Laminin-{alpha}1 globular domains 3 and 4 induce heterotrimeric G protein binding to {alpha}-syntrophin's PDZ domain and alter intracellular Ca2+ in muscle

Yan Wen Zhou, Shilpa A. Oak, Susan E. Senogles, and Harry W. Jarrett

Department of Biochemistry, University of Tennessee, Memphis, Tennessee

Submitted 11 June 2004 ; accepted in final form 21 September 2004


    ABSTRACT
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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{alpha}-Syntrophin is a component of the dystrophin glycoprotein complex (DGC). It is firmly attached to the dystrophin cytoskeleton via a unique COOH-terminal domain and is associated indirectly with {alpha}-dystroglycan, which binds to extracellular matrix laminin. Syntrophin contains two pleckstrin homology (PH) domains and one PDZ domain. Because PH domains of other proteins are known to bind the {beta}{gamma}-subunits of the heterotrimeric G proteins, whether this is also a property of syntrophin was investigated. Isolated syntrophin from rabbit skeletal muscle binds bovine brain G{beta}{gamma}-subunits in gel blot overlay experiments. Laminin-1-Sepharose or specific antibodies against syntrophin, {alpha}- and {beta}-dystroglycan, or dystrophin precipitate a complex with G{beta}{gamma} from crude skeletal muscle microsomes. Bacterially expressed syntrophin fusion proteins and truncation mutants allowed mapping of G{beta}{gamma} binding to syntrophin's PDZ domain; this is a novel function for PDZ domains. When laminin-1 is bound, maximal binding of Gs{alpha} and G{beta}{gamma} occurs and active Gs{alpha}, measured as GTP-{gamma}35S bound, decreases. Because intracellular Ca2+ is elevated in Duchenne muscular dystrophy and Gs{alpha} is known to activate the dihydropyridine receptor Ca2+ channel, whether laminin also altered intracellular Ca2+ was investigated. Laminin-1 decreases active (GTP-{gamma}S-bound) Gs{alpha}, and the Ca2+ channel is inhibited by laminin-1. The laminin {alpha}1-chain globular domains 4 and 5 region, the region bound by DGC {alpha}-dystroglycan, is sufficient to cause an effect, and an antibody that specifically blocks laminin binding to {alpha}-dystroglycan inhibits G{beta} binding by syntrophin in C2C12 myotubes. These observations suggest that DGC is a matrix laminin, G protein-coupled receptor.

Duchenne muscular dystrophy; protein G {beta}{gamma}-subunit; pleckstrin homology domain


SYNTROPHINS ARE A GROUP of peripheral membrane proteins first identified in Torpedo postsynaptic membranes (18). Syntrophins (Syn) have been found to be closely associated with dystrophin (Dys) (10), the protein product of the Duchenne muscular dystrophy gene locus (10, 31). In skeletal muscle, Dys and Syn are found in a complex with other proteins and glycoproteins, the dystrophin glycoprotein complex (DGC) (14, 64), whose defects cause Duchenne, Becker, various limb girdle, and other muscular dystrophies (52). DGC {alpha}-dystroglycan binds laminin and other extracellular matrix proteins, while DGC {beta}-dystroglycan, the sarcoglycans, and sarcospan traverse the lipid bilayer. On the inside of the cell, {beta}-dystroglycan and {alpha}-sarcoglycan bind dystrophin, and dystrophin binds F-actin microfilaments. Thus the DGC provides a link between laminin in the extracellular matrix and the cytoskeleton (38, 44).

Syntrophins are a multigene family of homologous proteins. Three Syn isoforms, {alpha}, {beta}1, and {beta}2, are found in muscle and are products of different genes (1, 2, 4, 62). The {alpha}-syntrophin is expressed primarily in striated muscles and brain, while the {beta}-syntrophins are ubiquitous in mammalian tissues (3). Each of the syntrophins contains two pleckstrin homology (PH) domains: an NH2-terminal PH1 domain and a PH2 domain (see Fig. 1). The PH1 domain of {alpha}-syntrophin binds phosphatidylinositol 4,5-bisphosphate (11). Recently, this domain also was shown to be involved in the oligomerization of syntrophin in vitro in a Ca2+-dependent manner. Calmodulin inhibits oligomerization in a Ca2+-independent manner (41). PH domains are also known to bind the G{beta}{gamma} dimer of heterotrimeric G proteins. The NH2-terminal PH domain is interrupted by an inserted PDZ domain. The PDZ domain is found in membrane proteins and was named for the first three proteins in which this ~90-amino acid motif was identified: the postsynaptic density protein, the Drosophila disks large protein, and the zonula occludens 1 protein (30). Syntrophin's PDZ domain has been shown to bind neuronal nitric oxide synthetase (nNOS) (2, 20), muscle and nerve voltage-gated Na+ channels (19), and the MAP kinase, stress-activated protein kinase 3 (24). Syntrophins also bind calmodulin (37). Thus syntrophins act as adapters between the dystrophin complex of proteins and components of the cell signaling apparatus.



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Fig. 1. Diagrammatic representation of fusion proteins containing {alpha}1-syntrophin (Syn) sequences. The shaded portions show the location of the PDZ domain in syntrophin. Numbers in parentheses represent the amino acid sequences present in each fusion protein. PH, pleckstrin homology domain; SU, syntrophin unique domain.

 
Heterotrimeric G proteins are components of signal transduction pathways, which are regulated by soluble signals and by contact with adjacent cells or with components of the extracellular matrix, in normal growth and differentiation. Heterotrimeric G proteins consists of a G{alpha}-subunit, which binds and hydrolyzes GTP, and a G{beta}{gamma} heterodimer. Two major mechanisms exist for transmembrane signaling in intercellular communication mediated by receptor tyrosine kinases and G protein-coupled receptors (7). For the G protein-coupled receptors, interaction of the {beta}{gamma} complex of heterotrimeric G proteins (G{beta}{gamma}) appears to use specific protein-protein interactions to localize key signaling components to appropriate membrane compartments. This interaction has been proposed to be mediated by the PH domain found in many signaling proteins that interact with G{beta}{gamma} (20, 55).

Among the heterotrimeric G proteins, Gs is known to activate the voltage-gated, dihydropyridine-inhibited Ca2+ channel in skeletal muscle (63). Intracellular Ca2+ is elevated in Duchenne muscular dystrophy and the mdx mouse model for this disease. This results in increased Ca2+-dependent calpain proteolysis and eventual muscle fiber destruction, and these changes can be reversed by decreasing intracellular Ca2+ or inhibition of calpain (5, 6, 17, 57). Thus, if heterotrimeric G proteins bind to the DGC and induce changes in intracellular Ca2+, this could have important implications for muscle physiology and pathogenesis.

