Article |
Address correspondence to Thomas C. Südhof, Center for Basic Neuroscience, UT Southwestern Medical Center, 6000 Harry Hines Blvd. NA4.118, Dallas, TX 75390-9111. Tel.: (214) 648-1876. Fax: (214) 648-1879. E-mail: thomas.sudhof{at}utsouthwestern.edu
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Abstract |
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Key Words: neurexin; dystroglycan; synapse; LNS domain; laminin
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Introduction |
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Like neurexins, dystroglycan is also a cell surface protein. However, unlike neurexins dystroglycan is expressed ubiquitously and is composed of two invariable subunits, - and ß-dystroglycan, that are derived by proteolytic cleavage from a single dystroglycan precursor and remain bound to each other on the cell surface (Ibraghimov-Beskrovnaya et al., 1992, 1993). The
-dystroglycan subunit contains an autonomously folded NH2-terminal domain and a highly glycosylated sequence (Brancaccio et al., 1997) and accounts for almost the entire extracellular part of dystroglycan. In contrast, the ß-dystroglycan subunit is composed of a single transmembrane region and a cytoplasmic tail with little extracellular sequence. The function of dystroglycan has been well defined in embryonic development and in mature muscle cells (for reviews see Hemler, 1999; Henry and Campbell, 1999). Extracellularly, dystroglycan binds to the LNS domains of several extracellular matrix proteins, namely laminin, agrin, and perlecan (Ervasti and Campbell, 1993; Bowe et al., 1994; Campanelli et al., 1994; Gee et al., 1994; Peng et al., 1998; Henry et al., 2001). Intracellularly, it binds to dystrophin and utrophin, which in turn are connected to the actin cytoskeleton (Ibraghimov-Beskrovnaya et al., 1992, 1993; Ahn and Kunkel, 1993). By virtue of these interactions, dystroglycan links the intracellular actin cytoskeleton to the extracellular matrix, especially the basal lamina, and thereby connects cells mechanically in the context of an overall tissue. Dystroglycan function is essential, since its deletion leads to early embryonic lethality (Henry and Campbell, 1998). In addition, muscle dystroglycan is required for normal neuromuscular junction formation (Peng et al., 1999; Grady et al., 2000).
Dystrophin and dystroglycan are expressed abundantly in neurons where their function is unknown (Lidov et al., 1990; Kim et al., 1992; Ibraghimov-Beskrovnaya et al., 1993; Tian et al., 1996; Blake and Kröger, 2000). The extracellular space between neurons in adult brain lacks a basal lamina and does not contain the classical extracellular matrix proteins laminin, perlecan, collagen, or agrin (Jucker et al., 1996; Ruoslahti, 1996). Basal lamina in brain delineate blood vessels, which are contacted by astrocytic "feet" extensions but have no direct connections with neurons. Thus, dystroglycan in neurons has no known extracellular-binding partner, and its role is obscure. However, the function of neuronal dystroglycan may potentially be important not only for insight into neuronal cell biology but also for understanding muscular dystrophies, since these are often associated with cognitive impairment (Bresolin et al., 1994; Davies, 1997). In the present study, in a search for endogenous ligands of -neurexins we identified dystroglycan as the major interacting partner. Extensive studies showed that dystroglycan binds tightly to single LNS domains in both
- and ß-neurexins in a manner regulated by alternative splicing. Our studies suggest a novel role for dystroglycan in neurons as intercellular cell adhesion molecules by binding to neurexins.
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Results |
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The binding of neurexins to dystroglycan resembles binding of laminin, which is also Ca2+-dependent and mediated by LNS domains (for review see Timpl et al., 2000) but differs from laminin binding in that the alternative splicing of the neurexin LNS domains controls dystroglycan binding (Fig. 7 A). In the LNS domains from laminin, a Ca2+-binding site was defined structurally, and the residues involved in Ca2+-binding are conserved in the neurexin LNS domains (Hohenester et al., 1999; Rudenko et al., 1999; Tisi et al., 2000). To examine if the second LNS domain of neurexin 1 contains an analogous Ca2+-binding site, we mutated one of the aspartate residues of the putative Ca2+-binding site to asparagine. We then used glutathione S-transferase (GST) fusion proteins of wild-type and mutant LNS domains to test
-dystroglycan binding (Fig. 7 B). As with the Ig fusion proteins, the GST fusion protein bound dystroglycan in a Ca2+-dependent manner; binding was completely abolished by the Ca2+-binding site mutant. Furthermore, GST fusion proteins of the splice variants of the second LNS domain that were inactive in the Ig fusion proteins were also inactive in the GST fusion proteins (Fig. 7 B). Among others, these experiments suggest that neurexin LNS domains have Ca2+-binding sites similar to those of laminin but do not have to be glycosylated in order to bind to dystroglycan, since the bacterial GST fusion proteins are unlikely to be glycosylated.
