Abstract
Cysteine-rich transmembrane bone morphogenetic protein regulator 1 (CRIM1) is a type I transmembrane protein involved in the organogenesis of many tissues via its interactions with growth factors including BMP, TGF-β, and VEGF. In this study, we used whole-exome sequencing and linkage analysis to identify a novel Crim1 mutant allele generated by ENU mutagenesis in mice. This allele is a missense mutation that causes a cysteine-to-serine substitution at position 140, and is referred to as Crim1C140S. In addition to the previously reported phenotypes in Crim1 mutants, Crim1C140S homozygous mice exhibited several novel phenotypes, including dwarfism, enlarged seminal vesicles, and rectal prolapse. In vitro analyses showed that Crim1C140S mutation affected the formation of CRIM1 complexes and decreased the amount of the overexpressed CRIM1 proteins in the cell culture supernatants. Cys140 is located in the internal region 1 (IR1) of the N-terminal extracellular region of CRIM1 and resides outside any identified functional domains. Inference of the domain architecture suggested that the Crim1C140S mutation disturbs an intramolecular disulfide bond in IR1, leading to the protein instability and the functional defects of CRIM1. Crim1C140S highlights the functional importance of the IR1, and Crim1C140S mice should serve as a valuable model for investigating the functions of CRIM1 that are unidentified as yet.
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Acknowledgements
We thank Drs, J. Terrig Thomas (The US Food and Drug Administration), Chisa Shukunami (Hiroshima University), and Aki Takimoto (University of Tokyo) for providing plasmids. This project was supported by the NOVARTIS Foundation (Japan) for the Promotion of Science, the research grant of Iwate University (TF), and was partially supported by the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP19am0101110 (KT), KAKENHI No. 21240043 (RF, YG), 25241016 (YG), and 17H00789 (RF, YG).
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All experimental procedures were approved by the Animal Experimentation Committee (Approval No. A201247, A201509 and A201809) and the Gene Recombination Experiment Safety Committee (Approval No. 201513 and 201819) at Iwate University. All animal experiments were conducted in accordance with institutional procedures, national guidelines, and the relevant national laws on the protection of animals.
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Supplemental Fig. S1
Western blot analyses using the cell membrane proteins from COS-7 cells transfected with an expression vector encoding wild-type or C140S mutant CRIM1 proteins. We used vector constructs encoding full-length proteins. The intensities of the two CRIM1bands were almost the identical. Na+/K+ATPase was used as a membrane marker. (TIFF 12256 kb)
Supplemental Fig. S2
Multiple sequence alignment of internal region 1 (IR1) among various vertebrate species. Conserved Cys residues in IR1 are shaded in yellow. Conserved residue portions are denoted by asterisks. Thirty Cys residues are completely conserved. (TIFF 25728 kb)
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Furuichi, T., Tsukamoto, M., Saito, M. et al. Crim1C140S mutant mice reveal the importance of cysteine 140 in the internal region 1 of CRIM1 for its physiological functions. Mamm Genome 30, 329–338 (2019). https://doi.org/10.1007/s00335-019-09822-3
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DOI: https://doi.org/10.1007/s00335-019-09822-3