Dystrophin, the product of the Duchenne muscular dystrophy gene, is tightly associated with the sarcolemmal membrane to a large glycoprotein complex. One function of the dystrophin-glycoprotein complex is to link the cytoskeleton to the extracellular matrix in skeletal muscle. However, the molecular interactions of dystrophin with the membrane components of the dystrophin-glycoprotein complex are still elusive. Here, we demonstrate and characterize a specific interaction between ␤-dystroglycan and dystrophin. We show that skeletal muscle and brain dystrophin as well as brain dystrophin isoforms specifically bind to ␤-dystroglycan. To localize and characterize the dystrophin and ␤-dystroglycan interaction domains, we reconstituted the interaction in vitro using dystrophin fusion proteins and in vitro translated ␤-dystroglycan. We demonstrated that the 15 C-terminal amino acids of ␤-dystroglycan constituted a unique binding site for the second half of the hinge 4 and the cysteine-rich domain of dystrophin (amino acids 3054 -3271). This dystrophin binding site is located in a proline-rich environment of ␤-dystroglycan within amino acids 880 -895. The identification of the interaction sites in dystrophin and ␤-dystroglycan provides further insight into the structure and the molecular organization of the dystrophin-glycoprotein complex at the sarcolemma membrane and will be helpful for studying the pathogenesis of Duchenne muscular dystrophy.Dystrophin is a large protein with a molecular mass of 427 kDa, which is absent in muscle from patients with Duchenne muscular dystrophy (1). Based on its primary structure, dystrophin can be divided into four domains: the N-terminal actin binding domain, the large triple helical spectrin-like domain, the cysteine-rich domain, and the C-terminal domain (2). Deletion of the cysteine-rich and C-terminal domains is associated with severe muscular dystrophy, which indicates that these domains play important roles in the stability of dystrophin (3, 4).Immunofluorescence microscopy has established that dystrophin is located at the plasma membrane of skeletal muscle (5).Biochemical studies have demonstrated that dystrophin is tightly associated through its cysteine-rich and C-terminal domains with the sarcolemmal membrane to a large glycoprotein complex (6 -10). Furthermore, cell membrane fractionation has shown that the dystrophin-glycoprotein complex can only be dissociated by alkaline treatment (6). Dystrophin-glycoprotein complex (DGC) 1 is composed of at least five transmembrane proteins (50-kDa adhalin, 43-kDa ␤-dystroglycan, 43-kDa dystrophin-associated glycoprotein A3b, 35-kDa dystrophin-associated glycoprotein, and 25-kDa dystrophin-associated protein), one extracellular protein (156 kDa ␣-dystroglycan), and four cytoplasmic proteins (syntrophin triplet and dystrophin) (6 -9, 11-15). In skeletal muscle, interactions between ␣-dystroglycan and laminin ␣2 (11, 12, 16) as well as between dystrophin and cytoskeletal actin filaments (17) have been identified, indicating that one fun...

Dystroglycan is a novel laminin receptor that links the extracellular matrix and sarcolemma in skeletal muscle. The dystroglycan complex containing alpha- and beta-dystroglycan also serves as an agrin receptor in muscle, where it may regulate agrin-induced acetylcholine receptor clustering at the neuromuscular junction. beta-Dystroglycan has now been expressed in vitro and shown to directly interact with Grb2, an adapter protein involved in signal transduction and cytoskeletal organization. Protein binding assays with two Grb2 mutants, Grb2/P49L and Grb2/G203R, which correspond to the loss-of-function mutants in the Caenorhabditis elegans sem-5, demonstrated that the dystroglycan-Grb2 association is through beta-dystroglycan C-terminal proline-rich domains and Grb2 Src homology 3 domains. Affinity chromatography has also shown endogenous skeletal muscle Grb2 interacts with beta-dystroglycan. Immunoprecipitation experiments have demonstrated that Grb2 associates with alpha/beta-dystroglycan in vivo in both skeletal muscle and brain. The specific dystroglycan-Grb2 interaction may play an important role in extracellular matrix-mediated signal transduction and/or cytoskeleton organization in skeletal muscle that may be essential for muscle cell viability.

Syntrophin represents three cytoplasmic components of the dystrophin-glycoprotein complex that links the cytoskeleton to the extracellular matrix in skeletal muscle. alpha-Syntrophin has now been translated in vitro and shown to associate directly with all three components of the syntrophin triplet and with dystrophin. The in vitro translated 71-kDa non-muscle dystrophin isoform, containing the cystein-rich/C-terminal domain, can also interact with the syntrophin triplet. The syntrophin binding motif in dystrophin was localized to exons 73 and 74 including amino acids 3447-3481 by comparing the interactions of alpha-syntrophin and seven overlapping human dystrophin fusion proteins. More than one syntrophin interaction site in this binding motif was suggested. alpha-Syntrophin also interacts directly with a C-terminal utrophin fusion protein. alpha-Syntrophin is localized to the muscle sarcolemma as well as to the neuromuscular junction in control mouse muscle. However, similar to utrophin, alpha-syntrophin is only present at the neuromuscular junction in mdx mouse muscle in which dystrophin is absent. Our data suggest that alpha-syntrophin binds all syntrophin isoforms, and syntrophin directly interacts with dystrophin through more than one binding site in dystrophin exons 73 and 74 including amino acids 3447-3481.