Our data indicate the possibility that dystroglycan, currently known as an anchor protein for the extracellular matrix in nonneuronal cells but not as a cell adhesion molecule, may function in brain by interacting with neuron-specific neurexins. This hypothesis implies that brain dystroglycan should be expressed in a developmental profile similar to that of neurexins if they functionally cooperate. Indeed, immunoblot analysis of rat brain proteins at different stages of postnatal development revealed parallel expression of dystroglycan and neurexins (Fig. 8)
. -Dystroglycan and
- and ß-neurexins were of low abundance prenatally, increased dramatically after birth, and experienced peak expression during the second postnatal week, a period of intense synaptogenesis. Thereafter, the levels of dystroglycan and neurexins declined together to a steady-state level that was approximately half of peak abundance at the end of the second postnatal week (Fig. 8, P15). This expression profile was different from that of laminin, a ligand for dystroglycan in basement membranes, which was expressed at a relatively constant low level similar to the ubiquitous control protein VCP. By contrast, synaptotagmin exhibited a cumulative increase in expression during development, in parallel with the continuous accumulation of synaptic vesicles in presynaptic nerve terminals as synapses mature.
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Discussion |
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The neurexindystroglycan complex has several potentially interesting characteristics. The second and sixth LNS domains of -neurexins and the single LNS domain of ß-neurexins specifically bind to
-dystroglycan in a manner that is regulated by alternative splicing in an all-or-none fashion (Fig. 7, A and B). Thus, a single
-neurexin molecule can potentially bind multiple dystroglycans, which may explain the patchy appearance of bound neurexin on the surface of dystroglycan-expressing cells (Fig. 5 C). The fact that only an alternatively spliced subset of neurexins is capable of complexing with dystroglycan on the cell surface indicates that use of splice variants regulates neuronal interactions with dystroglycan. Glycosylation of dystroglycan is required for neurexin to bind. Dystroglycan exhibits tissue-specific differences in glycosylation (Ibraghimov-Beskrovnaya et al., 1992, 1993) that may be functionally relevant, since native lung dystroglycan was unable to bind to neurexin 1
but was activated by partial deglycosylation (Fig. 6 B). However, neurexin glycosylation is not required for dystroglycan binding, since bacterially expressed LNS domain from neurexin 1
was fully active (Fig. 7 B).
Neurexins constitute the first neuronal dystroglycan ligands that are also cell surface proteins. Conversely, dystroglycan is the first transmembrane ligand for -neurexins that could involve
-neurexins in cellcell interactions similar to the binding of neuroligins to ß-neurexins (Ichtchenko et al., 1995, 1996; Irie et al., 1997; Nguyen and Südhof, 1997). The binding of neuroligins to ß-neurexins has been implicated in synaptogenesis because neuroligins are highly enriched in postsynaptic densities (Ichtchenko et al., 1995; Song et al., 1999), the interaction of neuroligins with ß-neurexins results in the formation of an intercellular junction that is coated on both sides by PDZ domain proteins (Irie et al., 1997; Butz et al., 1998), and expression of neuroligin in fibroblasts induces presynaptic specializations in nerve terminals that contact these fibroblasts (Scheiffele et al., 2000). It is possible that the interaction of dystroglycan with neurexins has a similar role in synaptogenesis or that this interaction serves in other contacts of neurons with each other or with astrocytes. Ultrastructural localization of neurexins and dystroglycan would be very helpful here but has proved exceedingly difficult especially for neurexins because we and others have been unable until now to raise antibodies with high enough affinity for EM.
In nonneuronal cells, the primary function of dystroglycan is to provide a transmembrane link between the extracellular matrix, especially basal lamina, and the intracellular actin cytoskeleton (for reviews see Hemler, 1999; Henry and Campbell, 1999). However, in fully developed brain neurons are not in direct contact with basement membranes or other types of extracellular matrixcontaining laminin, agrin, or perlecan, the currently known ligands for dystroglycan (Ervasti and Campbell, 1993; Gee et al., 1993; Campanelli et al., 1996; Gesemann et al., 1996; Jucker et al., 1996; Ruoslahti, 1996; Tian et al., 1996; Peng et al., 1998; Serpinskaya et al., 1999; Talts et al., 1999). Thus, the primary function of dystroglycan in nonneuronal cells, to attach cells to the extracellular matrix, does not apply to neurons. Our results suggest that one of the ligands with which dystroglycan interacts in brain may be neurexins, thereby providing an explanation for the abundant expression of dystroglycan in neurons where its biological role is unknown (Lidov et al., 1990; Ibraghimov-Beskrovnaya et al., 1992, 1993; Tian et al., 1996; Blake and Kröger, 2000).
