Stem cell antigen-1 localizes to lipid microdomains and associates with insulin degrading enzyme in skeletal myoblasts

Stem cell antigen-1 (Sca-1, Ly6A/E) is a glycosylphosphotidylinositol (GPI)-anchored protein that identifies many tissue progenitor cells with self-renewal capacity (Holmes and Stanford, 2007). We originally identified Sca-1 as a marker of myogenic precursor cells undergoing cell cycle withdrawal during differentiation (Shen et al., 2003). We subsequently demonstrated both in vitro and in vivo that Sca-1 regulates proliferation of activated muscle precursor cells (Epting et al., 2004, 2008), suggesting an important role for Sca-1 in tissue homeostasis as well as a potential function in maintenance of the muscle stem cell pool. Beyond its functional role in regulating proliferation, however, little is known about how Sca-1 may be regulated in myogenic precursor cells, or how its signaling input is translated into cellular activities. Other GPI-anchored proteins (GPI-APs) have been shown to localize within lipid microdomains in the plasma membrane (Kenworthy et al., 2000), and to couple with non-receptor tyrosine kinases (Stefanova et al., 1991; Clissold, 1994). More specifically, it has been suggested that Sca-1 might associate with the lipid microdomain fraction in lymphocytes (Bohuslav et al., 1993). Such lipid microdomains are believed to facilitate coalescence of surface, transmembrane, and intracellular proteins in the assembly of intracellular signaling complexes (Hoessli et al., 2000). Demonstrating a lipid microdomain association of Sca-1, therefore, should reveal potential transmembrane signaling partners and mechanisms for this historically enigmatic molecule. Whether Sca-1 sorts to this membrane compartment in myogenic precursors, how this may play a regulatory role in its function, and the identity of associated proteins that may serve as secondary messengers in Sca-1-mediated signaling pathways have not previously been addressed. We now report that lipid microdomain organization is essential for normal myogenic differentiation, that Sca-1 associates with these domains in myogenic precursor cells, and that although Sca-1 regulates myoblast proliferation through its temporally regulated expression (Epting et al., 2004), its spatial localization to lipid microdomains is static, suggesting that migration between lipid microdomains and non-lipid microdomain regions of the plasma membrane does not regulate its function. In the absence of regulation through spatial reorganization, we sought to identify binding partners through which Sca-1 signaling might be regulated. We demonstrate that Sca-1 associates with insulin degrading enzyme (IDE), a catalytic protein responsible for the cleavage of mitogenic peptides (Duckworth et al., 1998), in differentiating myoblasts. We hypothesized that IDE might participate in signaling pathways downstream of Sca-1, and indeed found that inhibition of IDE recapitulates the phenotype of Sca-1 interference, that is, sustained myoblast proliferation and delayed myogenic differentiation (Epting et al., 2004, 2008). These findings identify the first signaling protein that physically and functionally associates with Sca-1 in myogenic precursor cells, and suggest that IDE participates in Sca-1-mediated signaling events.