Vulval development is induced by activation of LET-23 EGFR in the basal membrane compartment of polarized Vulval Precursors Cells (VPCs) after interaction with the Anchor Cell secreted LIN-3 EGF. LET-23 is retained in this compartment by the conserved tripartite LIN-2/LIN-7/LIN-10 PDZ protein complex (Kaech et al. 1996). This interaction is mediated through a C-terminal PDZ binding domain in LET-23. Mutations in any of the components of this complex cause a loss of retention of LET-23 in the baso-lateral membrane compartment and result in the apical mislocalization of LET-23. This impairs the interaction of LET-23 with LIN-3 and leads to a reduced vulval induction. We have performed a screen to find novel regulators of LET-23 localization in the VPCs and found that LET-23 is miss-localized in
erm-1 mutants.
erm-1 encodes the homologue of mammalian Ezrin, Radixin and Moesin proteins (ERM), which in their open phosphorylated form link plasma membrane proteins to the actin cytoskeleton. Here, we show that
erm-1 acts as negative regulator of the
let-23/let-60/mpk-1 pathway, possibly by sequestering and stabilizing the LET-23 receptor in an inactive compartment at or near the baso-lateral plasma membrane of the VPCs. The following lines of evidence support our model: (1) An
erm-1(lf) mutant suppresses the Vulvaless phenotype caused by reduction-of-function mutations in the
let-23/let-60/mpk-1 pathway and enhances the Multivulva phenotype caused by a gain-of-function mutation in
let-60 ras. (2) An ERM-1::mCherry fusion protein as well as a constitutively active, phospho-mimicking ERM-1::T544D::mCherry mutant co-localize with a functional LET-23::GFP protein on the baso-lateral membrane, while the phosphorylation resistant ERM-1::T544A::mCherry mutant remains in the cytoplasm. (3) Recombinant ERM-1::GST interacts with LET-23 from worm extracts, this interaction is independent of LIN-7 and the PDZ binding motif at the LET-23 C-terminus, suggesting that ERM-1 interacts with LET-23 in a complex distinct from the LET-23/LIN-2/LIN-7/LIN-10 complex. (4)
erm-1(lf) mutants display reduced basal LET-23::GFP staining and Fluorescence Recovery After Photobleaching experiments show a significantly faster recovery of basal LET-23::GFP in
erm-1 mutants compared to the wild-type. In summary, we propose that ERM-1 retains a fraction of LET-23 in an inactive compartment, thereby competing with the activating LET-23/LIN-2/LIN-7/LIN-10 complex. ERM-1 may act as a buffer to prevent the immediate activation of the entire pool of baso-lateral LET-23 and thus allow prolonged LET-23 signaling to occur.