Purchase this article with an account.
Chi Zhang, Maria Lai, Harald J Junge; Role of endocytosis in Norrin/Frizzled4 signaling. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):54.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Norrin/Frizzled4 signaling plays an important role in the development of retinal blood vessels and blood-retina barrier formation. Although Norrin is structurally unrelated to Wnts, Norrin binding to the Frizzled4 receptor activates canonical beta-catenin signaling in vascular endothelial cells. However, the mechanism of signal transduction remains incompletely understood. Because we observe that Norrin binding to Frizzled4 triggers endocytosis of the ligand/receptor complex, we ask if endocytosis is positively or negatively coupled with signaling.
Hela cells were transfected with Frizzled4 receptor, LRP5 co-receptor and the essential co-activator tetraspanin12. Live cells were then incubated with conditioned medium containing a hybrid protein of Norrin and alkaline phosphatase on ice. After washing, endocytosis was triggered by subjecting cells to a 37°C incubation for 30-60 min, non-internalized Norrin was then removed during an acid wash step. Endocytosed Norrin, Frizzled4 and associated proteins were localized relative to markers of the endocytic pathway by immuno-staining and imaged using confocal microscopy. To quantify Norrin/Frizzled4 signaling, 293T cells were co-transfected with FZD4, LRP5, TSPAN12, a LEF/TCF luciferase reporter construct and Renilla luciferase as internal control. Dual luciferase assays were performed to determine levels of Norrin/Frizzled4 signaling in the presence and absence of dynasore (an inhibitor of Dynamin), siRNA targeting clathrin or caveolin (mediators of endocytosis), or dominant negative VPS4-E/Q (an inhibitor of multivesicular body formation).
30 minutes after endocytosis was triggered, Norrin, Frizzled4, and associated proteins co-localized in puncta in Hela cells. The vast majority of these puncta co-localized with the early endosomal marker EEA1. At later time points (60 minutes), Norrin and Frizzled4 co-localized with markers of the lysosomal degradation pathway (Rab7, Lamp1), while little or no co-localization was detected with markers of the recycling endosome (Rab11). In Topflash assays, Norrin/Frizzled4 signaling was impaired when treated with 40mM Dynasore, siRNA against clathrin, or VPS4-E/Q.
Norrin is a potent trigger of endocytosis of the Frizzled4 receptor complex. Endocytosis is positively coupled to Norrin/Frizzled4 signaling.
This PDF is available to Subscribers Only