Membrane targeting and stabilization of sarcospan is mediated by the sarcoglycan subcomplex

Abstract

The dystrophin-glycoprotein complex (DGC) is a multisubunit complex that spans the muscle plasma membrane and forms a link between the F-actin cytoskeleton and the extracellular matrix. The proteins of the DGC are structurally organized into distinct subcomplexes, and genetic mutations in many individual components are manifested as muscular dystrophy. We recently identified a unique tetraspan-like dystrophin-associated protein, which we have named sarcospan (SPN) for its multiple sarcolemma spanning domains (Crosbie, R.H., J. Heighway, D.P. Venzke, J.C. Lee, and K.P. Campbell. 1997. J. Biol. Chem. 272:31221-31224). To probe molecular associations of SPN within the DGC, we investigated SPN expression in normal muscle as a baseline for comparison to SPN's expression in animal models of muscular dystrophy. We show that, in addition to its sarcolemma localization, SPN is enriched at the myotendinous junction (MTJ) and neuromuscular junction (NMJ), where it is a component of both the dystrophin- and utrophin-glycoprotein complexes. We demonstrate that SPN is preferentially associated with the sarcoglycan (SG) subcomplex, and this interaction is critical for stable localization of SPN to the sarcolemma, NMJ, and MTJ. Our experiments indicate that assembly of the SG subcomplex is a prerequisite for targeting SPN to the sarcolemma. In addition, the SG- SPN subcomplex functions to stabilize alpha-dystroglycan to the muscle plasma membrane. Taken together, our data provide important information about assembly and function of the SG-SPN subcomplex.

Figure 1

SPN is a novel tetraspan expressed predominantly in muscle. (a) Multiple amino acid sequence alignment of SPN isolated from rabbit, human, and mouse skeletal muscle. Residues identical in two or more species are shaded with gray boxes. Predicted transmembrane domains are indicated with underlining. These sequence data are available from GenBank/EMBL/DDBJ under accession numbers AF016028 for human, U02487 for mouse, and AF120276 for rabbit. Mouse SPN cDNAs isolated from Y1 adrenal carcinoma cells (Scott et al., 1994) and skeletal muscle (this study) are identical. (b) Northern blots containing 2 μg of poly (A)⁺ RNA from the indicated mouse tissues were hybridized with probes representing the 3′ untranslated region (UTR) and the coding region of mouse SPN. Molecular size markers are indicated on the left (kb).

Figure 2

SPN is absent in dystrophin-deficient muscle. Quadriceps, diaphragm, and cardiac muscle cryosections from wt and mdx mice were stained with antibodies to SPN by indirect immunofluorescence. SPN staining is dramatically reduced in the dystrophin-deficient mdx muscles. Bars, 100 μm.

Figure 3

Restoration of SPN by dystrophin transgenic products. Various truncated and internally deleted dystrophin transgenes were expressed on an mdx background and previously analyzed for their ability to rescue the mdx phenotype. Skeletal muscle cryosections from Δ17–48 (becker), Δ1–62 (Dp71), Δ71–74 (Δ330), and Δ75–78 mice were stained with SPN antibodies and visualized by indirect immunofluorescence. The localization of the deleted exons is represented in the schematic diagram of the dystrophin transgene. Binding regions for F-actin have been identified in the NH₂ terminus, as well as in the mid-rod domain of dystrophin (Rybakova et al., 1996; Rybakova and Ervasti, 1997; Amann et al., 1998). Syntrophin and β-DG binding sites are located at the dystrophin COOH terminus. Bar, 100 μm.

Figure 4

Enrichment of SPN at the NMJ is mediated by dystrophin and utrophin. Sections from quadriceps femoris muscles of adult mice were doubly stained with α-bungarotoxin (α-BTX) and SPN antibodies. SPN expression at the NMJ was examined for wt, mdx, utrn^−/−, and mdx:utrn^−/− mice. SPN staining was visualized by indirect immunofluorescence with Cy3-conjugated secondary antibodies (red) and synaptic sites were identified by fluorescein– α-bungarotoxin staining (green). Merged images (yellow) are shown in the right panels.

Figure 5

SPN staining is preserved in the EOMs of mdx mice by utrophin upregulation. Cryosections from the EOMs of wt, mdx, and mdx:utrn^−/− were stained with antibodies to SPN, utrophin (Utrn), and laminin α2 chain (Lam). The mdx EOM are among the few muscles that are spared the pathological consequences of muscular dystrophy, likely due to its sarcolemma expression of utrophin. Shown are EOM rectus muscles. SPN expression is maintained in the mdx EOM. Bar, 50 μm.

