Abstract
BackgroundThe sarcoglycan complex (SC) is part of a network that links the striated muscle cytoskeleton to the basal lamina across the sarcolemma. The SC coordinates changes in phosphorylation and Ca++-flux during mechanical deformation, and these processes are disrupted with loss-of-function mutations in gamma-sarcoglycan (Sgcg) that cause Limb girdle muscular dystrophy 2C/R5.MethodsTo gain insight into how the SC mediates mechano-signaling in muscle, we utilized LC-MS/MS proteomics of SC-associated proteins in immunoprecipitates from enriched sarcolemmal fractions. Criteria for inclusion were co-immunoprecipitation with anti-Sgcg from C57BL/6 control muscle and under-representation in parallel experiments with Sgcg-null muscle and with non-specific IgG. Validation of interaction was performed in co-expression experiments in human RH30 rhabdomyosarcoma cells.ResultsWe identified 19 candidates as direct or indirect interactors for Sgcg, including the other 3 SC proteins. Novel potential interactors included protein-phosphatase-1-catalytic-subunit-beta (Ppp1cb, PP1b) and Na+-K+-Cl−-co-transporter NKCC1 (SLC12A2). NKCC1 co-localized with Sgcg after co-expression in human RH30 rhabdomyosarcoma cells, and its cytosolic domains depleted Sgcg from cell lysates upon immunoprecipitation and co-localized with Sgcg after detergent permeabilization. NKCC1 localized in proximity to the dystrophin complex at costameres in vivo. Bumetanide inhibition of NKCC1 cotransporter activity in isolated muscles reduced SC-dependent, strain-induced increases in phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2). In silico analysis suggests that candidate SC interactors may cross-talk with survival signaling pathways, including p53, estrogen receptor, and TRIM25.ConclusionsResults support that NKCC1 is a new SC-associated signaling protein. Moreover, the identities of other candidate SC interactors suggest ways by which the SC and NKCC1, along with other Sgcg interactors such as the membrane-cytoskeleton linker archvillin, may regulate kinase- and Ca++-mediated survival signaling in skeletal muscle.
Highlights
The sarcoglycan complex (SC) is part of a network that links the striated muscle cytoskeleton to the basal lamina across the sarcolemma
We report for the first time that Sodium potassium chloride co-transporter 1 (NKCC1) immunofluorescence overlaps with that of costameric dystrophin at the sarcolemma and that NKCC1 cotransporter activity is required for the eccentric contractions (ECC)-induced increases in activated ERK seen in Sgcg−/− mouse muscle
The RIPA1 extract was enriched in proteins associated with the dystrophin glycoprotein complex (DGC) (Fig. 2B–E), and proteins associated with the sarcoplasmic reticulum (SERCA1) and
Summary
The sarcoglycan complex (SC) is part of a network that links the striated muscle cytoskeleton to the basal lamina across the sarcolemma. The dystrophin glycoprotein complex (DGC) is a major sarcolemmal complex that tethers the extracellular matrix (ECM) to the intracellular actin cytoskeleton [1, 2]. The dominant DGC connection to actin is via dystrophin, which links the actin cytoskeleton to dystroglycan, and in turn binds laminin in the ECM [1, 2]. The sarcoglycan complex (SC) is a sub-complex of the DGC, comprised of α-, β-, δ-, and γ-sarcoglycans (Sgca, Sgcb, Sgcd, Sgcg) in skeletal muscle [7]. These proteins form a heterotetramer, each having a short intracellular domain, a single membrane-spanning α-helix, and a large extracellular domain. Sarcolemmal DGC proteins are concentrated at costameres, which are associated with the mouths of T-tubules and contain a multitude of signaling proteins [2, 8,9,10,11,12,13]
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