Although functionally different, the binding of dystroglycan to the LNS domains of neurexins follows the same paradigm as the binding of dystroglycan to the LNS domains in laminin and agrin, all of which bind to dystroglycan in a Ca2+-dependent reaction that requires glycosylation of dystroglycan and that is mediated by single LNS domains. The three-dimensional structures of LNS domains uncovered a similarity with lectins (Hohenester et al., 1999; Rudenko et al., 1999; Grishkovskaya et al., 2000), consistent with the notion that the carbohydrates on dystroglycan are bound directly by the LNS domain. Furthermore, a Ca2+-binding site was observed in the laminin LNS domain (Hohenester et al., 1999), and our mutational analysis indicates that neurexin LNS domains have a similar Ca2+-binding site (Fig. 7 B). Together, these data suggest a unitary structural mechanism by which LNS domains bind to dystroglycan; possibly other LNS domain proteins, such as NG2, CASPR, or slit (Rothberg et al., 1990; Nishiyama et al., 1991; Peles et al., 1997), may also bind. However, our results also reveal an unexpected versatility of LNS domains. The second LNS domain of -neurexins interacts with two proteins, dystroglycan and neurexophilin, via distinct mechanisms. Neurexophilin binds to the second LNS domain independent of Ca2+ and irrespective of alternative splicing (Missler and Südhof, 1998b; Missler et al., 1998), whereas dystroglycan binding requires Ca2+ and is regulated by alternative splicing. This suggests that neurexophilin and dystroglycan contact distinct sites in the second LNS domain. The sixth LNS domain of
-neurexins, simultaneously the only LNS domain of ß-neurexins, also participates in multiple interactions, namely with dystroglycan, neuroligins, and
-latrotoxin (Ichtchenko et al., 1995, 1996; Sugita et al., 1999). These interactions share the same Ca2+ requirement and regulation by alternative splicing, indicating that all three ligands may contact the same site on the surface of this LNS domain. Dystroglycan and
-latrotoxin bind to either
- and ß-neurexins without regard to flanking sequences, in agreement with the finding that a single LNS domain is sufficient for dystroglycan binding (Fig. 7). However, neuroligins only bind to the ß-neurexin LNS domain when it is preceded by the short sequence unique to ß-neurexins, which thus confers specificity (Ichtchenko et al., 1995, 1996).
What is the function of the dystroglycanneurexin complex? The stoichiometric nature and abundance of the complex suggest that it is physiologically important, but its localization is unclear. Indirect evidence links neurexins and dystroglycan to the synapse (Lidov et al., 1990; Ushkaryov et al., 1992; Hsueh et al., 1998; Song et al., 1999; Sugita et al., 1999; Blake and Kröger, 2000), an interesting possibility in view of the cognitive disorders associated with some cases of muscular dystrophy in which the dystroglycan-binding protein dystrophin is mutated (Bresolin et al., 1994). Both dystroglycan and -neurexins are essential genes required for viability (Henry and Campbell, 1998). Although in mice dystrophin is not necessary for normal neuronal function, possibly because of redundancy, dystrophin-deficient mice exhibit a mislocalization of synaptic
-aminobutyric acid receptors (Knuesel et al., 1999, 2001). This indicates that the dystrophin-related complex may be required for the proper localization of proteins to postsynaptic density in central synapses, similar to the role of postsynaptic dystroglycan in neuromuscular junctions (Grady et al., 2000). A synaptic cell adhesion complex composed of dystroglycan and neurexins would be ideally suited for synaptic interactions because similar to synapses this complex is asymmetric. Furthermore, the regulation of this complex by alternative splicing of neurexins could provide a mechanism of control whereby the type of alternative splicing would dictate the intercellular interactions of a neuron, and the polymorphism of neurexins would translate into a recognition code. Finally, our results suggest a mechanism by which some forms of muscular dystrophy could give rise to cognitive dysfunction, namely via a defective linkage of the dystroglycanneurexin complex to dystrophin. However, these are speculative hypotheses at present that have to be explored in future studies.
