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John McVey, Maria Lai, Harald J Junge; Forced interaction between FZD4 and LRP5 induces Norrin/Frizzled4 signaling. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):53.
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© ARVO (1962-2015); The Authors (2016-present)
Norrin/Frizzled4 signaling is required for retinal vascular development and blood-retina barrier formation. Frizzled4 (FZD4), low-density lipoprotein receptor-related protein 5 (LRP5), and Tetraspanin12 (TSPAN12) mediate signal initiation of this pathway; however, how these membrane molecules operate together to induce signaling in vascular endothelial cells is not well understood. Using the FKBP/FRB-tag (iDimerize) system, we forced homo- or hetero-interactions between TSPAN12, FZD4, and LRP5 to gain insight into their role in inducing Norrin/Frizzled4 signaling.
The iDimerize system was used to force interactions between proteins tagged with c-terminal tandem DmrA (FKBP domain from FKBP1A) or DmrC (FRB domain of mTOR) domains for hetero-interactions, or DmrB (FKBP domain with F36V mutation) domains for homo-interactions. A/C-inducer or B/B-inducer small molecules were partially dispensable as Dmr domains interacted constitutively. TOPflash reporter assays in 293T cells were employed to determine signaling levels for proteins with and without Dmr domains. All combination of hetero-interactions as well as FZD4 homo-interactions were tested.
Norrin-dependent signaling was 2.7 fold increased when FZD4-homo-interactions were forced and was 3 fold increased when FZD4/LRP5 hetero-interactions were forced (fold increase relative to signal of cells transfected with untagged FZD4 and LRP5). Forcing FZD4/LRP5 hetero-interactions not only enhanced Norrin-dependent signaling but also induced a high degree of Norrin-independent signaling. FZD4 M105V and FZD4 M157V point mutations strongly impaired Norrin-dependent Frizzled4 signaling and this defect was rescued by co-transfection of TSPAN12 but was not rescued by forcing FZD4 homo-interactions or FZD4/LRP5 hetero-interactions.
FZD4 homo-interactions and FZD4/LRP5 hetero-interactions enhance Norrin/FZD4 signaling. Forcing the FZD4/LRP5 hetero-interaction is sufficient to initiate signaling in the absence of exogenous Norrin. Distinct mechanisms appear to be responsible for the enhanced signaling mediated by the co-activator TSPAN12 and the enhanced signaling mediated by forcing FZD4 homo-interactions and FZD4/LRP5 hetero-interactions.
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