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Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 Downloaded from http://rupress.org/jcb/article-pdf/122/4/809/1258304/809.pdf by guest on 12 October 2022 several groups have noted an abnormal accumu- lation of collagen in Duchenne muscular dystrophy (Duance et al., 1980; Rampoldi et al., 1986; Marshall et al., 1989). These results suggest that the interaction of other extracellu- lar matrix molecules with the sarcolemmal membrane may be affected by the absence of dystrophin. Furthermore, these results raise the question of whether the dystrophin-glyco- protein complex can interact with extracellular matrix mole- cules other than laminin. With regard to F-actin binding to dystrophin, the experi- ments were performed with dystrophin polypeptide frag- ments (Hemmings et al., 1992; Way et al., 1992; Levine et al,, 1992) which may expose binding sites not present in na- tive dystrophin. For example, the targeting of the dystro- phin/a-actinin chimera to the actin fibers and adhesion plaques of COS cells (Hemmings et al., 1992) is contrasted by the diffuse cytoplasmic (Ascadi et al., 1991) or plasma membrane location (Lee et al., 1991) of full-length dystro- phin constructs expressed in COS cells. Furthermore, pro- teolytic cleavage of synapsin I results in fragments exhibiting threefold greater affinity for actin than native synapsin (Bah- ler et al., 1989). Since the NHe-terminal fusion protein of dystrophin binds F-actin with an estimated Kd of 44 #M et al., 1992), a threefold lower actin-binding affinity for native dystrophin corresponding to the difference be- tween synapsin I fragments and native synapsin would bring into question the physiological relevance of F-actin binding to dystrophin. address these issues, we tested the purified dystrophin- glycoprotein complex for interaction with several purified components of the extraceUular matrix as well as actin. Our results demonstrate that dystroglycan specifically binds laminin in a calcium- and ionic strength-dependent manner, whether alone or as part of the dystrophin-glycoprotein com- plex. Nonmuscle dystroglycan also binds laminin. However, the other proteins in the dystrophin-glycoprotein comp

lex in striated muscle tissues appear to be absent, antigenically dis- similar, or less tightly associated in nonmuscle tissues. Fi- nally, we show that the dystrophin-glycoprotein complex cosediments with F-actin but, unlike spectrin or a-actinin, does not bind calcium or calmodulin. Our results support a role for the striated muscle dystrophin-glycoprotein complex in linking the actin cytoskeleton with the extracellular matrix. and Methods Isolation of Rabbit Tissue Membranes rabbit skeletal muscle, cardiac muscle, brain and lung mem- branes, skeletal muscle triads, and surface membranes were prepared as previously described (Sharp et al., 1987; Ohlendieck et ai., 1991b). of Alkaline Extracts from Surface Membranes mg of skeletal muscle surface membranes were diluted to a volume of 2 mi with 50 mM Tris-HC1, pH 7.4, 0.1 mM PMSF, 0.75 mM benzamidine, 2.5/tg/mi aprotinin, 93/~g/mi iodeacetamide, 2.5/tg ml leupeptin and 0.5 /~g/ml pepstatin A, and titrated to pH 12 with 10 M NaOH. After a 1-h incu- bation at 22°C with mixing, the samples were centrifuged for 30 rain at 100,000 g. The resulting supernatant (alkaline surface membrane extract) was decanted from the membrane pellets and titrated to pH 7.4 with M of Dystrophin-Glycoprotein Complex dystrophin-glycoprotein complex was prepared from rabbit skeletal muscle membranes as previously described (Ervasti and Campbell, 1991). Alkaline-dissociated dystrophin-glycoprotein complex was prepared as pre- viously described (Ervasti et ai., 1991). Transfer Overlays laminin (Sigma Chemical Co., St. Louis, MO; Collaborative Research Inc., Lexington, MA, Upstate Biotechnology, or the kind gift of Dr. Hynda K. Kleinman), bovine plasma fibronectin (Sigma Chem. Co.), human placenta merosin (Telios), recombinant mouse entactin (Upstate Biotech- (Collaborative Research), and bovine brain calmodulin (Calbiochem-Novabiochem Corp., La Jolla, CA) were io- dinated with l~INal by the Diabetes, Endocrinology Research Center at the University of Iowa using a lactoperoxidase/glucose oxidase reaction. The iodinated extracellular matrix