Novel γ-sarcoglycan interactors in murine muscle membranes

Abstract

Background The 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. Methods To 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. Results We 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. Conclusions Results 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.

Multi-omics analysis of sarcospan overexpression in mdx skeletal muscle reveals compensatory remodeling of cytoskeleton-matrix interactions that promote mechanotransduction pathways

“…While many therapeutic genes that improve sarcolemma instability, decrease fibrosis, and increase force in mdx muscle have been identified [14][15][16][17][18][19][20][21], there are gaps in understanding whether their protective mechanisms affect overlapping molecular processes and signaling pathways. Furthermore, there is emerging evidence of an expanded network of DGC-interacting proteins, suggesting that it functions as a central unit to integrate cellular signaling, lateral force transmission, ion channel function, and cytoskeletal organization [22,23]. Sarcospan (SSPN), a core component of the DGC [24][25][26], prevents muscle degeneration and histopathology in mdx mice in a mechanism that is dependent on increased abundance of utrophin and integrin α7β1 at the sarcolemma [27][28][29][30][31][32].…”

Multi-omics analysis of sarcospan overexpression in mdx skeletal muscle reveals compensatory remodeling of cytoskeleton-matrix interactions that promote mechanotransduction pathways

“…While many therapeutic genes that improve sarcolemma instability, decrease fibrosis, and increase force in mdx muscle have been identified (14–21), there are gaps in understanding whether their protective mechanisms affect overlapping molecular processes and signaling pathways. Furthermore, there is emerging evidence of an expanded network of DGC interacting proteins, suggesting that it functions as a central unit to integrate cellular signaling, lateral force transmission, ion channel function, and cytoskeletal organization (22,23).…”

“…While many therapeutic genes that improve sarcolemma instability, decrease fibrosis, and increase force in mdx muscle have been identified (14)(15)(16)(17)(18)(19)(20)(21), there are gaps in understanding whether their protective mechanisms affect overlapping molecular processes and signaling pathways. Furthermore, there is emerging evidence of an expanded network of DGC interacting proteins, suggesting that it functions as a central unit to integrate cellular signaling, lateral force transmission, ion channel function, and cytoskeletal organization (22,23). Sarcospan (SSPN), a core component of the DGC (24)(25)(26) prevents muscle degeneration and histopathology in mdx mice in a mechanism that is dependent on increased abundance of utrophin and integrin α7β1 at the sarcolemma (27)(28)(29)(30)(31).…”