title: Biochemical analysis of a dimerization domain mutation in RetGC-1 associated with dominant cone-rod dystrophy.
abstract: Mutations in the photoreceptor membrane guanylyl cyclase RetGC-1 have been linked to autosomal dominant cone-rod dystrophy. Three mutations were identified that alter strictly conserved residues within the RetGC-1 dimerization domain, a region predicted to form an amphipathic alpha-helical coil. Here we report on a biochemical characterization of one of the mutations, a substitution of cysteine for arginine at residue 838. We generated this mutation in vitro and measured its catalytic activity and sensitivity to guanylyl cyclase activating protein 1 (GCAP-1) and GCAP-2. The R838C substitution has several effects. It reduces the overall catalytic ability of RetGC-1 and dramatically reduces stimulation by GCAP-2, although GCAP-2 still appears to interact with the protein. The R838C substitution also increases the apparent affinity of RetGC-1 for GCAP-1 and alters the Ca(2+) sensitivity of the GCAP-1 response, allowing the mutant to be stimulated by GCAP-1 at higher Ca(2+) concentrations than wild type. The diminished response to GCAP-2, which we propose is not likely the cause of cone-rod degeneration in these patients, is interesting mechanistically because it separates the ability to bind a specific GCAP from the ability to be stimulated by it, and it also discriminates between the mechanisms of activation of GCAP-1 vs. GCAP-2. We suggest that the gain-of-function effects of R838C on RetGC-1 stimulated by GCAP-1, which are dominant in vitro and may cause an abnormal increase in cGMP synthesis in dark-adapted photoreceptors, may be the cause of the cone-rod degeneration.
authors.affiliation: Howard Hughes Medical Institute and Department of Biochemistry, Box 357370, University of Washington, Seattle, WA 98195, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Tucker
authors.forename: S C
authors.initials: SC
authors.lastname: Woodcock
authors.forename: R E
authors.initials: RE
authors.lastname: Kelsell
authors.forename: V
authors.initials: V
authors.lastname: Ramamurthy
authors.forename: D M
authors.initials: DM
authors.lastname: Hunt
authors.forename: J B
authors.initials: JB
authors.lastname: Hurley
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D019281:Dimerization; D006162:Guanylate Cyclase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051604:Guanylate Cyclase-Activating Proteins; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000378:metabolism; D012174:Retinitis Pigmentosa / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
chemical_list: D002135:Calcium-Binding Proteins; C496736:GUCA1A protein, human; C496739:GUCA1B protein, human; D051604:Guanylate Cyclase-Activating Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C110715:guanylate cyclase 1; D006162:Guanylate Cyclase; D002118:Calcium