title: Identification of the cellular mechanisms that modulate trafficking of frizzled family receptor 4 (FZD4) missense mutants associated with familial exudative vitreoretinopathy.
abstract: OBJECTIVE
  Fifteen missense mutations in the frizzled family receptor 4 (FZD4) reported to cause familial exudative vitreoretinopathy (FEVR) were evaluated to establish the pathological cellular mechanism of disease and to explore novel therapeutic strategies.
  
  
  METHODS
  The mutations were generated by site-directed mutagenesis and expressed in HeLa and COS-7 cell lines. Confocal fluorescence microscopy and N-glycosylation profiling were used to observe the subcellular localization of the mutant proteins relative to wild-type (WT). Polyubiquitination studies were used to establish the involvement of the proteasome. Culturing at reduced temperatures and incubation in the presence of chemical compounds were used to enhance mutant protein processing and exit out of the endoplasmic reticulum (ER).
  
  
  RESULTS
  Confocal fluorescence microscopy of the mutants showed three distinct subcellular localizations, namely, a plasma membrane pattern, an ER pattern, and a mixed pattern to both compartments. Confocal fluorescence microscopy and N-glycosylation profiling established the predominant ER localization of P33S, G36N, H69Y, M105T, M105V, C181R, C204R, C204Y, and G488D mutants. Coexpression of these mutants with WT FZD4 showed the inability of the mutants to trap WT FZD4. Culturing the expressing cells at reduced temperatures or in the presence of chemical agents directed at ameliorating protein misfolding resulted in partial rescue of trafficking defects observed for M105T and C204Y mutants.
  
  
  CONCLUSIONS
  Defective trafficking resulting in haploinsufficiency is a major cellular mechanism for several missense FEVR-causing FZD4 mutants. Our findings indicate that this trafficking defect might be correctable for some mutants, which may offer opportunities for the development of novel therapeutics approaches for this condition.
authors.affiliation: Department of Pathology, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.
authors.forename: Reham M
authors.initials: RM
authors.lastname: Milhem
authors.affiliation: Department of Pathology, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.
authors.forename: Salma
authors.initials: S
authors.lastname: Ben-Salem
authors.affiliation: Department of Pediatrics, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.
authors.forename: Lihadh
authors.initials: L
authors.lastname: Al-Gazali
authors.affiliation: Department of Pathology, College of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates.
authors.forename: Bassam R
authors.initials: BR
authors.lastname: Ali
mesh_terms: D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004247:DNA / Q000235:genetics*; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000473:pathology; D005122:Exudates and Transudates; D051157:Frizzled Receptors / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D005838:Genotype; D006801:Humans; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense / Q000235:genetics*; D021381:Protein Transport / Q000235:genetics; D018630:Vitreoretinopathy, Proliferative / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D014822:Vitreous Body / Q000378:metabolism* / Q000473:pathology
chemical_list: C402930:FZD4 protein, human; D051157:Frizzled Receptors; D004247:DNA
keywords: FEVR; FZD4; endoplasmic reticulum quality control; endoplasmic reticulum–associated degradation; familial exudative vitreoretinopathy; frizzled family receptor 4; trafficking