protein overlay procedure used was previously described (lbraghimov-Beskrovnaya et al., 1992). In the case of laminin, for example, nitrocellulose transfers of SDS polyacrylamide gels containing the various samples were blocked overnight at room temperature in 140 mM NaC1, 1 mM CaC12, 1 mM MgC12, 10 mM triethanolamine, pH 7.6 (Ix LBB) containing 5% nonfat dry milk, rinsed briefly in lx LBB, and incubated for 2-3 h at room temperature in Ix LBB containing 3 % BSA and 0.090/~g/ml (0.1 nM) 125I-laminin. The nitrocellulose transfers were washed twice for 30 rain at room temperature with 25-50 ml of lx LBB, dried, and exposed to X-ray film. Lactose, IKVAV and YIGSR peptides, bovine trachea chondroitin sulfate A, bovine cornea keratan sulfate, and porcine intestinal mucosa heparin were all purchased from Sigma Chem. Co. and tested for their effects on laminin binding to dystroglycan by inclu- sion in the overlay medium at the indicated concentration or wt/wt ratio with respect to the 125I-laminin concentration. The effects of Jacalin, Maackia amurensis leetin H, peanut agglutinin, Con A, and wheat germ agglutinin (all purchased from Vector Labs Inc., Buriingeme, CA) on laminin binding to dystroglycan were tested at 1,000-fold (wt/wt) excess of the 125I-laminin concentration. Overlay of nitrocellulose transfers with 45CAC12 (Dupont New England Boston, was performed by the method to demonstrate calcium binding to erythrocyte and brain speetrin (Wallis et al., 1992). Overlay of nitrocellulo

se transfers with 125I-calmodulin in the presence of 1 mM CaCI2 or l mM EGTA was performed as previously described (Flanagan and Yost, 1984). Transfers overlaid with 125I-calmodulin also contained 0.3/~g rat brain Calmodulin kinase II (Hashimoto et al., 1987) which was the kind gift of Drs. Roger Colbran and Thomas Soderling. Precipitation and Chromatography tumor laminin (Upstate Biotechnology or the kind gift of Dr. Hynda K. Kleinman), bovine plasma fibronectin (Sigma Chem. Co.), gelatin from porcine skin (Sigma Chem. Co.), rat tail collagen I (Collaborative Re- search), and EHS tumor collagen IV (Collaborative Research) were coupled to CNBr-activated Sepharose 413 (Sigma Chem. Co.). 0.9 ml of alkaline sur- face membrane extracts were diluted twofold with 0.28 M NaCI, 2 mM CaC12, 2 mM MgC12, 20 mM triethanolamine, pH 7.6 (2x LBB) and 0.3 ml applied to 0.1 ml of iaminin-, fibronectin-, gelatin-, collagen I-, or colla- gen IV-Sepharose which had been preequllibrated with Ix LBB containing 3 % BSA and washed with three 0.3-ml aliquots of Ix LBB. After incubating overnight at 4°C with mixing, the Sepharose matrices were separated from the supernatants by a brief centrifugation and the supernatants (voids) re- moved. The Sepharose matrices were washed with three 0.3-ml aliquots of Ix LBB, and then sohibilized in 0.3 ml of Ix LBB plus sample buffer for gel analysis. Equal volumes of alkaline surface membrane extracts, Sepharose voids, washes, and Sepharose matrices were analyzed by SDS- PAGE and immunoblotting. The collagen matrices were determined to be functional by their ability to precipitate purified fibroneetin (all three ma- trices) as well as laminin (collagen IV-Sepharose) using the same method under the conditions described above. Dystroglycan binding to heparin was tested under identical conditions except heparin-agarose (Sigma Chem, Co.) was used as the affinity matrix and 8% beaded agerose (Sigma Chem. Co.) was included as a control. To test for dystrophin-glycoprotein complex binding to the various Seph- arose matrices, untreated or alkaline-dissociated dystrophin-glycoprotein complex (44/~g) was diluted fourfold such that the final buffer conditions were 0.1% digitonin, 44 mM NaCI, 1 mM CaCI2, 1 m_M MgCl2, I0/~g/ The Journal of Cell Biology, Volume 122, 1993 810 trans-epoxysuccinyl-L-leucylamido(4-guanidino)-butane (Sigma Chem. Co.), 50 mM Tris-HCl, pH 7.4 (DLB) I. 0.3 ml of the diluted dystrophin- glycoprotein complex was added to 0.1 ml of laminin-, flbronectin-, gelatin-, collagen I-, or IV-Sepharose had been first washed with 0.5 ml DLB containing 10 mM Tris-EDTA, pH 8.0, followed by three 0.5-ml washes with DLB. After incubation for 12 h with mixing at 4°C, the Sepharose matrices were washed three times with 0.3-ml aliquots of DLB and subsequently eluted with twu 0.3-mi