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Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function.

ABSTRACT: R-spondins (Rspos) potentiate Wnt/beta-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein,demonstrating for the first time specific amino acid residues in Rspos that are critical for beta-catenin signaling. The diminished activity of Rspo2/C78Y and Rspo2/C113R was attributable to a defect in their secretion, while Rspo2/Q70R exhibited a decrease in its intrinsic activity. Cysteine assignments in a Rspo2 derivative containing only the two furin-like domains (Rspo2-2F) provided the first information about the disulfide bonding pattern of this motif, which was characterized by multiple short loops and unpaired cysteine residues, and established that the loss-of-function cysteine mutants disrupted disulfide bond formation. Moreover, Rspo2-2F demonstrated potent activity and synergized strongly with Wnt-3a in a beta-catenin reporter assay. In contrast, an Rspo2-2F derivative containing the Q70R substitution showed significantly reduced activity, although it still synergized with Wnt-3a in the reporter assay. Rspo2-2F derivatives elicited an unusually sustained phosphorylation (20 h) of the Wnt co-receptor, low density lipoprotein receptor-related protein 6 (LRP6), as well as an increase in cell surface LRP6. Co-immunoprecipitation experiments involving LRP6 and Kremens suggested that these associations contribute to Rspo2 activity, although the lack of major differences between wild-type and Q70R derivatives implied that additional interactions may be important.

SUBMITTER: Li SJ

PROVIDER: S-EPMC2813491 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function.

Li Sheng-Jian SJ Yen Ten-Yang TY Endo Yoshimi Y Klauzinska Malgorzata M Baljinnyam Bolormaa B Macher Bruce B Callahan Robert R Rubin Jeffrey S JS

Cellular signalling 20090601 6

R-spondins (Rspos) potentiate Wnt/beta-catenin signaling, an important pathway in embryonic development that is constitutively active in many cancers. To analyze Rspo structure and function, we expressed full-length wild-type Rspo2 and Rspo2 point mutants corresponding to Rspo4 variants that have been linked to developmental defects. The Rspo2 mutants had markedly reduced potency relative to the wild-type protein,demonstrating for the first time specific amino acid residues in Rspos that are cri ...[more]

PMID: 19385064

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Project description:Missense mutations in coding region of PDX1 predispose to type-2 diabetes mellitus as well as cause MODY through largely unexplored mechanisms. Here, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying heterozygous missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/-). Using an in vitro β-cell differentiation protocol, we demonstrated that both PDX1P33T/+, PDX1C18R/+ and PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/- and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NEURONATIN, both involved in insulin synthesis and secretion. Our results reveal mechanistic details of how diabetes-associated PDX1 point mutations impair human pancreatic endocrine lineage formation and β-cell function and contribute to pre-disposition for diabetes. Methods: In this study, we have generated a novel induced pluripotent stem cell (iPSC) line that efficiently differentiates into human pancreatic progenitors (PPs). Furthermore, PDX1 and H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq) was used to identify PDX1 transcriptional targets and active enhancer and promoter regions. To address potential differences in the function of PDX1 during development and adulthood, we compared PDX1 binding profiles from PPs and adult islets. Moreover, combining ChIP-seq and GWAS meta-analysis data we identified T2DM-associated SNPs in PDX1 binding sites and active chromatin regions.

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Dataset Information

Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function.

Publications

Loss-of-function point mutations and two-furin domain derivatives provide insights about R-spondin2 structure and function.

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