While investigating whether the PH domain of {alpha}-syntrophin interacts with the {beta}{gamma} complex of the heterotrimeric G protein, we discovered that syntrophin does indeed interact with the G{beta}{gamma} complex. However, much to our surprise, the binding was found to be through the PDZ domain of syntrophin instead of through the PH domain. Laminin binds to DGC {alpha}-dystroglycan and muscle {beta}1 integrins. Recently, laminin binding was shown to affect both phosphatidylinositol 3-kinase (PI3K)/Akt (33) and a Rac1/JNK (43) signaling pathway. Because laminin binds the DGC, we also investigated whether laminin affected G{beta}{gamma} binding and found that it does. Furthermore, a specific {alpha}-dystroglycan antibody blocked the laminin effect, and the same effect of laminin could be obtained with a small region of the laminin-1 {alpha}-chain, which is known to bind to {alpha}-dystroglycan. These data strongly argue that laminin's effect is mediated by the DGC and not by integrins. Finally, given the importance of Ca2+ in muscular dystrophy and the involvement of Gs{alpha} in regulating a Ca2+ channel, we showed that Gs{alpha} was bound by syntrophin and that laminin binding alters the Gs{alpha} activation state. Laminin-1 also was shown to decrease the voltage-gated Ca2+ uptake of C2C12 myotubes, suggesting one potential role for the DGC-Gs interaction. These studies suggest that muscular dystrophy defects in the DGC, by altering laminin binding and consequently Gs{alpha} activity, also may contribute to the increased muscle cell Ca2+ that accompanies Duchenne muscular dystrophy and its pathology.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Antibody against mouse {alpha}-syntrophin was produced in rabbit and purified by performing affinity chromatography on Syn A-Sepharose as previously described (36, 43). Rabbit polyclonal antibody against Gs{alpha} (COOH terminus) was obtained from Calbiochem. Rabbit polyclonal G{beta} antibody was purchased from Upstate Biotechnology. Mouse monoclonal antibodies against {alpha}-sarcoglycan, dystrophin (NCL-Dys1), and {beta}-dystroglycan were purchased from Novocastra Laboratories (Newcastle upon Tyne, UK). Rabbit sera against {alpha}- and {beta}-dystroglycan were the generous gifts of Dr. Tamara Petrucci. Goat anti-mouse IgG (H+L)-horseradish peroxidase conjugate and goat anti-rabbit IgG (H+L)-horseradish peroxidase conjugate were purchased from Bio-Rad. Laminin-1 (laminin derived from Englebreth-Holm-Swarm sarcoma cells) was obtained from BD Life Sciences. Ni2+-nitrilotriacetic acid-agarose was obtained from Qiagen. Cyanogen bromide (CNBr)-preactivated Sepharose was purchased from Sigma. Laminin-1-Sepharose was prepared as described previously (43). GTP-{gamma}35S was obtained from PerkinElmer. Fura-2 AM and pluronic acid were purchased from Molecular Probes. Coverslips were obtained from EM Science. Nifedipine was purchased from Research Biochemicals. All other chemicals were of the highest purity available commercially.

DGC was partially purified from digitonin-solubilized rabbit skeletal muscle microsomes using the procedure of Ervasti et al. (16) with succinylated wheat germ agglutinin chromatography. Aliquots were stored frozen at –85°C. DGC was further fractionated by performing electrophoresis on 4–15% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and blotted onto nitrocellulose as previously described (36). The gel blots were then overlaid with G{beta}{gamma} as described in Overlay experiments. Gel blots were also stained with specific antibodies for {alpha}-syntrophin, dystrophin, and {alpha}-sarcoglycan to confirm the identity of bands interacting with G{beta}{gamma}.

Fusion proteins. The mouse {alpha}-syntrophin fusion proteins [His]6-Syn and [His]6-Syn A, as well as maltose-binding protein (MBP) fusions MBP-Syn B, MBP-Syn H, and MBP-Syn I, were prepared as described previously (40). pET32 plasmid constructs for mouse syntrophin PH1, PH2, and the PDZ domain were a generous gift from Drs. Steven Gee and Stan Froehner. pET32 plasmids encoding [His]6-thioredoxin-PH1, -PH2, and -PDZ were used to express proteins referred to as [His]6-PH1, -PH2, and -PDZ (19). The His-Tag fusion proteins were purified by using Ni2+-nitrilotriacetic acid-agarose from Qiagen as described previously (40). The MBP fusion proteins were purified using the batch method described previously with amylose resin by Jarrett and Foster (28). The purity of the proteins was determined by performing 12% SDS-PAGE (32). Protein concentration was determined (8) using bovine serum albumin (BSA) as the standard.

Laminin-1 E3 protein was expressed from stably transfected human embryonic kidney-293 cells (generous gift of Prof. Donald Gullberg) and purified on DEAE-Sepharose and heparin-Sepharose as described previously (53). The purified protein was of the expected size and was homogeneous on the basis of Coomassie blue-stained SDS-PAGE.

Overlay experiments. The fusion proteins (1 µg) were examined by performing electrophoresis on a 12% SDS-PAGE gel and electroblotted onto nitrocellulose paper. The blot was then blocked with 10 mg/ml BSA in TTBS (20 mM Tris·HCl, pH 7.5, 0.5 M NaCl, and 0.2% Tween-20). After washing extensively with 1 mM CaCl2 in BSA-TTBS (1 mg/ml BSA in TTBS), the blot was overlaid with 0.1 mg/ml bovine brain G{beta}{gamma} prepared as described previously (29) for 2 h in the same buffer. The blot was then washed extensively with Ca2+-BSA-TTBS and incubated with anti-G{beta} (1:1,000 dilution). Goat anti-rabbit IgG (H+L)-horseradish peroxidase conjugate (1:3,000 dilution; Bio-Rad) was used after the primary antibody. The blot was developed for enhanced chemiluminescence (ECL) by incubation in a mixture of 15 ml of solution A (0.4 mM coumaric acid and 2.5 mM luminol in 0.1 M Tris·HCl, pH 8.5) and 15 ml of solution B (7.2 µl of 30% H2O2 in 0.1 M Tris·HCl, pH 8.5) in the dark room and then exposure to Kodak scientific imaging film. MBP-LacZ{alpha} and [His]6-green fluorescent protein (GFP) were used as controls for maltose and His-Tag fusion proteins, respectively. A similar experiment was performed to test the binding of G{beta}{gamma} to syntrophin from partially purified DGC.

Skeletal muscle membrane preparation. Frozen rabbit skeletal muscle (2 g; back muscle, predominantly fast-twitch fibers) was homogenized in 7 volumes of pyrophosphate buffer (20 mM sodium pyrophosphate, 20 mM sodium phosphate, 1 mM MgCl2, 0.303 M sucrose, and 0.5 mM EDTA, pH 7.0) in the presence of a protease inhibitor cocktail (1 µg/ml pepstatin A, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM benzamidine, and 0.1 mM phenylmethylsulfonyl fluoride) to minimize protein degradation (64). The homogenate was centrifuged at 13,000 g for 15 min at 4°C. The supernatant was then centrifuged for 30 min at 32,500 g at 4°C to pellet crude muscle microsomes. The microsomes were suspended in 600 µl of 50 mM Tris, pH 7.5, and 100 mM NaCl. The muscle membrane preparation (10 µl) was saved and labeled as crude (C).

Heparin-Sepharose depletion of laminin from skeletal muscle microsomes was performed as previously described (43). Depletion of laminin was confirmed by probing the supernatant from heparin-Sepharose with antibodies against merosin (1:100 dilution; Novocastra), laminin {beta}1 chain (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and 2E8 anti-laminin (1:500 dilution; Developmental Studies Hybridoma Bank, Iowa City, IA). The presence of dystrophin, {alpha}-syntrophin, and dystroglycans ({alpha} and {beta}) in the supernatant microsomes was confirmed using their respective antibodies.

