Role of NDP- and FZD4-Related Novel Mutations Identified in Patients with FEVR in Norrin/β-Catenin Signaling Pathway

Figure 4

FEVR-linked NDP and FZD4 mutations impaired the binding of Norrin and Frizzled-4. (a) The schematic diagram showed that all the six mutations were located in the CTCK or CRD domain, respectively. (b, c) Transfected cells expressing Frizzled-4 were incubated with wild-type and mutant Norrin-conditioned medium before the Norrin-Frizzled-4 binding complex was immunoprecipitated. Mutant Norrin coprecipitated with Frizzled-4 to varying reduced degrees. It was noted that Norrin concentrations in the diluted lysate were below the detection limit of standard detection systems. (d, e) Transfected cells expressing wild-type and mutant Frizzled-4 were incubated with Norrin-conditioned medium before the Norrin-Frizzled-4 binding complex was immunoprecipitated. Norrin coprecipitated with mutant Frizzled-4 to a distinct reduced degree. It was noted that Norrin concentrations in the diluted lysate were below the detection limit of standard detection systems. (f) Integrated band density quantification of Frizzled-4 coprecipitated with Norrin. (g) Mutant Norrin was expressed at the same level as wild-type Norrin. (h) The secretion of extracellular mutant Norrin was expressed at the same level as wild-type Norrin. (i) Mutant Frizzled-4 was expressed at the same level as wild-type Frizzled-4. (j) Mutant Frizzled-4 was efficiently located to the plasma membrane the same as wild-type Frizzled-4.

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