Electrochemical cues regulate assembly of the Frizzled/Dishevelled complex at the plasma membrane during planar epithelial polarization.
Dishevelled (Dsh) is a cytoplasmic multidomain protein that is required for all known branches of the Wnt signalling pathway. The Frizzled/planar cell polarity (Fz/PCP) signalling branch requires an asymmetric cortical localization of Dsh, but this process remains poorly understood. Using a genome-wide RNA interference (RNAi) screen in Drosophila melanogaster cells, we show that Dsh membrane localization is dependent on the Na(+)/H(+) exchange activity of the plasma membrane exchanger Nhe2. Manipulating Nhe2 expression levels in the eye causes PCP defects, and Nhe2 interacts genetically with Fz. Our data show that the binding and surface recruitment of Dsh by Fz is pH- and charge-dependent. We identify a polybasic stretch within the Dsh DEP domain that binds to negatively charged phospholipids and appears to be mechanistically important. Dsh recruitment by Fz can be abolished by converting these basic amino-acid residues into acidic ones, as in the mutant, DshKR/E. In vivo, the DshKR/E(2x) mutant with two substituted residues fails to associate with the membrane during active PCP signalling but rescues canonical Wnt signalling defects in a dsh-background. These results suggest that direct interaction between Fz and Dsh is stabilized by a pH and charge-dependent interaction of the DEP domain with phospholipids. This stabilization is particularly important for the PCP signalling branch and, thus, promotes specific pathway selection in Wnt signalling.
Simons
Matias
M
Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.
Gault
William J
WJ
Gotthardt
Daniel
D
Rohatgi
Rajeev
R
Klein
Thomas J
TJ
Shao
Youming
Y
Lee
Ho-Jin
HJ
Wu
Ai-Luen
AL
Fang
Yimin
Y
Satlin
Lisa M
LM
Dow
Julian T
JT
Chen
Jie
J
Zheng
Jie
J
Boutros
Michael
M
Mlodzik
Marek
M
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D003463:Cues; D000072261:Dishevelled Proteins; D029721:Drosophila Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology* / Q000378:metabolism*; D004563:Electrochemistry; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005123:Eye / Q000166:cytology; D051157:Frizzled Receptors / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010641:Phenotype; D010743:Phospholipids; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D000072261:Dishevelled Proteins; D029721:Drosophila Proteins; D051157:Frizzled Receptors; D010743:Phospholipids; D010750:Phosphoproteins; C000606588:dsh protein, Drosophila