Abstract
The dystrophin-glycoprotein complex (DGC) links the extracellular matrix and actin cytoskeleton. Caveolae form membrane arrays on smooth muscle cells; we investigated the mechanism for this organization. Caveolin-1 and beta-dystroglycan, the core transmembrane DGC subunit, colocalize in airway smooth muscle. Immunoprecipitation revealed the association of caveolin-1 with beta-dystroglycan. Disruption of actin filaments disordered caveolae arrays, reduced association of beta-dystroglycan and caveolin-1 to lipid rafts, and suppressed the sensitivity and responsiveness of methacholine-induced intracellular Ca2+ release. We generated novel human airway smooth muscle cell lines expressing shRNA to stably silence beta-dystroglycan expression. In these myocytes, caveolae arrays were disorganized, caveolae structural proteins caveolin-1 and PTRF/cavin were displaced, the signaling proteins PLCbeta1 and G(alphaq), which are required for receptor-mediated Ca2+ release, were absent from caveolae, and the sensitivity and responsiveness of methacholine-induced intracellular Ca2+ release, was diminished. These data reveal an interaction between caveolin-1 and beta-dystroglycan and demonstrate that this association, in concert with anchorage to the actin cytoskeleton, underpins the spatial organization and functional role of caveolae in receptor-mediated Ca2+ release, which is an essential initiator step in smooth muscle contraction.
Highlights
Smooth muscle in hollow organs is subject to cellular deformation and mechanical stress during contraction and changes in transmural pressure
Results -Dystroglycan co-fractionates and co-precipitates with caveolin-1 We reported that dystrophin–glycoprotein complex (DGC) subunits are abundant in human airway smooth muscle tissue and cells (Sharma et al, 2008)
We have shown that M3 receptors (M3Rs) can be associated with caveolae in airway smooth muscle (Gosens et al, 2007a), we used sucrose gradient fractionation to compare the subcellular distribution of G q and PLC 1 in control and -dystroglycan-silenced human airway myocytes
Summary
Smooth muscle in hollow organs is subject to cellular deformation and mechanical stress during contraction and changes in transmural pressure. The complex is composed of several transmembrane, cytoplasmic and extracellular protein subunits, with dystrophin, a large intracellular rod-like protein, serving as a tether between cytoskeletal actin and -dystroglycan, the core transmembrane subunit of the DGC (Ervasti and Campbell, 1993; Lapidos et al, 2004). Because they are needed to facilitate contraction and Ca2+ mobilization mediated by some G-protein-coupled receptors (GPCRs) in airway smooth muscle (Gosens et al, 2007b; Prakash et al, 2007). These observations suggest that the spatial organization of caveolae and the repertoire of proteins that localize to them are fundamental determinants of the contractile response of smooth muscle
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