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Biochemical analysis of phenotypic diversity associated with mutations in codon 244 of the retinal degeneration slow gene.

ABSTRACT: Mutations in the protein product of the retinal degeneration slow (RDS) gene cause both rod-dominant retinitis pigmentosa and different forms of cone-dominant macular dystrophies. In particular, mutations in codon 244 can cause either of these types of disease. In this study, we examine the biochemical effects of N244H and N244K in an effort to understand the mechanism underlying rod- and cone-dominant defects, respectively. COS-1 cells were cotransfected with either wild-type (WT) RDS or RDS containing an N244H or N244K mutation along with its binding partner, ROM-1 (rod outer segment membrane protein 1). Cell extracts were analyzed for mutant protein stability by Western blot, and localization was examined by immunocytochemistry. Interactions between transfected proteins were assessed by reciprocal co-immunoprecipitation, and nonreducing velocity sedimentation was used to identify the pattern of RDS complex assembly. Interactions were confirmed using GST fusion constructs of WT and mutant RDS in GST pull-down assays from WT mouse retinal extract. In COS-1 cells, recombinant N244H RDS had a weakened ability to assemble into higher-order complexes but retained the ability to co-immunoprecipitate with ROM-1 as well as localize properly throughout the cells. In contrast, recombinant N244K protein did not associate with ROM-1, showed signs of protein aggregation, and colocalized with an ER marker. These experiments support the hypothesis that RDS mutations that interrupt higher-order oligomer formation but still interact with ROM-1 and fold properly in membranes may cause dominant, gain-of-function disease phenotypes while mutations that cause RDS misfolding (and thus incorrect trafficking and assembly) may be associated with a loss-of-function haploinsufficiency phenotype.

SUBMITTER: Conley SM

PROVIDER: S-EPMC2937569 | biostudies-other | 2010 Feb

REPOSITORIES: biostudies-other

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Biochemical analysis of phenotypic diversity associated with mutations in codon 244 of the retinal degeneration slow gene.

Conley Shannon M SM Stricker Heidi M HM Naash Muna I MI

Biochemistry 20100201 5

Mutations in the protein product of the retinal degeneration slow (RDS) gene cause both rod-dominant retinitis pigmentosa and different forms of cone-dominant macular dystrophies. In particular, mutations in codon 244 can cause either of these types of disease. In this study, we examine the biochemical effects of N244H and N244K in an effort to understand the mechanism underlying rod- and cone-dominant defects, respectively. COS-1 cells were cotransfected with either wild-type (WT) RDS or RDS co ...[more]

PMID: 20055437

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Project description:Inherited photoreceptor degenerations (IPDs) are the most genetically heterogeneous of Mendelian diseases. Many IPDs exhibit substantial phenotypic variability, but the basis is usually unknown. Mutations in MERTK cause recessive IPD phenotypes associated with the RP38 locus. We have identified a murine genetic modifier of Mertk-associated photoreceptor degeneration, the C57BL/6 (B6) allele of which acts as a suppressor. Photoreceptors degenerate rapidly in Mertk-deficient animals homozygous for the 129P2/Ola (129) modifier allele, whereas animals heterozygous for B6 and 129 modifier alleles exhibit an unusual intermixing of degenerating and preserved retinal regions, with females more severely affected than males. Mertk-deficient mice homozygous for the B6 modifier allele display degeneration only in the far periphery, even at 8 months of age, and have improved retinal function compared to animals homozygous for the 129 allele. We genetically mapped the modifier to an approximately 2-megabase critical interval that includes Tyro3, a paralog of Mertk. Tyro3 expression in the outer retina varies with modifier genotype in a manner characteristic of a cis-acting expression quantitative trait locus (eQTL), with the B6 allele conferring an approximately three-fold higher expression level. Loss of Tyro3 function accelerates the pace of photoreceptor degeneration in Mertk knockout mice, and TYRO3 protein is more abundant in the retinal pigment epithelium (RPE) adjacent to preserved central retinal regions of Mertk knockout mice homozygous for the B6 modifier allele. Endogenous human TYRO3 protein co-localizes with nascent photoreceptor outer segment (POS) phagosomes in a primary RPE cell culture assay, and expression of murine Tyro3 in cultured cells stimulates phagocytic ingestion of POS. Our findings demonstrate that Tyro3 gene dosage modulates Mertk-associated retinal degeneration, provide strong evidence for a direct role for TYRO3 in RPE phagocytosis, and suggest that an eQTL can modify a recessive IPD.

Dataset Information

Biochemical analysis of phenotypic diversity associated with mutations in codon 244 of the retinal degeneration slow gene.

Publications

Biochemical analysis of phenotypic diversity associated with mutations in codon 244 of the retinal degeneration slow gene.

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