Immunoprecipitation. To the laminin-depleted portion of the microsomes, either 0 or 3 µl of 1 mg/ml exogenous laminin was added to buffer K containing 1 mM CaCl2 for 1 h at 4°C, with gentle mixing. For some experiments, untreated microsomes were used. Microsomes were then "precleared" with protein G-Sepharose (25 µl) by being incubated for 30 min at 4°C and then with 2 µl of dystrophin (NCL-Dys1), syntrophin, {beta}-dystroglycan (either monoclonal or the polyclonal NCL-b-DG; gift from Dr. Tamara Petrucci), {alpha}-dystroglycan (VIA4 monoclonal antibody; gift from Dr. Kevin P. Campbell), or Na+-K+-2Cl cotransporter (NKCC) (monoclonal antibody; gift from Dr. Donald B. Thomason, Department of Physiology, College of Medicine, University of Tennessee Health Science Center, Memphis, TN) antibody. Microsomes were then solubilized by adding 1% Triton X-100, 0.5% Igepal, and 0.5% sodium deoxycholate. Incubation was continued for another 1 h at 4°C with gentle mixing. The immune complexes were then incubated with protein G-Sepharose (25 µl) for 1 h, precipitated, and washed extensively with buffer K. The bound proteins were detected, typically with the G{beta} antibody (1:2,000 dilution) or with others as specified in the figures, and visualized using ECL.

Solid-phase binding assays. [His]6-Syn (1.6 mg; 0.8 mg/ml) was coupled with 1 g of CNBr-activated Sepharose (Sigma) using procedures recommended by the manufacturer (Pharmacia). The support was then washed with the coupling buffer (0.1 M NaHCO3, pH 8.3, 0.5 M NaCl) and blocked for 2 h with 0.1 M Tris·HCl, pH 8, 0.5 M NaCl. The amount of the protein coupled (0.2 mg Syn/g of Sepharose) was determined by the difference in the ultraviolet absorption of the added protein and that recovered from coupling in the wash fractions. Similarly, 3 mg of [His]6-PDZ was coupled to 2 ml of CNBr-activated Sepharose, yielding 1.5 mg PDZ/ml of Sepharose (100% coupled). For negative controls, CNBr-activated Sepharose was treated the same, but no protein was coupled.

Syn-Sepharose (or PDZ-Sepharose) (200 µl of a 50% slurry) was equilibrated with buffer K (in mM: 20 HEPES, pH 7.4, 100 KCl, and 10 MgCl2) containing 1 mM CaCl2 and then centrifuged, and the pelleted beads were incubated with either purified bovine brain G{beta}{gamma} or 100 µl of rabbit skeletal muscle microsomes solubilized with 1% digitonin in buffer K with 1 mM CaCl2. Incubation was for 1 h on ice with gentle mixing in a final volume of 200 µl. For some experiments, 1 mM GTP-{gamma}S was included. For control, Sepharose without any protein coupled to it was used. After the incubation, Syn- or PDZ-Sepharose was washed three times with 0.5 ml of buffer K. The protein was eluted using 60 µl of 2x Laemmli sample buffer (32). Samples were heated for 5 min at 95°C. The samples were then centrifuged for 5 min at room temperature to remove the resin, subjected to electrophoresis on a 12% SDS-PAGE gel (32), and electroblotted onto nitrocellulose (56). The filter was then blocked with 5% nonfat dry milk in TTBS. After being washed extensively with 1 mg/ml BSA/TTBS, the blot was incubated with G{beta} (1:1,000 dilution) or Gs{alpha} (1:1,000 dilution) antibody. Goat anti-rabbit IgG (H+L)-horseradish peroxidase conjugate (1:3,000 dilution) and ECL were used for detection.

GTP-{gamma}35S binding experiments. Microsomes either were treated with Sepharose or heparin-Sepharose (43) to deplete endogenous laminin or were left untreated. Four samples were used: two samples of microsomes were treated with Sepharose (endogenous laminin), one sample was heparin-Sepharose treated (referred to as "minus" in the figures), and one sample was heparin-Sepharose treated with 1.8 µg of laminin-1 (exogenous). For each, 100 µl of microsomes were used. Each sample was precleared by mixing with 50 µl of pelleted protein G-Sepharose (washed in buffer K containing 1 mM CaCl2 and 1% digitonin) and 100 µl of buffer K containing 1 mM CaCl2, 2% digitonin, and then 2.9 µCi of GTP-{gamma}35S was added. This solution was gently mixed for 60 min at 4°C, the resin was pelleted (1,500 g, 1 min), and the supernatant microsomes were carefully removed. MBP antibody (3 µl) was added to one endogenous laminin sample to serve as a negative control, and 3 µl of either the affinity-purified syntrophin antibody or Gs{alpha} antibody were added to the other samples. Tubes containing 33 µl of fresh protein G-Sepharose (1:1 slurry) were prepared and washed with buffer K containing 1 mM CaCl2 and 1% digitonin. The microsome-antibody-GTP-{gamma}35S mixture (33 µl) was added to the pelleted resin in such a way that triplicates of each sample condition were obtained. These were gently mixed for 60 min at 4°C, and the resin was pelleted as described above and washed four times with 200 µl of buffer K containing 1 mM CaCl2 and 1% digitonin. The resin was then suspended in 100 µl of 125 mM Tris·HCl, pH 6.8, and 4% SDS and transferred to a scintillation vial. Scintillation fluid (4 ml of Ecosafe) was added, and the samples were counted for 35S.

Cell culture. C2C12 myoblasts were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS). Myotubes were differentiated by culturing in DMEM with 1% FCS for 5 days.

Antibody blockade with IIH6 antibody. C2C12 myotubes (1 x 106 cells) in 2 ml of DMEM containing 1% FCS were grown overnight in 35-mm plates at 37°C with anti-{alpha}-dystroglycan antibody VIA41 (1:200 dilution) or IIH6 (1:200 dilution). Both antibodies were the generous gift from Dr. Kevin P. Campbell. Cells were harvested without trypsin by scraping and counted in Trypan blue, and both contained the same number of cells and the same proportion of viable cells. Cells (7 x 105) were suspended in 200 µl of pyrophosphate buffer, homogenized using a Dounce homogenizer with type B pestle, and centrifuged for 15 min at 13,000 g at 4°C, and then the supernatant was centrifuged for 30 min at 4°C at 400,000 g to pellet microsomes. The pellets were suspended in 100 µl of buffer K with 1 mM CaCl2 and 1 mM GTP-{gamma}S, and 2 µl of additional VIA41 or IIH6 was added on ice for 1 h. The incubation was continued for another 1 h after adding 1 µg of laminin-1, 100 µl of PDZ-Sepharose, and then digitonin to produce a final concentration of 1%. After washing three times with 500 µl of buffer K with 1 mM CaCl2 and 1% digitonin, the bound protein was eluted using SDS-PAGE sample buffer. After electrophoresis and electroblotting, samples were probed with G{beta} antibody (1:1,000 dilution).