Abstract. Dystrophin plays an important role in skeletal muscle by linking the cytoskeleton and the extracellular matrix. The amino terminus of dystrophin binds to actin and possibly other components of the subsarcolemmal cytoskeleton, while the carboxy terminus associates with a group of integral and peripheral membrane proteins and glycoproteins that are collectively known as the dystrophin-associated protein (DAP) complex. We have generated transgenic/mdx mice expressing "full-length" dystrophin constructs, but with consecutive deletions within the COOH-terminal domains. These mice have enabled analysis of the interaction between dystrophin and members of the DAP complex and the effects that perturbing these associations have on the dystrophic process. Deletions within the cysteine-rich region disrupt the interaction between dystrophin and the DAP complex, leading to a severe dystrophic pathology. These deletions remove the [3-dystroglycan-binding site, which leads to a parallel loss of both [3-dystroglycan and the sarcoglycan complex from the sarcolemma. In contrast, deletion of the alternatively spliced domain and the extreme COOH terminus has no apparent effect on the function of dystrophin when expressed at normal levels. The proteins resulting from these latter two deletions supported formation of a completely normal DAP complex, and their expression was associated with normal muscle morphology in mdx mice. These data indicate that the cysteine-rich domain is critical for functional activity, presumably by mediating a direct interaction with [3-dystroglycan. However, the remainder of the COOH terminus is not required for assembly of the DAP complex.UCHENNE muscular dystrophy (DMD) 1 and the milder Becker muscular dystrophy (BMD) are caused by mutations in the dystrophin gene (24). Although dystrophin is expressed from a variety of promoters in a wide array of tissues, disruption of dystrophin function in striated muscle leads to the most devastating effects of these diseases (for review see reference 2). The complete function of the dystrophin protein in skeletal muscle has not yet been fully elucidated, although it is thought to provide a crucial link between the intracellular actin cytoskeleton and the extracellular matrix (15). The mdx mouse contains a nonsense mutation in the dystrophin gene that leads to a complete absence of dystrophin in muscle, which causes a similar muscle degeneration as is Address all correspondence to J.S. Chamberlain, Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109. Tel.: (313) 763-4171; Fax: (313) 763-3784. The current address for Gregory A. Cox is The Jackson Laboratories, 600 Main Street, Bar Harbor, ME 04609. Abbreviations used in this paper:BMD, Becker muscular dystrophy; DAP, dystrophin-associated protein; DMD, Duchenne muscular dystrophy; GMA, glycol methacrylate; LGMD, limb girdle muscular dystrophy. seen in DMD patients. As a result, the mdx mouse is a useful system for studying the function of the dystrophin protein.The full...

Autosomal recessive limb girdle muscular dystrophy (LGMD2) is a clinically and genetically heterogenous group of diseases involving at least six different loci. Five genes have already been identified: calpain-3 at LGMD2A (15q15), and four members of the sarcoglycan (SG) complex, alpha-SG at LGMD2D (17q21), beta-SG at LGMD2E (4q12), gamma-SG at LGMD2C (13q12), and delta-SG at LGMD2F (5q33-q34). The gene product at LGMD2B (2p13-p16) is still unknown and at least one other gene is still unmapped. We investigated 20 Turkish families (18 consanguineous) diagnosed as having LGMD2. Most of our patients had onset of symptoms before age 10. The phenotypes varied from severe to benign. We analyzed the SG complex by immunofluorescence and/or western blot. Genotyping was performed using markers defining the six known loci and the suspected genes were screened for mutations. Six of 17 index cases showed deficiency of the SG complex, by immunofluorescence and/or western blot. Seven cases involved one of the known genes of the SG complex (alpha, 2; beta, 1; and gamma, 4 cases), and five mutations were documented in the alpha- and gamma-SG genes. After linkage analysis, 10 families were characterized as having LGMD2A (calpain-3 deficiency), and all mutations were eventually identified. One family was classified as having LGMD2B and 1 family that has normal SGs was linked to the chromosome 5q33-q34 locus (LGMD2F). In 1 family there was no linkage to any of the known LGMD2 loci. It appears that in Turkey, there is a broad spectrum of genes and defects involved in LGMD2. It may be possible to correlate genotype to phenotype in LGMD2. All severe cases belonged to the gamma-SG-deficiency group. Nine calpain-3-deficient cases had intermediate and 1 had moderate clinical courses. The LGMD2B patient had a moderate clinical expression, whereas the LGMD2F case was truly benign.

