Abstract
Neph proteins are evolutionarily conserved membrane proteins of the immunoglobulin superfamily that control the formation of specific intercellular contacts. Cell recognition through these proteins is essential in diverse cellular contexts such as patterning of the compound eye in Drosophila melanogaster, neuronal connectivity in Caenorhabditis elegans, and the formation of the kidney filtration barrier in mammals. Here we identify the PDZ and BAR domain protein PICK1 (protein interacting with C-kinase 1) as a Neph-interacting protein. Binding required dimerization of PICK1, was dependent on PDZ domain protein interactions, and mediated stabilization of Neph1 at the plasma membrane. Moreover, protein kinase C (PKCα) activity facilitated the interaction through releasing Neph proteins from their binding to the multidomain scaffolding protein zonula occludens 1 (ZO-1), another PDZ domain protein. In Drosophila, the Neph homologue Roughest is essential for sorting of interommatidial precursor cells and patterning of the compound eye. RNA interference-mediated knockdown of PICK1 in the Drosophila eye imaginal disc caused a Roughest destabilization at the plasma membrane and a phenotype that resembled rst mutation. These data indicate that Neph proteins and PICK1 synergistically regulate cell recognition and contact formation.
ACKNOWLEDGMENTS
We thank Stefanie Keller, Bettina Maar, Ruth Herzog, Manuela Hochberger, Christina Engel, and Charlotte Meyer for excellent technical assistance and members of the laboratories for helpful discussions. We also thank Ross Cagan, the Vienna Drosophila RNAi Center, and Developmental Studies Hybridoma Bank, University of Iowa, for flies and antibodies.
H.C.R. received funding through the Deutsche Forschungsgemeinschaft (RE2246/1-1, RE2246/2-1, and SFB832), the Deutsche Nierenstiftung, and the Köln Fortune Program. T.B.H. received funding through the Deutsche Forschungsgemeinschaft and through exc 294. T.B. received funding through the Deutsche Forschungsgemeinschaft (BE2212, SFB572, and SFB635).