Intracellular Ca2+ measurement with fura-2. Ca2+ flux experiments were performed using a modification of the method of Parmentier et al. (45). C2C12 myoblasts were transformed to myotubes by incubation for 5 days in DMEM-1% FCS as described previously (34). On the fourth day, they were removed from the plate with trypsin and replated in dishes (100 mm diameter; Falcon) containing six coverslips (9 x 35 mm) and grown in DMEM-1% FCS for an additional day. The plates were then washed with Krebs-Ringer solution (in mM: 120 NaCl, 5 KCl, 0.62 MgSO4, 1.8 CaCl2, 10 HEPES, and 6 glucose, pH 7.4) and then incubated in 5 ml of Krebs-Ringer solution containing 0.05% BSA, 5 µM fura-2 AM, and 0.025% pluronic acid for 30 min at 37°C. Coverslips were incubated for an additional 30 min after being washed with fresh Krebs-Ringer solution. Coverslips were then placed in a PerkinElmer LS50B fluorescence spectrometer in a cuvette thermostatted at 37°C, which allowed solutions to be poured over the coverslip and the attached cells. The program Flwinlab was used to control the spectrophotometer and collect data. Intracellular Ca2+ concentrations were measured by 510-nm emission as the ratio of emission at excitation 340 nm/380 nm. The flow rate was 0.8 ml/min. Each solution (1.5 ml) containing an elevated concentration of KCl with or without laminin-1 or nifedipine was pumped over the cells, and then the cells were returned to HA solution (in mM: 20 HEPES-Tris, pH 7.4, 5 glucose, 1 CaCl2, and 1 MgCl2) containing 140 mM NaCl and 5 mM KCl before the next experiment. When laminin-1 was used, it was added to both the high- and low-KCl solutions. For other experiments in which KCl was varied, the solutions were prepared mixing HA containing 140 mM NaCl and 5 mM KCl with HA containing 145 mM KCl to obtain the desired KCl concentration while maintaining constant osmolarity. For most experiments, the elevated KCl solution was HA containing 60 mM KCl and 85 mM NaCl.


    RESULTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
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A schematic representation of mouse {alpha}-syntrophin sequences expressed as fusion proteins is shown in Fig. 1. The regions of mouse {alpha}-syntrophin's amino acid sequence in each construct are shown in parentheses. The boundaries of the PH1 domain are not well defined; our PH1 construct contains some additional sequences, which are shown in Fig. 1. The fusion proteins containing the PDZ domain are shaded because this domain is involved in binding to G{beta}{gamma}. Syn, Syn A, PH1, PH2, and PDZ were produced as His-Tag fusion proteins, while Syn B, Syn H, and Syn I were produced as maltose-binding fusion proteins. Each fusion protein was affinity purified.

To determine whether any of the DGC proteins interact with G{beta}{gamma}, a gel blot overlay experiment was performed. Gel blots of partially purified DGC bind purified bovine brain G{beta}{gamma} as shown in Fig. 2. Coomassie blue staining of the purified DGC is also shown. Clearly, the DGC is only partially purified and other proteins are present, but it is sufficient to determine G{beta}{gamma} binding. The two arrows show the position, in descending order, of {alpha}-syntrophin and {alpha}-sarcoglycan; specific staining with {alpha}-syntrophin and {alpha}-sarcoglycan antibodies confirms these identities. The G{beta}{gamma} binding at the position of {alpha}-sarcoglycan was very weak in the DGC preparations that we tested. Therefore, the remainder of this article is focused on this newly discovered interaction (Fig. 2), which we show is {alpha}-syntrophin, an intracellular peripheral membrane protein.



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Fig. 2. Protein G{beta}{gamma} binds syntrophin in the partially purified dystrophin glycoprotein complex (DGC) from rabbit skeletal muscle. Partially purified DGC was subjected to electrophoresis using Coomassie blue (CB) on a 4–15% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and electroblotted onto nitrocellulose paper. The blot was then overlaid with 0.1 mg/ml bovine brain G{beta}{gamma} for 2 h, and binding was detected with G{beta} antibody. Syn and {alpha}-sarcoglycan ({alpha}SG) show the bands detected by the {alpha}-syntrophin and {alpha}-sarcoglycan antibodies, respectively. Molecular mass values are shown at left.

 
In other experiments, the partially purified DGC was probed with other antibodies (data not shown). The top band in the Coomassie blue-stained gel in Fig. 2 is dystrophin, which was detected with a dystrophin antibody (NCL-Dys2; Novocastra) and was discussed previously (42).

To investigate whether a syntrophin-G{beta} complex is present in skeletal muscle, two different immunoprecipitation experiments were performed (data not shown). Microsomes prepared as described in EXPERIMENTAL PROCEDURES represent an early starting material used for the purification of the DGC (16). They represent impure sarcolemma and are useful for probing the bulk G{beta}{gamma} of skeletal muscle membranes and its associations with the DGC. Microsomes were detergent solubilized and immunoprecipitated with either the syntrophin polyclonal antibody or the {beta}-dystroglycan polyclonal antibody. As a negative control, the irrelevant anti-MBP antibody was also used. The gel blot of the bound proteins was probed with the G{beta}{gamma} antibody. The negative control showed no staining for G{beta}, while immunoprecipitates from the syntrophin or {beta}-dystroglycan antibodies clearly showed G{beta}. These experiments also showed that G{beta} is present in the crude rabbit skeletal muscle extract (data not shown) and is coimmunoprecipitated by these two different antibodies against DGC proteins.

Perhaps more informative are the results shown in Fig. 3. Figure 3A shows G{beta} added as a marker, crude microsomes, and immunoprecipitates with Dys and {alpha}-dystroglycan monoclonal antibodies. The precipitates clearly contain G{beta}, while the bulk of the G{beta} remains in the supernatant. Densitometry revealed that 30% of the G{beta} is precipitated by either the {alpha}-dystroglycan or the dystrophin antibody, suggesting that a large portion of the G{beta} in microsomes is associated with the DGC. Neither antibody apparently blocks G{beta}-DGC binding (see also Fig. 5D).



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Fig. 3. Immunoprecipitates (IP) with DGC antibodies specifically retain G{beta}. Rabbit skeletal muscle microsomes in (in mM) 50 Tris, pH 7.5, 100 NaCl, 1 CaCl2, 1 MgCl2 and 1 GTP{gamma}S [crude (C)] were used. The samples were precleared with protein G-Sepharose, and 2 µl of mouse antibodies against dystrophin (Dys), {alpha}-dystroglycan (VIA4) in A, and Na+-K+-2Cl cotransporter (NKCC) in B and C, were added. After 1-h incubation at 4°C, the microsomes were detergent solubilized (0.5% deoxycholate, 1% Triton X-100, and 0.5% Igepal final concentrations), and incubation continued for another 1 h. A fresh portion of protein G-Sepharose was then added and incubated overnight with gentle mixing. After centrifugation, the supernatants (S) were saved, and the pellets (P) were washed extensively with buffer K. The samples were mixed with SDS-PAGE sample buffer, and the same relative amount of microsomes was then added to each well for electrophoresis and electroblotting. The blots were probed with antibodies against G{beta} or Syn and visualized using enhanced chemiluminescence (ECL). Purified G{beta}{gamma} was applied as a positive control. HC, antibody heavy chain.

 


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Fig. 5. Syntrophin's G{beta}{gamma}-binding region is localized to the PDZ domain. Molecular mass values are shown at left. A: syntrophin fusion proteins and control proteins (5 µg except for G{beta}{gamma}, which was 3 µg) were electrophoresed on 12% SDS-PAGE gel and stained with Coomassie blue. Arrowheads indicate the position of the predicted size of each protein (for G{beta}{gamma}, G{beta} is indicated) at left of each band. PH1 is subject to proteolysis and oligomerization as reported previously (41) and appears complex; the position of the monomer is indicated. PDZ migrated as a dimer under these conditions as observed previously (41). B: Syn fusion proteins (1 µg) were separated by electrophoresis, blotted onto nitrocellulose, and overlaid with purified bovine brain G{beta}{gamma} (0.1 mg/ml), and binding was detected with anti-G{beta} antibody. [His]6-green fluorescent protein (GFP) and pMalC were used as controls for His-Tag and MBP, respectively. G{beta}{gamma} also was applied to lane 3 of the gel to serve as a positive control. The faint staining seen for PH2 is not PH2 (which migrates to a molecular mass near that of Syn A) and is an artifact not seen in other experiments. C: solid-phase binding assay of G{beta} from muscle microsomes was performed using either Sepharose (S) or PDZ-Sepharose (PDZ), as described in EXPERIMENTAL PROCEDURES, in the presence or absence of 1 mM GTP-{gamma}S as indicated. Crude microsomes (C) are also shown. The position of G{beta} is indicated with an arrow in D. D: C2C12 myotubes were grown in the presence of IIH6 or VIA4 antibodies overnight as described in EXPERIMENTAL PROCEDURES. IIH6 is an {alpha}-dystroglycan antibody that binds to the polysaccharides of this protein and is known to block laminin binding, while VIA4 also binds to {alpha}-dystroglycan but does not block laminin binding. Microsomes were then solubilized in SDS-PAGE sample buffer, electrophoresed, and electroblotted, and the blot was probed with the G{beta} antibody.

