title: Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23.
abstract: BACKGROUND
  The gene for the renal phosphate wasting disorder autosomal-dominant hypophosphatemic rickets (ADHR) is FGF23, which encodes a secreted protein related to the fibroblast growth factors (FGFs). We previously detected missense mutations R176Q, R179W, and R179Q in FGF23 from ADHR kindreds. The mutations replace R residues within a subtilisin-like proprotein convertase (SPC) cleavage site 176RHTR-179 (RXXR motif). The goal of these studies was to determine if the ADHR mutations lead to protease resistance of FGF-23.
  
  
  METHODS
  The ADHR mutations were introduced into human FGF-23 cDNA clones with or without an N-terminal FLAG tag by site-directed mutagenesis and were transiently transfected into HEK293 cells. Protein expression was determined by Western analyses.
  
  
  RESULTS
  Antibodies directed toward the C-terminal portion of FGF-23 revealed that the native FGF-23 protein resolved as 32 kD and 12 kD species in HEK293 conditioned media; however, the three mutated proteins were detected only as the 32 kD band. An N-terminal FLAG-tagged native FGF-23 resolved as two bands of 36 kD and 26 kD when detected with a FLAG antibody, whereas the R176Q mutant resolved primarily as the 36 kD protein species. Cleavage of FGF-23 was not enhanced by extracellular incubation of FGF-23 with HEK293 cells. Native and mutant FGF-23s bound heparin.
  
  
  CONCLUSIONS
  FGF-23 proteins containing the ADHR mutations are secreted, and produce polypeptides less sensitive to protease cleavage than wild-type FGF-23. Therefore, the ADHR mutations may protect FGF-23 from proteolysis, thereby potentially elevating circulating concentrations of FGF-23 and leading to phosphate wasting in ADHR patients.
authors.affiliation: Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: K E
authors.initials: KE
authors.lastname: White
authors.forename: G
authors.initials: G
authors.lastname: Carn
authors.forename: B
authors.initials: B
authors.lastname: Lorenz-Depiereux
authors.forename: A
authors.initials: A
authors.lastname: Benet-Pages
authors.forename: T M
authors.initials: TM
authors.lastname: Strom
authors.forename: M J
authors.initials: MJ
authors.lastname: Econs
mesh_terms: D002460:Cell Line; D004355:Drug Stability; D005110:Extracellular Space / Q000378:metabolism; D000089703:Fibroblast Growth Factor-23; D005346:Fibroblast Growth Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005799:Genes, Dominant; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007015:Hypophosphatemia, Familial / Q000235:genetics*; D007425:Intracellular Membranes / Q000378:metabolism; D020125:Mutation, Missense / Q000502:physiology*
chemical_list: C000717427:FGF23 protein, human; D005346:Fibroblast Growth Factors; D000089703:Fibroblast Growth Factor-23; D006493:Heparin