aliquots (1 h each elution) Of DLB containing 10 mM Tris-EDTA, pH 8.0 (in the case of laminin-Sepharose), or with one 0.5-ml aliquot of DLB containing 10 mM Tris-EDTA, pH 8.0, and 0.5 M NaCI (all other Sepharose matrices). The Sepbarose matrices were then solubilized in 0.3 ml of lx LBB plus sample buffer for gel analy- sis. Equal volumes of dystrophin-glycoprotein complex, Sephasose voids, washes, and Sepharose matrices were analyzed by SDS-PAGE and immuno- blotting. Laminin affinity chromatography of detergent-solubflized membranes was performed under conditions identical to those used for affinity precipi- tation of the dystrophin-glycoprotein complex. Twenty-five mg of rabbit KCl-washed skeletal muscle, brain, cardiac muscle, and lung membranes were sol

ubflized in 5 ml of 1% digitonin, 0.44 M NaCI, 0.1 mM PMSF, 0.75 mM benzamidine, 2.5 t,g/rrd aprotinin, 2.5 t~g ml leupeptin, and 0.5 /~g/ml pepstatin A and 50 mM Tris-HC1, pH 7.4.3.0 ml of the solubilized membranes were diluted tenfold with 1.33 mM CaCl2, 1.33 mM MgCI2, 13.3 /tg/ml trans-epoxysuccinyl-E-leucylamido(4-guanidino)-hutane, 50 rnM Tris-HCl, pH 7.4, to reduce the digitonin and NaCl concentrations to 0.1% and 44 mM, respectively. After incubation overnight with mixing at 4°C with 0.75 mi of laminin-Sepbarose which had been preequllibrated with DLB, the laminin-Sepharose was separated from the supernatant (void) by a brief centrifugation, and then washed with five 0.75-mi aliquots of DLB. The laminin-Sepharose was subsequently eluted with two 0.75-ml aliquots (1 h each solution) of DLB containing 10 mM Tris-EDTA, pH 8.0. The two EDTA eluates were pooled and 15 ml of each laminin-Sepharose void was concentrated tenfold in a Centriprep 30 for gel analysis. Equal volumes of solubilized membranes, Sepharose voids, and EDTA eluates were analyzed by SDS-PAGE and immunoblotting. and Enzymatic Treatments deglycosylation of alkaline surface membrane extracts using trifluoromethanesulfonic acid (TFMS) was performed as previously de- scribed (Burgess and Norman, 1988). Briefly, 1 ml of alkaline surface mem- brane extracts were lyophilized in a 5-ml Reactivial (Pierce, Rockford, IL) and incubated under nitrogen for 4 h on ice with 0.392 ml anisole and 0.588 ml TFMS (Sigma Chem. Co.). The reaction was terminated with 1.568 ml ice-cold pyridine/H20 (3:5 vol/vol) and dialyzed at 4°C overnight alter 4 liters of H20. The dialyzed sample was extracted with anhydrous ether, lyophilized, and resolubilized in 1 ml of H20. Dystrophin-glycoprotein complex was treated with neuraminidase as previously described (Ervasti and Campbell, 1991). The dystrophin-glycoprotein complex was digested with alkalase (Novo, New York, NY) by incubating 72 pg of dystrophin- glycoprotein complex which had been titrated to pH 9 using 1 M NaOH, for 2 h at 60°C in the presence of 0.1% alkalase (Linhardt et al., 1992). Cosedimentation Assay buffer conditions in the actin cosedimentation assay were based on those recently used to demonstrate actin cosedimentation with a fusion pro- tein corresponding to the IqH2-terminal domain of dystrophin (Hemmings et al., 1992). 0.5 mi of dystrophin-glycoprotein complex (0.116 mg/ml) was applied to a Pharmacia PD-10 column (Sephadex G-25 M), which had been preequilibrated with 25 ml of 0.1% digitonin, 0.2 mM CaCI2, 0.2 mM ATP, 0.2 mM DTT, I0 mM Tris-HCl, pH 7.4 (ABB), and eluted according to the manufacturer's instructions. The PD-10 elnate was concentrated to 0.25 ml in a Centricon 100 (Amicon, Beverly, MA) for use in the actin cosedimentation assay. Various amounts of rabbit muscle actin (Sigma Chem. Co.) dissolved in ABB were added to 40 ml of the concentrated PD- 10 elnate and actin polymerization was initiated by the addition of NaCi and MgCl2 to the final concentrations of 100 mM and 3 raM, respectively, in Abbreviations used in thispaper: actin binding buffer (0.1% digi- tonin, 0.2 mM CaCI2, 0.2 mM ATE 0.2 mM DTT, 10 mM Tris-HC1, pH 7.4); DLB, digitonin laminin binding buffer (0.1% digitonin, 44 mM NaC1, 1 mM CaC12, 1 mM MgCI2, 10/~g/ml trans-epoxysuceinyl-L-leucylamido- (4-guanidino)-butane, 50 mM Tris-HC1, pH 7.4); LBB, laminin binding buffer (140 mM NaC1, 1 mM CaCI2, 1 mM MgCI2, 10 mM triethanol- amine, pH 7.6); TFMS, trifluoromethanesulfonic acid. a total volume of 0.1 ml. After incubation at room temperature for 1 h, the samp

