, however, requires further investigation.
In contrast to the above findings, in rodent and chick myotubes AChR agonists reduce surface AChR clusters [12, 13] and muscle electrical activity suppresses extra-synaptic AChR synthesis [60, 61]. Other studies have revealed a more direct role of ACh in AChR cluster disassembly, as summarized by this recent model : ACh elevates muscle intracellular Ca2+ and stimulates the protease calpain, which cleaves a protein named p35 to generate a p25 fragment ; p25 potently activates the protein kinase Cdk5, which disperses AChR clusters [17–19]. At the NMJ agrin promotes calpain's interaction with rapsyn, which inhibits calpain and suppresses p25 production to locally limit Cdk5 activity and block AChR dispersal . Thus agrin is thought to protect synaptic AChRs against dispersal by ACh . However, as discussed in these studies, some aspects of this dispersal pathway warrant further investigation. For example, calpain is activated in muscle more rapidly than Cdk5, suggesting that additional unknown factors regulate Cdk5 during dispersal . Since Cdk5 phosphorylation sites have not been found in AChR subunits, it is also unclear exactly how Cdk5 disperses AChR clusters . Intriguingly, AChR activity, which favors dispersal in embryonic muscle, stabilizes AChRs during synaptic competition , which could be due to changes in the molecular makeup of AChR subunits or clusters during development . And, the elevation of intracellular Ca2+ by agonists of AChRs and L-type calcium channels actually promote AChR clustering [63, 64], suggesting that Ca2+ can either favor AChR cluster assembly and stabilization or cluster destabilization and dispersal (through calpain/Cdk5).
Roles of Shp2 or other tyrosine phosphatases in ACh-dependent AChR dispersal have not been described, but phosphatase inhibition blocks ACh-independent dispersal caused by defects in src signaling [29, 65, 66] or intracellular Ca2+ flux . Shp2's function at the NMJ in vivo also remains elusive, although one study  has reported that NMJs appear normal around birth in mice in which Shp2 expression is reduced in muscle. In that study the presence of residual Shp2 in muscle was not ruled out and the formation of pre-patterned AChR clusters ~E14 and the dispersal of these clusters by ~E17 were not examined . Several other findings, however, argue in favor of Shp2's involvement in NMJ development: Shp2 regulates neuregulin/ErbB signaling and AChR gene expression , Shp2 limits agrin/MuSK-dependent formation of new AChR clusters [25, 43], and Shp2 promotes the disassembly of pre-patterned (this study). Interestingly, Shp2 associates with MuSK and is able to stabilize AChR clusters in rodent myotubes , which could be due to its ability to activate src. However, excessive src kinase or tyrosine phosphatase activity also destabilizes AChR clusters in these cells , which is compatible with our findings vis-à-vis SIRPα1 and Shp2 and the inhibition of AChR clustering [25, 43] and the facilitation of dispersal (this study). Lastly, tyrosine phosphatases have recently also been found to regulate the membrane insertion of AChRs at the NMJ  suggesting that additional novel functions of Shp2 and other phosphatase in NMJ development await discovery.
During the earliest stages of NMJ establishment, motor innervation of muscle induces the clustering of AChRs at incipient synapses as well as the disassembly of pre-patterned AChR aggregates formed in muscle before innervation. In amphibian muscle, synaptogenic stimuli direct both of these processes independently of AChR activity, with the former process being suppressed by tyrosine phosphatases and the latter being mediated by phosphatases. In this study we found that pharmacological or molecular manipulation of one specific phosphatase – Shp2 – produced quantifiable changes in the dispersal of pre-patterned AChR clusters in Xenopus muscle cells: whereas dispersal was blocked by inhibitors of Shp2 signaling, it was accelerated under conditions elevating muscle Shp2 protein or activity levels. Our results suggest a role of Shp2 (and its regulators such as SIRPα1) in not only limiting AChR cluster formation [25, 43] but also in promoting the disassembly of pre-patterned AChR clusters by synaptogenic stimuli. Functioning in these two distinct ways Shp2 may enhance the efficiency with which AChRs are concentrated at developing NMJs and depleted from non-synaptic regions of muscle. The participation of other muscle tyrosine phosphatases in the dispersal of pre-patterned AChR clusters, by synaptogenic stimuli or by ACh, and the potential involvement of Shp2 signaling in the dispersal of AChR clusters by ACh remain to be tested.
We thank Drs. Benjamin Neel and David Clemmons for cDNA constructs and Ms. Frances Chan for excellent technical help. This work was supported by RGC grants HKUST6280/03M, HKUST6401/05M and AoE grant B-15/01.
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