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Materials and methods |
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Affinity chromatography experiments
For Ig fusion protein affinity chromatography, two frozen rat brains were homogenized in 11 ml of 20 mM Hepes-NaOH, pH 7.4, 1 mM EGTA, and 0.1 g/liter PMSF, and an equal volume of the same buffer containing 0.2 M NaCl, 2% Triton X-100 was added. The homogenate was extracted for 1 h at 4°C, insoluble material was removed by centrifugation (30 min at 100,000 g), and 2.5 mM MgCl2 and 3.0 mM CaCl2 were added. Protein ASepharose containing IgN1-1 or Igcontrol (
80 µg protein in 0.2 ml) were preequilibrated with buffer A (20 mM Hepes-NaOH, pH 7.4, 0.1 M NaCl, 1% Triton X-100, 2.5 mM MgCl2, 2 mM CaCl2), incubated overnight at 4°C with 10 ml of the brain extract, centrifuged (800 g for 2 min), and washed with 15 ml buffer A. Washed Sepharose was packed into polypropylene columns with paper discs (Quick-Sep; Isolab), washed again with buffer A (5 ml), and sequentially eluted with 4 ml of buffer A containing 0.2 M NaCl, 1.0 M NaCl, and 1.0 M NaCl with 5 mM EGTA instead of CaCl2. Eluted proteins were TCA precipitated, resuspended in 200 µl sample buffer, and 20 µl were analyzed by SDS-PAGE and silver staining. The 120-kD protein that was affinity purified on the IgN1
-1 was cut out of the gel, digested with trypsin, and tryptic fragments were purified by high performance liquid chromatography and analyzed by Edman degradation as described (Hata et al., 1993), identifying
-dystroglycan. The identity of both dystroglycan subunits was then confirmed by immunoblotting with specific antibodies. For analysis of how much of the dystroglycan in brain extracts bound to Igneurexins, 1 ml each of the brain extract was incubated with 20 µg of IgN1
-1 or Igcontrol protein immobilized on protein ASepharose, affinity chromatography was performed essentially as above, and samples were analysed by SDS-PAGE and immunoblotting with antibodies to
- and ß-dystroglycan (VIA4-1, Upstate Biotechnologies; and 43DAG/8D5, Novocastra) and to neuroligin (4C12; Song et al., 1999), with equal amounts of the total samples to allow precise comparisons. For domain analyses, 1 ml of brain extract containing 1 mM EGTA or 2 mM Ca2+ was incubated overnight with 30 µl protein ASepharose containing
8 µg of the various Ig neurexin fusion proteins (Fig. 1). The Sepharose beads were washed five times with 1 ml of the incubation buffers, resuspended in 250 µl of SDS-sample buffer, and 40 µl were analyzed by SDS-PAGE and Coomassie blue staining or immunoblotting. GST affinity chromatography experiments were carried out with GST fusion proteins immobilized on glutathione agarose (Sigma Aldrich) essentially as described above for the Ig affinity chromatography procedures. To test if
-latrotoxin can displace dystroglycan bound to neurexin, 10 ml brain extract were incubated for 5 h at 4°C with 200 µl of protein ASepharose containing 5 µg of IgN1
-1 to saturate binding of dystroglycan to neurexin 1
. After washing, the Sepharose beads were divided into nine tubes containing 0.1 ml buffer A with 0.5 µg of BSA and 0.0140 nM
-latrotoxin. After overnight incubation, the supernatant was recovered by centrifugation (800 g), the Sepharose was washed three times with 0.5 ml buffer A, resuspendend in 80 µl sample buffer, and 10 µl of the supernatant and the bound material were analyzed by SDS-PAGE and immunoblotting.
Immunoprecipitations
Eight frozen rat brains were homogenized in 80 ml of 10 mM Hepes-NaOH, pH 7.2, 0.32 M sucrose, and 1 mM CaCl2, centrifuged 10 min at 800 g to remove debris, and recentrifuged for 20 min at 14,000 g. The resulting crude synaptosomes were resuspended for 30 min at 4°C in 40 ml buffer B (20 mM Hepes-NaOH, pH 7.4, 0.1 M NaCl, 1% Triton X-100, 1 mM CaCl2, and 2.5 mM MgCl2) with protease inhibitors (0.1 g/liter PMSF, 10 mg/liter leupeptin, 10 mg/liter aprotinin, and 1 mg/liter pepstatin A). Insoluble material was removed by centrifugation (30 min at 100,000 g), and 20 ml of the synaptosome extract were incubated overnight with 150 µl of a mixture of antibodies directed against the COOH terminus of all neurexins (A473, B186, G393, G394) (Ushkaryov et al., 1992) or of the preimmune serum for B186, followed by 2 h incubation with 200 µl of protein ASepharose preequilibrated with buffer B. Afterwards, samples were centrifuged (800 g for 2 min), the Sepharose beads were washed with 15 ml of buffer B, and packed into a polypropylene column. The column was washed with 5 and 4 ml buffer B, and eluted with 4 ml buffer B containing 1.0 M NaCl and 5 mM EDTA. The final wash and eluted materials were TCA precipitated, resuspended in 160 µl sample buffer, and 40 µl were analyzed by SDS-PAGE and immunoblotting using ECL detection.