les were centrifuged at 100,000 g for 30 rain, the supernatants re- moved, and the acfin pellets re, suspended in ABB plus SDS sample buffer. Equal volumes of the supernatants and resuspended actin pellets were ana- lyzed by SDS-PAGE and immunoblotting. of sheep polyclonal antisera against the dystrophin-glycoprotein complex was previously described (Ohlendieck and Campbell, 1991b). Polyclonal antibodies specific for fusion protein D, which corresponds to the core protein of dystroglycan, were afffinity-purified from the sheep poly- clonal antisera a~ainst the dystrophin-glycoprotein complex as previously described (Ibraghimov-Beskrovnaya et al., 1992). The preparation and characterization of monoclonal antibodies IIH6 and VIA41, specific for dystroglycan and the dystrophin-specific monoclonai antibody XIXC2 have previously been described (Ervasti and Campbell, 1991; Ervasti et al., 1990; Jorgensen et al., 1990; Ohiendieck et al., 1991/7). Monoclonal anti- body HH6 was purified from tissue culture media by a previously described method (Imagawa et al., 1987) except Sephacryl S-400 was used instead of Sepharose CL-4B. Monoclonal antibodies specific for chondroitin sulfate and keratin sulfate were obtained from Sigma Chem. Co. and ICN Biomedi- cals, Inc., Costa Mesa, CA, respectively. and Immunoblotting (Laemmli, 1970) was carried out on 3-12 % gradient gels in the presence of 1% 2-mercaptoethanol and stained with Coomassie blue, Stains-All (Campbell et al., 1983), Alcian Blue (AI-Hakim and Linhardt, 1991), or transferred to nitrocellulose (Towbin et al., 1979). Molecular weight standards shown in the figures were purchased from GIBCO BRL (Gaithersburg, MD). Nitrocellulose transfers were stained with polyclonal antisera, affinity-purified polyclonal antibodies, or monoclonal antibodies as previously described (Campbell et al., 1987). Coomassie blue-stained gels and autoradiograms were analyzed densitometrically using a model 300S scanning densitometer (Molecular Dynamics, Inc., Sunnyvale, CA). Laminin-binding Properties of Dystroglycan number of commercially available purified extracellular matrix components were radiolabeled and tested for binding to dystrophin-glycoprotein complex which had been electro- phoretically separated on SDS polyacrylamide gels and trans- ferred to nitrocellulose. As previously reported (Ibraghimov- Beskrovnaya et al., 1992), 125I-laminin bound to a protein band in crude skeletal muscle surface membranes and purified dystrophin-glycoprotein complex corresponding to 156 kD dystroglycan (Fig. 1 A). t25I-Merosin also labeled dystroglycan, albeit more weakly than EHS laminin (not shown). Overexposed autoradiograms revealed additional laminin-binding proteins of 100 and 60 kD in the crude sur- face membrane preparation (not shown). However, these ad- ditional laminin-binding proteins were less abundant in pure sarcolemma than in crude muscle membranes, suggesting that they are either peripheral proteins which were removed by the KCI wash step or were a component of a distinct vesi- cle population. The binding of ~I-laminin to dystroglycan was inhibited by the inclusion of 10 mM EDTA (Fig. 1 A). The absence of CaCI2, but not MgCl2 from the overlay medium also in- hibited mI-laminin to dystroglycan (not shown). ~2sI- laminin binding to dystroglycan was also completely in- hibited by inclusion of NaC1 to the overlay medium (Fig. 1 A) with an average half-maximal concentration for inhibition (ICs0) of 250 raM. The binding of '2q-laminin to dystroglycan was inhibited by the inclusion of an excess of unlabeled laminin but a

nd Campbell Complex Binds Laminin Laminin-binding properties dystroglycan. Shown in A rabbit skeletal muscle surface membranes dystrophin-glycoprotein complex which were elec- trophoretically separated a 3-12% polyacrylamide gel and stained with Coomassie blue Also shown in A is a nitrocellu- lose transfer stained with monoclonal antibody IIH6 corresponding autoradiograms transfers over- laid with nSI-laminin in the absence mM EDTA M NaCI Shown in B are the cor- responding autoradiograms nitrocellulose transfers containing 4 electrophoretically separated dystrophin-glycoprotein com- plex which were overlaid with excesses (wt/wt) laminin (+/_AM), fibronectin YIGSR peptide in the presence M lactose