title: Functional analyses of mutant recessive GUCY2D alleles identified in Leber congenital amaurosis patients: protein domain comparisons and dominant negative effects.
abstract: OBJECTIVE
  Recessive mutations in GUCY2D, the gene encoding the retinal guanylyl cyclase protein, RetGC-1, have been shown to cause Leber Congenital Amaurosis (LCA), a severe retinal dystrophy. The purpose of this study was to determine the functional consequences of selected mutations in GUCY2Dlinked to LCA. The mutations investigated in this study map to the catalytic domain (P858S, L954P) and the extracellular domain (C105Y, L325P) of RetGC-1.
  
  
  METHODS
  All four mutations were introduced into the in vitro expression plasmid, pRC-CMV human RetGC-1, and expressed in HEK-293 cells. We assayed the abilities of the mutant cyclases to generate cGMP (basal activity), and to be activated by guanylyl cyclase activating proteins (GCAP-1 and GCAP-2). Additionally, we co-expressed the catalytic domain mutations (P858S and L954P) with a wild-type allele to test for dominant negative effects on wild-type RetGC-1.
  
  
  RESULTS
  The P858S and L954P mutations, both in highly conserved residues of the catalytic domain of RetGC-1, severely impair basal, GCAP-1, and GCAP-2 stimulated catalytic activity of the enzyme. In addition, when co-expressed with the wild-type allele, both catalytic domain mutations act as dominant negative proteins and reduce the activity of wild-type RetGC-1. The basal activities of the C105Y and L325P mutants are unaltered, but GCAP-1 and GCAP-2 stimulated cyclase activities are reduced approximately 50%.
  
  
  CONCLUSIONS
  GUCY2D mutations from LCA patients have distinct functional consequences on RetGC-1 catalytic activity in vitro. Our analyses showed that the catalytic domain mutations cause a marked reduction in cyclase activity, while the extracellular domain mutations moderately reduce activity. The catalytic domain mutant alleles cause dominant negative effects, indicating that the functionality of RetGC-1 is compromised even in heterozygotes. This is consistent with abnormalities in cone electroretinograms (ERGs) detected in obligate heterozygous GUCY2D parents that carry the L954P mutation.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA, USA.
authors.forename: Chandra L
authors.initials: CL
authors.lastname: Tucker
authors.forename: Visvanathan
authors.initials: V
authors.lastname: Ramamurthy
authors.forename: Ana-Luisa
authors.initials: AL
authors.lastname: Pina
authors.forename: Magali
authors.initials: M
authors.lastname: Loyer
authors.forename: Sharola
authors.initials: S
authors.lastname: Dharmaraj
authors.forename: Yingying
authors.initials: Y
authors.lastname: Li
authors.forename: Irene H
authors.initials: IH
authors.lastname: Maumenee
authors.forename: James B
authors.initials: JB
authors.lastname: Hurley
authors.forename: Robert K
authors.initials: RK
authors.lastname: Koenekoop
mesh_terms: D000483:Alleles; D001766:Blindness / Q000151:congenital*; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D005799:Genes, Dominant; D005808:Genes, Recessive / Q000502:physiology*; D006162:Guanylate Cyclase / Q000235:genetics* / Q000378:metabolism*; D051604:Guanylate Cyclase-Activating Proteins; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000201:enzymology; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D018000:Receptors, Peptide / Q000235:genetics*; D012162:Retinal Degeneration / Q000235:genetics*; D014162:Transfection
chemical_list: D002135:Calcium-Binding Proteins; C496736:GUCA1A protein, human; C496739:GUCA1B protein, human; D051604:Guanylate Cyclase-Activating Proteins; D018000:Receptors, Peptide; D006162:Guanylate Cyclase; D000074261:Receptors, Enterotoxin; D050705:Receptors, Guanylate Cyclase-Coupled; D006152:Cyclic GMP