Novel gamma-sarcoglycan interactors in murine muscle membranes
Smith, Tara C. ; Vasilakos, Georgios ; Shaffer, Scott A ; Puglise, Jason M. ; Chou, Chih-Hsuan ; Barton, Elisabeth R. ; Luna, Elizabeth J.
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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.
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Smith TC, Vasilakos G, Shaffer SA, Puglise JM, Chou CH, Barton ER, Luna EJ. Novel γ-sarcoglycan interactors in murine muscle membranes. Skelet Muscle. 2022 Jan 22;12(1):2. doi: 10.1186/s13395-021-00285-2. PMID: 35065666; PMCID: PMC8783446. Link to article on publisher's site