molecular weight standards are indicated the left. not fibronectin 12q-fibronectin, ~25I-entactin, nitrocellulose transfer in 1,000-fold et al., et al., laminin binding 3), two Kinne and reactive with not responsible dyes with Neuraminidase treatment (Fig. 3) as amurensis lectin assessed by moieties that are not proteoglycans (Soroka Shown in #g of in the ab- 1,000-fold excesses (wt/wt) sulfate (KS). IIH6 to line surface and the washed laminin-Sepharose (/.AM -3 ) (Alcian Blue) identical nitrocellulose transfers stained antibodies specific fusion protein a transfer overlaid protein predicted body IIH6 (Wu et 43/156-kD dystrophin-associated Ervasti and Campbell Dystrophin-Glycoprotein Complex Binds Laminin Chemically degly- cosylated dystroglycan corresponding fusion proteins not bind laminin. Shown in A are nitrocellulose transfers and TFMS-treated affinity-purified polyclonal antibodies specific protein D corresponding the core protein sequence the corresponding autoradio- a transfer overlaid B is the ing autoradiogram a trans- fer containing fusion proteins B (FIB), quences present in the core dystroglycan over- laid with nSI-laminin right mark the migration the fusion were detected with Ponceau S before the procedure. The molecular weight standards (×10 ) are the left. for the protein complex hardt et smear detected did not D-specific antibodies and the 59-kD dystrophin- the laminin-Sepharose bound to interaction between is selective the RGD recognition reported to be it is the dystrophin-glycoprotein with high laminin-Sepharose has laminin binding ionic strength us to the dystrophin-glycoprotein complex 122, 1993 Laminin-Sepharose binding extracts. Shown in A is a identical nitrocellulose transfer stained with monoclonal antibody RI-I6 equal volumes alkaline surface laminin-Sepharose void three washes the laminin- the washed laminin-Sepharose in B is a nitrocellulose transfer containing equal volumes alkaline surface voids (It') and Sepharose pellets alkaline surface incubation with laminin-, fibronectin-, gelatin-, colla- collagen IV-Sepharose which was stained with monoclo- hal antibody HI-I6 weight standards the left. Laminin-Sepharose binding the dystrophin-glycopm- tein complex. Shown a Coomassie identical nitrocellulose transfer stained with monoclonal antibody containing equal volumes alkaline-dissociated dystrophin-glycoprotein complex incubation with laminin-Sepharose and the two subsequent the laminin-Sepharose 1 and contaminant typically found in dystrophin-glycoprotein complex molecular weight standards the left. the dystro- was not data demonstrate sociated with no longer et al., alkaline dissociation. between laminin and dystroglycan ble for void and Ervasti and Campbell Complex Binds Laminin-Sepharose binds dystmglycan various tis- sues. Shown are Coomassie blue-stained gels and portions tical nitrocellulose transfers staine

d with monoclonal antibody polyclonal antibod- ies specific fusion protein D corresponding containing equal volumes digitonin-solubilized skeletal muscle, brain, cardiac muscle, lung membranes the solubi- lized membranes with laminin-Sepharose the EDTA each laminin-Sepharose extensive washing Because the laminin-Sepharose a vol- one-half that the original solubilate, the EDTA eluate loaded the gels represents twice the pro- tein equivalents loaded the solubilates and voids. the molecular weight standards (224, 109, 72, and 29 x are indicated the left the Coomassie blue-stained gel. Only the nitrocellulose transfers that lies between the 224,000 and 109,000 molecular weight stan- 50, 43, Because dystroglycan to be sues. To brain, cardiac muscle, and incubated with EDTA Fig. cardiac muscle, and void. However, et al., dieck et al., cardiac muscle, and membranes were tenfold and (Fig. 8). the concentrated laminin-Sepharose elu- ates from various tissues. Shown are the lam- eluates described in 7 concentrated lO-fold, electrophoreti- cally separated on an and stained with Coomassie blue identical nitrocellulose transfer stained with sheep antiserum against the and monoclonal antibody the corresponding autoradiogram a transfer lecular weight standards are indicated the left. as by is specific 59, 50, and cardiac laminin-Sepharose the 43-kD in skeletal tissues, it composition than Sites on antibody IIH6 other hand, able with antibody