Figure 6

SPN's absence from the sarcolemma of δ-SG-deficient BIO 14.6 hamster can be restored with recombinant δ-SG adenovirus. Quadriceps muscle of the BIO 14.6 hamster was injected with 10⁹ particles of δ-SG adenovirus or α-SG adenovirus particles. Tissue was harvested 7 d after injection. Muscle cryosections from these injected animals, as well as from the F1B and BIO 14.6 (uninjected) hamsters were examined for SPN expression by indirect immunofluorescence with SPN antibodies.

Figure 7

SPN is absent from the NMJ and MTJ of Sgca-null mice. (a) Cryosections from Sgca-null muscle were analyzed for SPN expression at the sarcolemma, NMJ, and MTJ. SPN is completely absent in the SG deficient muscle. (b) Skeletal muscle membranes from wt, mdx, and Sgca-null mice were analyzed by 3–15% SDS-PAGE and immunoblotted using antibodies against SPN. SPN isolated from mouse skeletal muscle membranes migrates at 20 kD. The level of SPN expression is dramatically reduced in the mdx membranes and is completely absent from the Sgca-null muscle. Molecular weights are indicated (× 10³ D).

Figure 7

SPN is absent from the NMJ and MTJ of Sgca-null mice. (a) Cryosections from Sgca-null muscle were analyzed for SPN expression at the sarcolemma, NMJ, and MTJ. SPN is completely absent in the SG deficient muscle. (b) Skeletal muscle membranes from wt, mdx, and Sgca-null mice were analyzed by 3–15% SDS-PAGE and immunoblotted using antibodies against SPN. SPN isolated from mouse skeletal muscle membranes migrates at 20 kD. The level of SPN expression is dramatically reduced in the mdx membranes and is completely absent from the Sgca-null muscle. Molecular weights are indicated (× 10³ D).

Figure 8

Isolation of the SG–SPN subcomplex from skeletal muscle. (a) The DGC, purified from rabbit skeletal muscle membranes, was titrated to pH 11 and centrifuged through 5–30% linear sucrose gradients. Fractions from the gradients were separated by SDS-PAGE and separately immunoblotted with antibodies to α- and β-DG, SGs, dystrophin, and SPN. Alkaline treatment dissociates the SG subcomplex from the DG subcomplex. SPN comigrates with the SG subcomplex. (b) Glycoproteins from skeletal muscle of dystrophin-deficient mdx mice were prepared and centrifuged through 5–30% linear sucrose gradients. Protein fractions were immunoblotted with the indicated antibodies. Without dystrophin, the DG and SG subcomplexes are no longer associated, and migrate separately during sucrose gradient centrifugation. SPN preferentially associates with the SG subcomplex. Molecular size standards are indicated on each panel (× 10³ D).

Figure 9

Reconstitution of the SG–SPN subcomplex. The SG– SPN subcomplex was reconstituted in vivo using a heterologous cell expression system. CHO cells were transfected with expression vectors encoding myc-tagged human α-, β-, γ-, and δ-SGs with either SPN (SGs + SPN) or Grb2 (SGs + Grb2). Mock transfected CHO cells are shown. To demonstrate similar expression of proteins, whole cell lysates are shown (lysates). Note that Grb2, which serves as a negative control, and SPN migrate at similar molecular weights. Cells transfected with all four SGs and SPN were treated with NHS-biotin and recovered from detergent extracts as avidin precipitates to demonstrate surface localization of these proteins (NHS). Immunoprecipitation of β-SG from lysates prepared from cells expressing all four SGs and SPN results in coprecipitation of the SG–SPN subcomplex (β-SG IP). Grb2 does not immunoprecipitate with the SGs. Proteins are detected by immunoblotting with an anti-myc mAb, which recognizes the 9E10 tag on each protein construct. The 50-kD protein band in the mock transfected cell represents the β-SG immunoprecipitating antibody. Molecular weights are indicated on each blot (× 10³ D).

Figure 10

Schematic diagram representing interactions among DGC subcomplexes. Evidence presented in the current report demonstrates that assembly and membrane localization of the SG subcomplex are prerequisites for SPN localization to the sarcolemma, NMJ, and MTJ. This dependence is likely mediated through direct interaction of SPN with the SGs, as suggested by cofractionation and coimmunoprecipitation of SPN with the SG subcomplex. In the absence of the SG–SPN subcomplex, α-DG is not properly anchored to the muscle plasma membrane. This suggests that there may be direct interactions between α-DG and the SG–SPN subcomplex, which stabilize α-DG to the cell membrane (dashed arrows). The molecular basis of the interaction between the SG–SPN and DG subcomplexes is not thoroughly understood. Clearly, proper structural alignment of the two subcomplexes along with dystrophin is required for DGC function and prevention of muscular dystrophy.