 
The specificity of these microsome immunoprecipitations is shown in Fig. 3, B and C. Like the DGC, NKCC is found in the sarcolemma. While it is known to be activated indirectly by Gs and Gi, it is activated through the {beta}-adrenergic receptor, and NKCC is not known to have any direct binding to G proteins (21). As shown in Fig. 3B, immunoprecipitates with NKCC antibody do not contain detectable G{beta} or the DGC component Syn (Fig. 3D). Other experiments show that the immunoprecipitates with the dystrophin and {alpha}-dystroglycan antibodies do not contain detectable NKCC, while immunoprecipitates with the NKCC antibody do contain most of the NKCC (data not shown). Thus microsome immunoprecipitates show a great deal of specificity only for proteins associated with the antibody used, and other sarcolemma proteins do not coprecipitate in these assays. Thus a significant fraction (30%) of sarcolemma G{beta} is associated with the DGC and not with an unrelated sarcolemma protein, NKCC.

Figure 4A shows that laminin-1-Sepharose binds to a complex containing G{beta} and that the addition of a syntrophin antibody or an irrelevant antibody had no effect on this interaction. Thus, unlike the case with the Rac1 complex previously characterized (43), the {alpha}-syntrophin antibody does not block the G{beta} complex (Fig. 4A), suggesting that different regions of syntrophin function in these two complexes. Laminin binds to {alpha}-dystroglycan and thus to the DGC containing syntrophin. DGC syntrophin may be responsible for the G{beta} binding shown in Fig. 4A. Alternatively, laminin-1 also binds to integrin-containing complexes.



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Fig. 4. Laminin affects G{beta} binding. In all images shown, brain G{beta}{gamma} was applied as a positive control in adjacent wells, and arrow in A, C, and D shows the positions of G{beta} on the gel blots. A: microsomes in buffer K with 1 mM CaCl2 solubilized with 1% digitonin were incubated with either the anti-maltose-binding protein (MBP) antibody or with anti-syntrophin and either Sepharose (CTL) or laminin-1-Sepharose. After washing, the resins were eluted with SDS-PAGE sample buffer, electrophoresed, and electroblotted, and the blot was probed with the G{beta} antibody. BD: microsomes in (in mM) 50 Tris, pH 7.5, 100 NaCl, and 1 CaCl2 either were treated with Sepharose alone and contained endogenous laminin (Endo) or were depleted of laminin using heparin-Sepharose (minus). The microsomes were then solubilized with digitonin and further treated. B shows the crude microsomes (C) and microsomes treated with Sepharose (S) or heparin-Sepharose (H)-treated fractions; these are the same microsomes labeled "Endo" and "minus," respectively, in C and D and in other figures. The microsomes were dissolved in SDS-PAGE sample buffer, electrophoresed, electroblotted, and probed with the 2E8 antibody (1:500 dilution) against the laminin {beta} chain. The results show that laminin (arrow in B) is depleted by the heparin-Sepharose treatment. C and D: "endo" refers to microsomes treated with Sepharose alone (same as S in B) that contain endogenous laminin. Minus refers to heparin-Sepharose-treated microsomes depleted of laminin (i.e., the same as "H" in B), and Exo refers to these depleted microsomes to which 3 µg of exogenous laminin-1 were added. The treated microsomes were incubated with syntrophin-Sepharose (C) or with an anti-dystrophin antibody (2 µl, 0.4 µg) followed by protein G-Sepharose (D). After being washed, the resins were then eluted with SDS-PAGE sample buffer, electrophoresed, electroblotted, and then probed with the anti-G{beta} antibody. The position of G{beta} is shown by the arrows in C and D.

 
To determine whether laminin binding affects G{beta} binding, it was first necessary to deplete the microsomes of laminin, as shown in Fig. 4B. Treatment of microsomes with heparin-Sepharose but not Sepharose alone was previously shown to deplete microsome laminin (41), and this is shown in Fig. 4B as well. This treatment has little effect on DGC proteins (41). Microsomes treated only with Sepharose contained endogenous laminin (Fig. 4B). Heparin-Sepharose treatment depletes laminin (Fig. 4B). Exogenous laminin-1 (3 µg) was added to heparin-Sepharose-treated microsomes.

Because syntrophin forms oligomers (41), syntrophin-Sepharose can also be used to bind complexes such as the DGC that contain syntrophin (43). We next used these observations to determine whether laminin binding to the DGC affects the association of heterotrimeric G proteins with the DGC. As shown in Fig. 4C, syntrophin-Sepharose binds a complex containing G{beta} from microsomes containing endogenous laminin (extracted with Sepharose alone); when laminin is depleted with heparin-Sepharose, much less G{beta} is found and readdition of laminin-1 reconstitutes G{beta} binding. Similar results with somewhat higher background were obtained when the complex was precipitated with a dystrophin antibody (Fig. 4D). As mentioned above, when complexes were precipitated with the {beta}-dystroglycan or syntrophin polyclonal antibodies, they also contained G{beta} (data not shown), and Fig. 3 shows that {alpha}-dystroglycan and dystrophin complexes contain G{beta}. Thus a complex that binds laminin-1 and contains dystrophin, {alpha}- and {beta}-dystroglycan, and syntrophin binds G{beta}. All of these results are consistent with laminin binding to the DGC to alter G{beta} binding to {alpha}-syntrophin. However, the possibility remains that integrins could have some role in this interaction.

To determine the region of {alpha}-syntrophin that binds G{beta}, an overlay experiment was performed in which a gel blot of syntrophin fusion proteins was overlaid with purified bovine brain G{beta}{gamma}. Figure 5 shows the G{beta}{gamma} interaction observed for all of the syntrophin fusion proteins tested, namely, Syn, Syn A, Syn B, Syn H, Syn I, PH1, PH2, and PDZ. The purity of these fusion proteins was previously demonstrated (40) and also is shown in Fig. 5A. [His]6-GFP and MBP-LacZ{alpha} (pMalC) were used as controls for His-Tag and maltose-binding fusion proteins, respectively, and as expected, neither showed any binding to G{beta}{gamma} (Fig. 5B). Purified bovine brain G{beta}{gamma} (0.6 µg) was used as a positive control for the antibody (Fig. 5B, lane 3). Syn, Syn A, Syn H, and PDZ all bind G{beta}{gamma}, while Syn B, Syn I, PH1, and PH2 do not. Note that the PH1 domain is present along with the PDZ domain in Syn H (see Fig. 1) and binds G{beta}{gamma} (Fig. 5B). However, this PH domain by itself does not show any binding to G{beta}{gamma}, while the PDZ domain alone shows strong binding to G{beta}{gamma}. Thus those fusion proteins containing the PDZ domain bind G{beta}{gamma}. Furthermore, the PDZ, PH1, and PH2 fusion proteins all contain a thioredoxin tag, but only the former binds G{beta}{gamma}, showing that binding resides in the PDZ sequences and not in the tag.