IIH6 the binding antibody IIH6 and laminin plex were either monoclonal Preincubation with but Not 1992; Way 1992). To the dystrophin- with actin, assay based the absence 100,000 g the actin As reviewed by tisera to (Anti-DGC + glycoprotein complex the indicated rabbit muscle 100,000 g ~ 109- cated on binding sites dystrophin-glycoprotein complex with tissue (IIH6), VIA41 100 tzg the absence Shown is a Coomassie rabbit brain and dystrophin-glyco- protein complex (DGC). Also shown are identical nitrocellu- transfers overlaid Caimodulin in the presence (~Zsl-CaM + rat brain kinase II (CaM/t). The molecular are indicated the left. calcium and between dystrophin, the possibility phin-actin interactions this hypothesis, bound to presumably brain bound to three known muscle; the did not Neither did the dystrophin- in skeletal may not laminin binding through laminin the complex extracellular matrix. sarcolemmal membrane. glycoproteins are which the the dystrophin-glycoprotein complex subunit led sarcolemma through et al., laminin but the dystrophin-glycoprotein and the experiments in from mouse designated B1 and B2 (Timpl et al., 1987). Recent studies have shown that laminin is a member of a family of proteins which vary in their subunit structure and composi- tion. Skeletal muscle predominately expresses a protein named merosin, which differs from laminin in that the A chain is replaced by a structurally homologous M chain (Sanes et al., 1990; Engvall et al., 1990). Laminin, and merosin to a lesser extent, can be further substituted by replacement of the B1 chain with a homologous subunit named S-laminin (Sanes et al., 1990; Engvall et ai., 1990). More recently, it was shown that cardiac muscle laminin con- tains a 300-kD heavy chain which is immunochemicaUy similar to the M chain of merosin (Paulsson et al., 1991). We previously noted that both S-laminin (Sanes et al., 1990) and dystrophin-rdated protein are specifically localized to the neuromuscular junction (Ohiendieck et al., 1991a) and it is apparent that merosin (Sanes et al., 1990) and dystro- phin (Ohlendieck et al., 1991a) exhibit similar distributions throughou

t the sarcolemmal membrane, including the neuro- muscular junction. Recently, we demonstrated (Matsumura et al., 1992a) that dystrophin-related protein is associated with a complex of glycoproteins that is identical or antigeni- cally similar to the complex associated with dystrophin (Er- vasti and Campbell, 1991). In the present work, we have noted that merosin purified from human placenta also bound dystroglycan as detected by the blot overlay method. Curi- ously, the autoradiographic intensity of ~25I-merosin bind- ing to dystroglycan did not appear as strong as that observed for 125I-laminin when performed in parallel. While this is not the anticipated result, there are several possible explana- tions for our observation. SDS-polyacrylamide gel dec- trophoretic analysis has demonstrated that purified merosin preparations contain polypeptides of 600, 300, and 180-200 kD as well as minor components of 60-90 kD (Ehrig et al., 1990; Ervasti, J. M., and K. D. Campbell, unpublished ob- servations). Thus, it is possible that the commercial merosin preparation is impure or proteolyzed, either of which could account for its reduced signal intensity (in comparison to laminin) in the blot overlay assay. Furthermore, there is precedent for an integrin binding to a nonnative laminin with greater affinity than it exhibits for its native ligand (Sonnen- berg et al., 1991). Additional experiments will be necessary to understand the nature of this apparent difference between laminin and merosin in binding dystroglycan. The emerging importance of laminin in skeletal muscle development (Goodman et al., 1989; yon der Mark et al., 1991) raises the question of whether dystroglycan plays a role in skeletal muscle differentiation. In cultured muscle cells, dystrophin expression is not evident in myoblasts before fu- sion (Lev et al., 1987). Thus, laminin binding by dystrogly- can would not be expected to mediate any early events in skeletal muscle differentiation, assuming that expression of the components of the dystrophin-glycoprotein complex is coordinately regulated. However, our finding that monoclo- hal antibody lItt6 blocks laminin binding to dystroglycan (Fig. 