The sarcoglycan complex is known to be involved in limb-girdle muscular dystrophy (LGMD) and is composed of at least three proteins: ␣-, ␤-, and ␥-sarcoglycan. ␦-Sarcoglycan has now been identified as a second 35-kDa sarcolemmal transmembrane glycoprotein that shares high homology with ␥-sarcoglycan and is expressed mainly in skeletal and cardiac muscle. Biochemical analysis has demonstrated that ␥-and ␦-sarcoglycan are separate entities within the sarcoglycan complex and that all four sarcoglycans exist in the complex on a stoichiometrically equal basis. Immunohistochemical analysis of skeletal muscle biopsies from patients with LGMD2C, LGMD2D, and LGMD2E demonstrated a reduction of the entire sarcoglycan complex in these muscular dystrophies. Furthermore, we have mapped the human ␦-sarcoglycan gene to chromosome 5q33-q34 in a region overlapping the recently linked autosomal recessive LGMD2F locus.The dystrophin-glycoprotein complex (DGC) 1 (1-4) in skeletal muscle is a complex of sarcolemmal proteins and glycoproteins. It is composed of dystrophin, a cytoskeletal actin-binding protein (5-7); the syntrophins, a 59-kDa triplet of intracellular proteins that bind the C-terminal domain of dystrophin (8 -12); ␣-dystroglycan, a 156-kDa extracellular proteoglycan that binds the G domain of laminin (13-15); ␤-dystroglycan, a 43-kDa transmembrane glycoprotein (2, 3, 13) that binds the cysteine-rich region of dystrophin (16, 17); ␣-, ␤-, and ␥-sarcoglycan, transmembrane glycoproteins of 50, 43, and 35 kDa, respectively (18 -24); and a 25-kDa transmembrane protein (1-4). Recent experiments have demonstrated the existence of two complexes within the DGC (24, 25): the dystroglycan complex, composed of ␣-and ␤-dystroglycan, and the sarcoglycan complex, consisting of ␣-, ␤-, and ␥-sarcoglycan.Defects in DGC components lead to muscle fiber necrosis, the major pathological event in muscular dystrophies (26). In Duchenne muscular dystrophy (DMD), mutations in the dystrophin gene cause the loss of dystrophin and a reduction of the dystrophin-associated proteins (2, 5). One form of congenital muscular dystrophy has recently been characterized as being caused by mutations in the laminin ␣2-chain gene (27, 28). Limb-girdle muscular dystrophy (LGMD) represents a clinically and genetically heterogeneous class of disorders (29, 30). They are inherited as either autosomal dominant or recessive traits. An autosomal dominant form, LGMD1A, was mapped to 5q31-q33 (31, 32), while six genes involved in the autosomal recessive forms were mapped to 15q15.1 (LGMD2A) (33), 2p16-p13 (LGMD2B) (34), 13q12 (LGMD2C) (23,35,36),20), 4q12 (LGMD2E) (21,22), and most recently 5q33-q34 (LGMD2F) (37). Patients with LGMD2C, -2D, and -2E have a deficiency of components of the sarcoglycan complex resulting from mutations in the genes encoding ␥-, ␣-, and ␤-sarcoglycan, respectively (19, 21-23, 38 -40). The gene responsible for LGMD2A has been identified as the musclespecific calpain (41), whereas the genes responsible for LGMD1A, -2B, and -2F are still unknow...

Several human histo-blood groups are glycosphingolipids, including P/P1/P k . Glycosphingolipids are implicated in HIVhost-cell-fusion and some bind to HIVgp120 in vitro. Based on our previous studies on Fabry disease, where P k accumulates and reduces infection, and a soluble P k analog that inhibits infection, we investigated cell surface-expressed P k in HIV infection. HIV-1 infection of peripheral blood-derived mononuclear cells (PBMCs) from otherwise healthy persons, with blood group P 1 k , where P k is overexpressed, or blood group p, that completely lacks P k , were compared with draw date-matched controls. Fluorescenceactivated cell sorter analysis and/or thin layer chromatography were used to verify P k levels. P 1 k PBMCs were highly resistant to R5 and X4 HIV-1 infection. In contrast, p PBMCs showed 10-to 1000-fold increased susceptibility to HIV-1 infection. Surface and total cell expression of P k , but not CD4 or chemokine coreceptor expression, correlated with infection. P k liposome-fused cells and CD4 ؉ HeLa cells manipulated to express high or low P k levels confirmed a protective effect of P k . We conclude that P k expression strongly influences susceptibility to HIV-1 infection, which implicates P k as a new endogenous cell-surface factor that may provide protection against HIV-1 infection. (Blood. 2009;113:4980-4991)

We have partially sequenced rabbit skeletal muscle T-sarcoglycan an integral component of the dystrophin-glycoprotein complex. Specific antibodies were produced against a 7-sarcoglycan peptide and used to examine the expression of T-sarcoglycan in skeletal muscle of patients with severe childhood autosomal muscular dystrophy linked to chromosome 13q12 (SCARMD). We show by immunofluorescence and Western blotting that in skeletal muscle from these patients T-sarcoglycan is completely absent and c~-and ~-sarcoglycan are greatly reduced in abundance, whereas other components of the DGC are preserved. In addition, we show that in normal muscle ~-, ~-, and T-sarcoglycan constitute a tightly associated sarcolemma complex which can not be disrupted by SDS treatment.