This result was confirmed in the experiment shown in Fig. 5C. The PDZ fusion protein was coupled to Sepharose and added to digitonin-solubilized microsomes in the presence or absence of GTP-{gamma}S, a nonhydrolyzable GTP analog. Clearly, PDZ-Sepharose but not Sepharose binds G{beta}, and GTP-{gamma}S has no discernible effect. Purified G{beta}{gamma} also was applied to the gel as a positive control to positively locate the G{beta}-subunit. The crude microsomes in this, but not in all, experiments showed a band at 45,000 mol wt that bound the G{beta} antibody. The identity of this band is unknown, but it clearly did not bind syntrophin's PDZ domain.

To further probe the laminin receptor responsible for G{beta} binding to syntrophin, this time using cultured C2C12 myotubes, two {alpha}-dystroglycan antibodies were used (Fig. 5D). The IIH6 antibody specifically blocks laminin binding to {alpha}-dystroglycan, while the VIA41 antibody does not (15). The IIH6 antibody also greatly attenuates G{beta} binding, while the VIA41 antibody does not. We conclude that {alpha}-dystroglycan serves as a laminin receptor for G{beta} binding to syntrophin.

Syntrophins also bind calmodulin (37), and one site lies at the NH2 terminus of the PDZ domain (27). Syntrophins also bind Ca2+, and Ca2+ causes syntrophin oligomerization (41). To determine whether calmodulin has any effect on this interaction, {alpha}-syntrophin-Sepharose was incubated with purified bovine brain G{beta}{gamma} in the presence or absence of calmodulin in 1 mM CaCl2 and 1 mM EGTA. Calmodulin had no effect on syntrophin binding to G{beta}{gamma} in the presence or absence of Ca2+. Other experiments confirmed that syntrophin binding to G{beta} was independent of the presence of Ca2+ or EGTA (data not shown)

One early occurrence in dystrophic muscle cell death is an increase in intracellular Ca2+ (6), which enters from the extracellular space and triggers muscle proteolysis (17, 57). Because G{beta} is a subunit of heterotrimeric G proteins and because there is considerable evidence that Gs binds to the voltage-gated Ca2+ channel of skeletal muscle (23), we next investigated whether the Gs protein binds, as well as how the nonhydrolyzable GTP analog GTP-{gamma}S affects syntrophin binding. For these experiments, we used two antibodies: one specific for G{beta} and another specific for Gs{alpha}. As shown in Fig. 6, Gs{alpha} and G{beta} were associated with DGC containing endogenous laminin or with heparin-Sepharose-depleted DGC when exogenous laminin-1 was added. However, when laminin was depleted and not readded, little binding of Gs{alpha} or G{beta} was observed. The buffer used for these experiments (buffer K) contained 10 mM MgCl2 (necessary for GTP-{gamma}S binding) and 1 mM CaCl2 (necessary for laminin binding). Under these conditions, the presence or absence of GTP-{gamma}S had little effect, suggesting that the activation state of Gs{alpha} had little discernible effect on the association with syntrophin. Similar results were observed when heparin, an inhibitor of the laminin-DGC interaction, was added to microsomes to inhibit laminin binding (data not shown). Thus Gs{alpha} and G{beta} showed a laminin-dependent interaction with the DGC that was independent of the GTP-bound state of Gs{alpha}.



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Fig. 6. Syntrophin binds G{beta} and Gs{alpha}, regardless of GTP-{gamma}S. Rabbit skeletal muscle microsomes were treated for 1 h at 4°C with Sepharose (Endo) or with heparin-Sepharose (minus). Laminin-1 (Exo; 1.8 µg) or E3 protein (0.25 µg) was added to some heparin-Sepharose-treated microsomes. The microsomes were then incubated for 1 h at 4°C with syntrophin-Sepharose in buffer K containing 1 mM CaCl2 and 1% digitonin in the presence and absence of 1 mM GTP-{gamma}S, a nonhydrolyzable GTP analog. After being washed, the proteins were eluted in SDS-PAGE sample buffer, resolved by electrophoresis, and electroblotted. The blots were then detected with either the G{beta} or Gs{alpha} antibody as shown. G{beta}{gamma} was also included as a positive control in an adjacent well.

 
Figure 6 also shows the effect of the E3 protein. Laminin is an {alpha}{beta}{gamma}-heterotrimer. Laminin {alpha} chain contains five globular domains called laminin globular (LG) domains. E3 comprises the expressed LG4–5 domains of the laminin {alpha}1 chain, and LG4 is the specific region of laminin-1 that binds to {alpha}-dystroglycan (13, 54), while tested integrins bind to LG1–3, sequences not present in E3. Figure 6 shows that E3 and laminin both increased G{beta} binding to laminin-depleted microsomes, localizing the region of laminin-1 responsible to the LG4–5 domains. This also strongly suggests laminin binding to {alpha}-dystroglycan rather than to integrins to induce G{beta} binding to syntrophin.

The effect of laminin binding on the activation state (measured as GTP-{gamma}35S binding) of Gs{alpha} and the protein G{alpha}-subunits bound by {alpha}-syntrophin is shown in Fig. 7, again using heparin-Sepharose to deplete microsome laminin. After incubation with GTP-{gamma}35S and detergent solubilization, Gs{alpha} was immunoprecipitated with its antibody or the complex with syntrophin was immunoprecipitated with the {alpha}-syntrophin antibody. For a negative control, the anti-MBP antibody was used. In the presence of either endogenous or exogenous laminin, very little Gs{alpha} was active (i.e., GTP-{gamma}S bound) and the amount was comparable to that obtained with the irrelevant antibody control. Laminin depletion resulted in a statistically significant (P < 0.05) doubling of the activation. The results with the syntrophin and Gs{alpha} antibodies were similar, suggesting that much of the active microsomal Gs{alpha} was associated with syntrophin.



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Fig. 7. The activation state of Gs{alpha} is altered by laminin binding. Microsomes that were treated with Sepharose ("endo"), heparin-Sepharose ("minus"), or heparin-Sepharose with addition of 1.8 µg of laminin-1 ("exo") were subjected to the GTP-{gamma}35S binding assay. After binding and detergent solubilization, either Gs{alpha} (shaded bars) or syntrophin (solid bars) antibodies were used for immunoprecipitation. As a negative control (CTL), the MBP antibody was used. The average of 3 determinations is shown, and the error bars show the standard deviation. *P < 0.05, significantly different from "endo" samples (2-tailed Student's t-test).

 
The experiments shown in Fig. 6 and 7 were performed under similar conditions, and by comparison, laminin binding decreased the activation of Gs{alpha} (Fig. 7), but the activation state did not affect the association of Gs{alpha} or G{beta}{gamma} with syntrophin (Fig. 6). Rather, these latter associations were dependent on laminin binding (Fig. 6). Thus, in the presence of laminin, little activation of Gs{alpha} occurred (Fig. 6) and G{alpha}{beta}{gamma} remained associated with syntrophin, while in the absence of laminin, Gs{alpha} was activated (Fig. 7) and Gs{alpha} and G{beta}{gamma} were mostly dissociated from syntrophin (Fig. 6), but some of the activated Gs{alpha} remained bound by syntrophin (Fig. 7).

