The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones.
The E-Cadherin-catenin complex plays a critical role in epithelial cell-cell adhesion, polarization, and morphogenesis. Here, we have analyzed the mechanism of Drosophila E-Cadherin (DE-Cad) localization. Loss of function of the Drosophila exocyst components sec5, sec6, and sec15 in epithelial cells results in DE-Cad accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits DE-Cad delivery to the membrane. Furthermore, Rab11 and Armadillo interact with the exocyst components Sec15 and Sec10, respectively. Our results support a model whereby the exocyst regulates DE-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located.
The mechanisms underlying functional interactions between ERM (ezrin, radixin, moesin) proteins and Rho GTPases are not well understood. Here we characterized the interaction between ezrin and a novel Rho guanine nucleotide exchange factor, PLEKHG6. We show that ezrin recruits PLEKHG6 to the apical pole of epithelial cells where PLEKHG6 induces the formation of microvilli and membrane ruffles. These morphological changes are inhibited by dominant negative forms of RhoG. Indeed, we found that PLEKHG6 activates RhoG and to a much lesser extent Rac1. In addition we show that ezrin forms a complex with PLEKHG6 and RhoG. Furthermore, we detected a ternary complex between ezrin, PLEKHG6, and the RhoG effector ELMO. We demonstrate that PLEKHG6 and ezrin are both required in macropinocytosis. After down-regulation of either PLEKHG6 or ezrin expression, we observed an inhibition of dextran uptake in EGF-stimulated A431 cells. Altogether, our data indicate that ezrin allows the local activation of RhoG at the apical pole of epithelial cells by recruiting upstream and downstream regulators of RhoG and that both PLEKHG6 and ezrin are required for efficient macropinocytosis. INTRODUCTIONThe membrane-cytoskeleton linker ezrin is mainly expressed in epithelial cells where it associates to the apical actin-rich structures such as microvilli (Berryman et al., 1993(Berryman et al., , 1995. Recent genetic analyses revealed that ezrin is essential for the morphogenesis of epithelial cells (Fiévet et al., 2007). In ezrin Ϫ/Ϫ mice, morphological defects in the apical domain of intestinal and retinal pigment epithelial cells have been observed (Saotome et al., 2004;Bonilha et al., 2006). In parietal cells, ezrin knockdown impairs the formation of canalicular apical membrane, resulting in severe achlorhydria (Tamura et al., 2005).How ezrin participates in the assembly of the apical actinrich structures such as microvilli is still poorly understood. The association of ezrin and the highly related proteins radixin and moesin with the cortical actin cytoskeleton is strictly regulated. ERM (ezrin, radixin, moesin) proteins contain a conserved globular N-terminal domain, called the FERM domain (Four point one ezrin, radixin, moesin), involved in the binding to both phosphatidylinositol 4,5 bisphosphate (PIP 2 ; Barret et al., 2000) and plasma membrane proteins and a C-terminal F-actin-binding domain that resides in the last 34 amino acids (Turunen et al., 1994;Bretscher et al., 2002). In the cytoplasm, ERM proteins exist in a closed conformation because of an intramolecular interaction between the N-terminal domain and the last 100 amino acids called N-and C-ERMAD (ERM association domain), respectively . This intramolecular association masks the binding sites for plasma membrane proteins and F-actin. An activation step is required to disrupt this association that occurs through conformational changes induced by sequential binding to PIP 2 and phosphorylation of a conserved C-terminal threonine residue (T567 in ezrin; Matsui et al., 1998;Fié...
Gem is a Ras-related protein whose expression is induced in several cell types upon activation by extracellular stimuli. With the aim of isolating the cellular partners of Gem that mediate its biological activity we performed a yeast two-hybrid screen and identified a novel protein of 970 amino acids, Gmip, that interacts with Gem through its N-terminal half, and presents a cysteine-rich domain followed by a Rho GTPase-activating protein (RhoGAP) domain in its C-terminal half. The RhoGAP domain of Gmip stimulates in vitro the GTPase activity of RhoA, but is inactive towards other Rho family proteins such as Rac1 and Cdc42; it is also specific for RhoA in vivo. The same is true for the full-length protein, which is furthermore able to down-regulate RhoA-dependent stress fibres in Ref-52 rat fibroblasts. These findings suggest that the signalling pathways controlled by two proteins of the Ras superfamily, RhoA and Gem, are linked via the action of the RhoGAP protein Gmip (Gem-interacting protein).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.