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Maria Lai, John McVey, Chi Zhang, Lavan Khandan, Harald J Junge; FEVR-associated mutations in TSPAN12 affect Norrin/Frizzled4 signaling. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1094.
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© ARVO (1962-2015); The Authors (2016-present)
Norrie disease and familial exudative vitreoretinopathy (FEVR) are rare inherited diseases caused by mutations impairing Norrin/Frizzled4 (FZD4) signaling in retinal vascular endothelial cells. Defective signaling disrupts retinal angiogenesis, the blood-retina barrier, and can cause blindness. We previously identified Tetraspanin12 (TSPAN12) as an essential co-activator of Norrin/FZD4 signaling. Human genetic studies identified mutations in the tspan12 gene in human FEVR patients; however, if these mutations functionally impair TSPAN12 remains unknown. We hypothesized that FEVR-associated TSPAN12 mutations impair Norrin/FZD4 signaling and that the mutations can be used to identify functionally important regions of TSPAN12.
We generated 10 HA-tagged TSPAN12 constructs with point mutations that have been isolated from FEVR patients. We also generated a series of HA-tagged TSPAN12/TSPAN11 chimeras, with TSPAN11 having no detectable effect on Norrin/FZD4 signaling. We tested the function of these constructs in TOPflash reporter assays in HEK 293T cells stimulated with Norrin. Western blot analysis was performed in 293T cells to determine protein levels of TSPAN12 point mutations and chimeras.
Of the 10 FEVR-associated TSPAN12 point mutation constructs tested to enhance Norrin/FZD4 signaling, 5 point mutations (C105R, M210R, L223P, A237P, and L245P) appeared to completely abolish enhancement, 3 mutations (T49M, L101H, and Y138C) showed partial reduction, while 2 point mutations (G188R and L201F) displayed functional impairment only in the context of TSPAN12-mediated rescue of FZD4-M157V signaling defects. None of the TSPAN12 mutations affected Wnt3a-or Wnt7b-induced signaling. All constructs did express, albeit at variable levels. Functional analysis of TSPAN12/TSPAN11 chimeras indicated that intracellular portions of TSPAN12 are dispensable for function, whereas, extracellular regions are required for function.
Our results indicate that TSPAN12 is required for enhancing Norrin-but not Wnt-induced FZD4 signaling. Signaling defects of TSPAN12 point mutations indicate that these mutations are disease causing. FEVR-associated TSPAN12 point mutations overlap with functionally critical TSPAN12 regions identified in the chimera approach. Intracellular domains of TSPAN12 are not required for function, suggesting that TSPAN12 does not directly connect to intracellular signal transduction machinery.
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