Gs is known to activate the L-type voltage-gated Ca2+ channel (i.e., the dihydropyridine receptor) in muscle (23, 63). The DGC is defective in Duchenne and other muscular dystrophies, and intracellular Ca2+ is elevated. Thus we investigated whether these laminin-DGC interactions affect muscle cell Ca2+. Changes in extracellular K+ were used to alter the sarcolemma membrane potential in C2C12 myotubes loaded with the fluorescent Ca2+ indicator fura-2 to monitor the effect on intracellular Ca2+. Figure 8A shows the effect of different K+ concentrations on Ca2+-induced fura-2 fluorescence. A maximal effect was observed at K+ concentrations >103 mM, but a substantial increase was noted at 61 mM K+. At all K+ concentrations, 50 µM nifedipine effectively inhibited the Ca2+ increase, suggesting that Ca2+ entered the myotubes via the L-type Ca2+ channel (data not shown). Further experiments were performed at either 60 or 103 mM K+. Figure 8B shows the effect of laminin-1 and the dihydropyridine antagonist nifedipine on voltage-gated Ca2+ uptake in 60 mM K+. The inhibitory effect of laminin-1 observed in our various experiments ranged from ~17 to 40% (for 23 µg/ml laminin-1) and varied somewhat from experiment to experiment; however, inhibition was observed in every case, regardless of whether 60 or 103 mM K+ was used to induce the membrane voltage change. Figure 8B shows a typical experiment using 60 mM K+. Laminin-1 shows a dose-dependent inhibition of the Ca2+ uptake: 21.6 µg/ml laminin-1 caused 17% inhibition, while 43.8 µg/ml led to 34% inhibition. Nifedipine effectively inhibited this uptake (by 89%), as reported by others (35), showing the involvement of the L-type channel. All of these changes were statistically significant. Furthermore, after treating the cells with laminin-1 and nifedipine and washing, the response to K+ alone returned to its previous value, showing that the myotubes were undamaged and that these treatments were fully reversible. Similar results were obtained when the E3 protein was used instead of laminin (data not shown), strongly suggesting the involvement of the DGC. Thus the presence of laminin diminished the amount of active Gs (Fig. 7) and reduced Ca2+ uptake via L-type Ca2+ channels (Fig. 8B).



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Fig. 8. Laminin-1 inhibits the uptake of Ca2+ by myotubes. Myotubes grown on quartz coverslips were loaded with fura-2 AM, an intracellular Ca2+ indicator, and buffer HA (in mM: 20 HEPES-Tris, pH 7.4, 5 glucose, 1 CaCl2, and 1 MgCl2) containing 140 mM NaCl and 5 mM KCl (resting solution) was flowed over them at 37°C. A: bathing solution was changed so that 1.5 ml of solution containing the indicated KCl concentrations was flowed over the cells. The K+ concentrations were made by mixing the resting solution with buffer HA containing 145 mM KCl. After each 1.5-ml K+ treatment, the cells were returned to 1.5 ml of the resting solution. B: Ca2+ uptake was induced in each case by changing from the resting solution to 1.5 ml of buffer HA containing 60 mM KCl and 85 mM NaCl. The experiment was begun by taking 3 measurements with no additions (No, before). Both the resting solution and the 60 mM KCl solution were made to contain the indicated concentrations of laminin-1, and the experiment was repeated. To test the effect of nifedipine, the 60 mM KCl buffer was made with 50 µM nifedipine. Finally, 3 additional measurements were obtained with no additions to the 60 mM KCl buffer (No, After). All are means of 3 determinations, except the 43.8 µg/ml laminin-1 data, which represent a duplicate determination. The error bars show the standard deviation, and above the bars are the probabilities that the data do not differ from No, before (2-tailed Student's t-test).

 
Finally, it should be pointed out that experiments in cultured C2C12 myotubes (Figs. 5D, 7, and 8) produced results entirely consistent with the other experiments in microsomes or purified DGC, suggesting that these observations are not dependent on the experimental models used.


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
PH domains have been found in 71 proteins (20). The G{beta}{gamma} binding region of {beta}-adrenergic receptor kinase overlaps with its PH domain (55). Thus PH domains are known to bind G{beta}{gamma}. {alpha}-Syntrophin has two PH domains, and neither binds G{beta}{gamma}. Rather, binding is to the PDZ domain (Fig. 5, B and C). Numerous proteins are known or suspected to interact with G{beta}{gamma} (12), but domains that interact with G{beta}{gamma} have been identified in only a handful of these proteins. In the present study, we have shown that {alpha}-syntrophin binds to the G{beta}{gamma}-subunit of the heterotrimeric G protein (Figs. 2, 4, and 6) and that the PDZ domain-containing sequence of {alpha}-syntrophin is required for binding G{beta}{gamma} (Fig. 5). This is a novel function for the PDZ domain.

PDZ domains bind proteins through both COOH-terminal and internal sequence motifs. The PDZ domain of syntrophin binds COOH termini with the consensus sequence S/TXV (Ser- or Thr-X-Val, where X is an intervening amino acid), found at the COOH terminus of the Na+ channel of muscle (49), making it a class I PDZ domain (26). For class I PDZ domains, S/TXL, in addition to S/TXV, is also bound (26). This may also be the mechanism of G{beta}{gamma} binding to syntrophin. While G{beta}-subunits typically have LWL or IWN at the COOH termini, several G{gamma}-subunits have TIL (e.g., G{gamma}4, G{gamma}12, G{gamma}13) as the COOH terminus, which fits this motif and may bind. The tissue distribution of all G{gamma}-subunits is not known, but G{gamma}4 mRNA is present in skeletal muscle (47). However, PDZ domains also bind to internal (nonterminal) sequences of proteins, presumably including the brain Na+ channel (19), nNOS (9), and other proteins (60) lacking this terminal motif. Syntrophin PDZ binds an internal sequence in nNOS that forms a {beta}-finger of sequence ETTF, which binds to the pocket of the PDZ domain used for COOH-terminal binding (25). Recently, another such internal sequence, KTXXXW, was found to bind to the mammalian Dvl1 PDZ domain (61). Thus these internal sequences have the structural feature of a {beta}-finger, can have quite different sequences, and at present are hard to identify by sequence alone. Thus it is possible that binding may be via the COOH terminus of certain G{gamma} chains containing the TIL sequence or could be to G{beta} or G{gamma} by way of internal sequences. How G{beta}{gamma} binds to syntrophin's PDZ domain is currently unknown and awaits further investigation. In the present study, we focused instead on how laminin affects this binding, the activation of Gs, and cellular Ca2+.

There are five syntrophin isoforms, all of which are products of different genes and contain a PDZ domain. The two {gamma}-syntrophins are found in nerve and other nonmuscle tissues (46). Among the syntrophins found in muscle, {alpha}-syntrophin is predominantly expressed in striated muscle, while {beta}1- and {beta}2-syntrophins are ubiquitously expressed in muscle and other tissues (3). Our muscle studies have focused on the muscle-specific {alpha}-syntrophin; however, the PDZ domain (amino acids 96–167 in human {alpha}-syntrophin) is 80% identical (94% conserved) in the three muscle syntrophins, and it is likely that all muscle syntrophins share the G{beta}{gamma} binding property.

The interactions between laminin, the DGC, and G proteins investigated in the present study are quite complex, and Fig. 9 shows a schematic of these interactions. Many details, such as regions of the {alpha}-dystroglycan polysaccharide or integrin subunits that bind laminin, are currently poorly understood, and the model therefore is not meant to depict such specifics but rather to provide a general framework for discussion. First, we use it to discuss the binding site for laminin-1.