Partial purification of dystroglycan
Brain, skeletal muscle, heart, and lung tissues from SVB/NJFVB mice were disrupted with a polytron followed by Dounce homogenization in 50 mM Tris-HCl, pH 7.4, 500 mM NaCl, 1% Triton X-100, 0.6 µg/ml pepstatin A, 0.5 µg/ml leupeptin, 0.5 µg/ml aprotinin, 0.75 mM benzamidine, 0.1 mM PMSF, 0.4 µg/ml calpain inhibitor 1, and 0.4 µg/ml calpeptin (buffer C). The homogenate was incubated for 2 h at 4°C, centrifuged at 140,000 g for 30 min at 4°C, and the supernatant was incubated with WGA agarose (Vector Laboratories) overnight at 4°C. The WGA agarose was washed with buffer C containing 0.1% Triton X-100 and eluted with 50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100, and 0.3 M N-acetyl glucosamine. The eluate was concentrated with Centricon-30 (Amicon). Extraction of dystroglycan from ES cells was performed accordingly except that 50 mM Hepes, pH 7.4, 0.2 M NaCl, 1% NP-40, and protease inhibitor mixture was used instead of buffer C.
Deglycosylation
For enzymatic deglycosylation, partially purified dystroglycan (30 µl) was boiled for 5 min in the presence of 0.7% SDS. 1% Triton X-100 was added, and the pH was adjusted to 5.5 with 50 mM sodium-acetate. The preparation was incubated with 100 mU of sialidase and 2 mU of O-glycosidase (Roche) at 37°C for 16 h, the pH was neutralized with 0.1 M sodium-phosphate buffer, and the mixture was incubated with 10 mU of N-glycosidase F (Glyko) at 37°C for 16 h and boiled for 3 min. Control samples were treated identically without enzymes. For chemical deglycosylation (Ervasti and Campbell, 1993), dystroglycan (30 µl) was lyophilized, resuspended under nitrogen in 0.9 ml TFMS (Sigma-Aldrich) and 0.6 ml anisole (Sigma-Aldrich), and incubated 5 h on ice. The reaction was quenched with 1.6 ml ice-cold pyridin/H2O (3:5), dialyzed against cold distilled water, and lyophilized. Control samples were treated identically without TFMS. Samples were analyzed with the neurexin overlay assay or dystroglycan immunoblotting using mouse monoclonal antibody IIH6 and affinity purified rabbit and goat polyclonal antibodies to ß- and -dystroglycan, respectively (Ibraghimov-Beskrovnaya et al., 1992; Ervasti and Campbell, 1993).
Neurexin overlay assays
Proteins were separated by SDS-PAGE on 312% gradient gels and transferred to PVDF membranes. Membranes were blocked for 1 h in 5% dry milk buffer D (10 mM triethanolamine, pH 7.6, 140 mM NaCl, 1 mM CaCl2, 0.1% Tween 20), incubated overnight at room temperature in buffer D containing 0.9 mg/liter IgN1-1 in 5% BSA, and washed three times with buffer D. Membranes were then incubated with HRP-conjugated protein A (Roche) for 1 h at room temperature and washed again three times with buffer D.
Cell culture and immunofluorescence analysis
Dystroglycan heterozygous (DG+/-) and homozygous mutant (DG-/-) R1 ES cells were cultured (Henry and Campbell, 1998) on coverslips coated with 5 µg/cm2 human fibronectin (Collaborative Biomedical Products). After 24 h, the culture medium was replaced with medium containing 10 mM CaCl2 and 75 nM IgN1-1 or Igcontrol, incubated overnight, washed with PBS, and fixed in 4% paraformaldehyde. Bound Ig fusion proteins were detected by Texas redconjugated antihuman (Fc domain) antibody (Jackson ImmunoResearch Laboratories), and viewed by confocal microscopy (Henry and Campbell, 1998; Henry et al., 2001).
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Footnotes |
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Acknowledgments |
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This study was supported by grants from the National Institutes of Health (RO1-MH52804 to T.C. Sudhof), by investigatorships from the Howard Hughes Medical Institute to K. Cambell and T.C. Sudhof, and by a postdoctoral fellowship from the Muscular Dystrophy Association to S. Sugita.
Submitted: 1 May 2001
Revised: 7 June 2001
Accepted: 11 June 2001
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References |
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