9) suggests that this antibody may be useful in further delineating the function of the dystrophin-glycoprotein com- plex through the possible perturbation of laminin-dystro- glyean interactions in vivo. Until recently, the actin-binding properties of dystrophin have largely been speculated from its sequence homologies with well characterized actin-binding proteins CKoenig et al., 1988; Karinch et al., 1990; Bresnick et al., 1990). Hem- mings et al. (1992) demonstrated that a chimera comprised of the first 233 amino acids of dystrophin and the last 645 amino acids of smooth muscle alpha-actinin localized to actin-containing structures when expressed in COS cells. In addition, bacterially expressed fusion proteins correspond- ing to the putative actin-binding domain of dystrophin have been shown to cosediment with F-actin (Hemmings et al., 1992; Way et al., 1992). However, the apparent dissociation constant of actin binding for one of these fusion proteins was estimated at 44 #M (Way et al., 1992) which the authors noted was ten times greater than the K~ value obtained for filamin and two orders of magnitude greater than the appar- ent K~ ~f ot-actinin dimer binding to actin. Although the concentration dependence of dystrophin-glycoprotein com- plex binding to F-actin was not rigorously determined, we observed significant (50%) dystrophin-glycoprotein com- plex cosedimentation with F-actin using a

n effective dystro- phin concentration of 0.1/zM (Fig. 10), suggesting that the native dystrophin-glycoprotein complex binds F-actin with an affinity similar to native a-actinin. Whether the difference between dystrophin fusion proteins and the dystrophin- glycoprotein complex with respect to F-actin binding affinity is due to an intact dystrophin molecule, dystrophin dimeriza- tion or a modulatory effect by one of the dystrophin- associated glycoproteins will require further investigation. Furthermore, dystrophin does not appear to be directly as- sociated with the myofibrillar actin filaments (Watkins et al., 1988; Bonilla et al., 1988) which raises the issue of what actin-based structures skeletal muscle dystrophin may inter- act with in vivo. Peripheral actin filaments emanating from the Z lines and M lines of skeletal muscle myofibers have re- cently been identified (Bard and Franzini-Armstrong, 1991) while 3,-actin (Craig and Pardo, 1983) and dystrophin (Por- ter et al., 1992) are two of several cytoskeletal proteins which exhibit discrete, lattice-like organizations comprised of a longitudinal dement and transverse elements coincident with the I bands and M lines. The low abundance of 7-actin in adult skeletal muscle would also favor its interaction with dystrophin from the standpoint of stoichiometry. In addition, the recent identification of novel actin-rdated proteins (lees-Miller et al., 1992; Clark and Meyer, 1992) raises the possibility for discovery of a unique actin-like protein in skeletal muscle which specifically binds dystrophin. In the meantime, our present results demonstrate that the dystro- phin-glycoprotein complex has the capacity to bind F-aetin. The cysteine-rich region of dystrophin shows significant homology to a domain of that con- talns two Ca2+-binding sites (Koenig et al., 1988). Thus, like nonmuscle ot-actinin and some spectrins (Dubreuil et al., 1991), dystrophin-actin interactions are conceivably affected by calcium. However, calcium was not found to bind any component of the skeletal muscle dystrophin-glycoprotein complex (Fig. 11) under 4sCaCl2 overlay conditions identi- cal to those used in demonstrating direct calcium binding to spectrin (Wallis et al., 1992). At variance with Madhavan et al. (1992), we have detected no interaction between any component of the dystrophin-glycoprotein complex and calmodulin (Fig. 11). Since calmodulin is a eytosolic pro- tein, the observation that biotinylated calmodulin interacts with 156 kD dystroglycan (Madhavan et al., 1992) conflicts strikingly with the proposition that it is wholly extracellular Journal of Cell Biology, Volume 122, 1993 820 on its extensive glycosylation (Fig. 7 and Ervasti et al., 1990; Ervasti and Campbell, 1991), laminin-binding proper- ties (Figs. 1-9), membrane extraction properties (Ohlen- dieck and Campbell, 1991a; Ervasti and Campbell, 1991), and lack of a predicted