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Fig. 9. Model of the laminin-DGC-G protein interactions and their effect on the dihydropyridine receptor (DHPR). Shown are the DGC (light gray) and the dihydropyridine receptor ({alpha}1{alpha}2{beta}{gamma}{delta}; in dark gray). Gs{alpha}{beta}{gamma} heterotrimeric G protein is shown in white, as are the integrins. The membrane bilayer is depicted as 2 parallel lines, and the outside and inside faces are labeled. In the absence of laminin (A), a greater portion of Gs{alpha} is in the GTP-bound active form and activates Ca2+ entry into the muscle cell. In the presence of laminin (B), Gs{alpha}{beta}{gamma} is bound by syntrophin and can be in either the GTP- or GDP-bound form, although the latter is shown because it predominates under most conditions. By interacting with syntrophin instead of the DHPR, Ca2+ entry is lessened. Also shown is the laminin globular (LG) domain LG1–5 region of the laminin {alpha}-chain, with the LG4–5 modules, those present in the E3 protein, shaded gray. This region binds to the polysaccharide (shown as the branched lines attached to the protein ovals) of {alpha}-dystroglycan ({alpha}DG), while other modules LG1–3 are thought to bind integrins, although the sites of interaction are only poorly characterized. Also shown are the other DGC constituent proteins {beta}-dystroglycan ({beta}DG), the {alpha}-, {beta}-, {gamma}-, and {delta}-sarcoglycans (SG), sarcospan (SSPN), dystrophin (Dys), and syntrophins (Syn). Syntrophins are known to oligomerize and to be approximately twice the abundance of other DGC proteins and so are shown as a dimer. Dystrophin's interaction with microfilament F-actin is also depicted.

 
Laminin binding increases G{beta} binding to sarcolemma syntrophin (Figs. 4 and 6), and the LG4–5 region of the laminin-1 {alpha}-chain is sufficient to cause the same effect (Fig. 6), localizing laminin-1 binding to this region. This is also the region of laminin-1 that binds to the polysaccharides of {alpha}-dystroglycan and thus the DGC (13, 54). Laminin binding to integrins is also localized to the LG domains but is localized primarily to the LG1–3 region (51, 65). Furthermore, the IIH6 antibody also binds to the polysaccharide of {alpha}-dystroglycan and specifically blocks laminin binding; this antibody prevents most of the G{beta} binding (Fig. 5D). These observations argue strongly that laminin binding to {alpha}-dystroglycan causes the greatest portion of the increased G{beta} binding by syntrophin and suggest that the DGC or some similar complex is involved. This laminin binding also inhibits the activation of Gs{alpha} (Fig. 7). All of these observations are depicted in Fig. 9; the evidence is quite good that the Gs{alpha}{beta}{gamma}-syntrophin interactions occur in response to laminin-{alpha}-dystroglycan binding.

Gs{alpha} activates L-type Ca2+ channels (the dihydropyridine receptor). The experiment shown in Fig. 8 demonstrated that laminin-1 inhibited Ca2+ entry into myotubes. Experiments such as these in whole cells are more difficult to ascribe to a single receptor, and laminin may well bind to more than one receptor to cause these effects. However, because the E3 protein had similar effects, it is likely that the DGC or some similar complex plays a role. Laminin binding increases the amount of Gs{alpha} and G{beta} binding to syntrophin (Fig. 6) and decreases the amount of active Gs{alpha} (Fig. 7). However, it should be recognized that laminin binding to {beta}1-integrins also affects L-type Ca2+ channels in cardiac myocytes (59) and may act at least in part through integrins. This possibility is indicated by denoting integrins with a question mark in Fig. 9. Regardless of the receptor, by altering Ca2+ levels in the cell, laminin binding can profoundly affect muscle contractility and function and may be germane to its pathology in muscular dystrophies.

Perhaps most interesting is the role of Ca2+ in dystrophic muscle degeneration. In Duchenne muscular dystrophy or the mdx mouse model of this disease, intracellular Ca2+ is elevated and cellular proteolysis is initiated by Ca2+-activated protease calpain (5, 6, 17, 57). In Duchenne muscular dystrophy and the mdx mouse model, dystrophin is mutated and is not expressed in significant amounts, and most of the components of the DGC, including the laminin receptor {alpha}-dystroglycan are not present in normal amounts in the sarcolemma. Thus laminin binding to the DGC would be expected to be defective, and laminin would no longer work to lessen intracellular Ca2+ (Figs. 8 and 9). Thus the signaling that we observed, with laminin binding decreasing the amount of active Gs{alpha} and decreasing Ca2+ entry, presumably through the L-type Ca2+-channel, may be defective and seminal to the process of muscular dystrophy. Defects in this laminin-induced signaling may trigger muscle destruction via inappropriate Ca2+ entry. In this regard, laminin mutation in congenital muscular dystrophy and sarcoglycan deficiency in limb-girdle muscular dystrophies may also represent signaling defects in this laminin-Ca2+-proteolysis cascade. Although we did not investigate calpains in the present study, Fig. 9 shows them to be a potential target of Ca2+.

G{beta}{gamma} itself can interact with and modulate the activities of kinases, cyclases, phospholipases, or ion channels, and we did not probe these other potential interactions. It was recently shown that laminin binding to {alpha}-dystroglycan activates PI3K and Akt and increases cell survival in myocytes (33). The precise way in which laminin binding results in PI3K activation is not known. G{beta}{gamma}, however, is known to activate both PI3K{gamma} and PI3K{beta} isoforms directly (reviewed in Ref. 50), and thus the G{beta}{gamma} interactions with syntrophin described in the present study could also potentially regulate PI3K. Thus the binding of G{beta}{gamma}-subunits by syntrophin could also be important to other kinds of cell signaling and could potentially recruit G{beta}{gamma} interactors (e.g., PI3K) to the DGC.

Laminin binding is important to the differentiation and proliferation of muscle cells (22, 34, 48, 58), and G{beta}{gamma} inhibits differentiation of myoblasts to myotubes (39). Laminin binding, by inducing G{beta}{gamma}-syntrophin binding, may sequester G{beta}{gamma} and prevent the negative effects of G{beta}{gamma} on differentiation (39). The signaling arising from laminin binding, including that in the present study involving the heterotrimeric G proteins, is thus likely to be quite important to the development of muscle, its physiology, and its pathologies.


    GRANTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by the Muscular Dystrophy Association and National Institutes of Health Grants GM-43609 and NS-28811.


    ACKNOWLEDGMENTS
 
We thank F. Darlene Robinson, Jasmine McNeill, and Wallace McCloy for excellent technical assistance. We greatly appreciate the antibodies provided by Dr. Tamara Petrucci (Laboratorio di Biologia Cellulare, Instituto Superiore di Sanita, Rome, Italy) and Dr. Kevin P. Campbell (Howard Hughes Medical Institute, University of Iowa, Iowa City, IA), the plasmids provided by Drs. Steven Gee and Stan Froehner (Department of Physiology, University of North Carolina, Chapel Hill, NC), and the E3-expressing cells supplied by Dr. Donald Gullberg (Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden).


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. W. Jarrett, Dept. of Biochemistry, Univ. of Tennessee, 858 Madison Ave., Memphis, TN 38163 (E-mail: hjarrett{at}utmem.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 RESULTS
 DISCUSSION
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