transmembrane domain (lbraghimov- Beskrovnaya et al., 1992). While the sum of these results provide support for calcium independent dystrophin-actin interactions in skeletal muscle, they leave open the possibil- ity that nonmuscle isoforms of dystrophin or its autosomal homologue dystrophin-related protein (Love et al., 1989) may function in a calcium-dependent manner as is the case for nonmuscle o~-actinin. While the distribution of dystrophin in skeletal muscle (Porter et al., 1992) supports a role for dystrophin in stabiliz- ing the sarcolemmal membrane, its distribution in cardiac and smooth muscle (Byers et al., 1991), cortical neurons (Lidov et al., 1990), and (Yeadon e

t al., 1991; Sealock et al., 1991) suggest that dystrophin may play more varied roles in noncontractile tissues. The 43-kD dystrophin-associated glycoprotein and 156-kD dystrogly- can are also expressed in nonmuscle tissues (Ibraghimov- Beskrovnaya et al., 1992). However, while nonmuscle dys- troglycan binds laminin (Fig. 7), it does not appear to form a complex with full-length dystrophin as in cardiac and skeletal muscle (Fig. 8). Nonmuscle dystroglycan may bind to the novel dystrophin isoforms which are expressed in some nonmuscle tissues (Rapaport et al., 1992; Blake et al., 1992; Lederfein et al., 1992) at levels comparable to that of full-length dystrophin in striated muscle (Hoffman et al., 1987). On the other hand, actin binding as a universal func- tion of dystrophin must also be reconsidered in light of these novel dystrophins, which completely lack the putative actin- binding domain (Rapaport et al., 1992; Blake et al., 1992; Lederfein et al., 1992). In conclusion, our results demonstrate that the skeletal muscle dystrophin-glycoprotein complex can bind both actin and laminin and suggest that dystrophin serves as a special- ized link between the actin cytoskeleton and the extracellular matrix. It is clear, however, that the same function cannot be immediately extrapolated to all tissues. Clarification of the role of dystrophin in nonmuscle tissues awaits further inves- tigation. gratefully acknowledge Steven Kahl for expert technical assistance, Dr. Oxana Ibraghimov-Beskrovnaya for providing the dystroglycan fusion proteins used in this study and Dr. Robert Linhardt for providing his exper- tise as well as alkalase, purified heparatinase, chondroitinase ABC, and keratanase. We would also like to thank Dr. Hynda K. Kleinman (National Institutes of Health) for the kind gift of laminin; Drs. Roger Colbran (Van- derbilt, Nashville, TN) and Thomas Soderling (Vollum Institute, Portland, OR) for purified Calmodulin kinase H, and Drs. Kiichiro Matsurnura and Steven Roberds for many helpful discussions and comments on the manu- script. K. P. Campbell is an investigator of the Howard Hughes Medical Institute. J. M. Ervasti was the Carl M. Pearson Fellow of the Muscular Dystrophy Association while conducting this study. This work was also supported by a grant from the Muscular Dystrophy Association. Received for publication 18 March 1993. A., and R. J. Linhardt. 1991. Electrophoresis and detection of nano- gram quantities of exogenous and endogenous glycosaminoglyeans in bio- logical fluids. Theor. Electrophor. Anderson, M. S., and L. M. Kunkel. 1992. The molecular and biochemical ba- sis of Ducharme muscular dystrophy. Biochem. Sci. Ascadi, G., G. Dickson, D. R. Love, A. Jani, F. S. Walsh, A. Gurusinghe, J. A. Wolff, and K. E. Davies. 1991. Human dystroplfin expression in mdx mice after intramuscular injection of DNA constructs. (Lond.). Aumailley, M., M. Gerl, A. Sonnenberg, R. Deutzmann, and R. Timpl. 1990. Identification of the Arg-Gly-Asp sequence in laminin A chain as a latent cell- binding site being exposed in fragment P1. (Fed. Eur. Biochem. Soc.) 262:82-86. Bahler, M., F. Benfenati, F. Valtorta, A. J. Czernik, and P. Greengard. 1989. Characterization of synapsin I fragments produced by cysteine-specific cleavage: a study of their interactions with F-actin. Cell Biol. 1841-1849. Bard, F., and C. Franzini-Armstrong. 1991. Extra actin filaments at the periph- ery of skeletal muscle myofibrils. Cell. Blake, D. J., D. R. Love, J. Tinsley, G. E. Morris, H. Turley, K. Gatter, G. Dickson, Y. H. Edwards, and K. E. Davies. 1992. Character

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