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Mammal-restricted elements predispose human RET to folding impairment by HSCR mutations

Nature Structural & Molecular Biology volume 17, pages 726–731 (2010)Cite this article

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Abstract

The maturation of human RET is adversely affected by a range of missense mutations found in patients with Hirschsprung's disease (HSCR), a complex multigenic disease. Here we show that two N-terminal cadherin-like domains, CLD1 and CLD2 (CLD(1-2)), from human RET adopt a clam-shell arrangement distinct from that of classical cadherins. CLD1 structural elements and disulfide composition are unique to mammals, indicating an unexpected structural diversity within higher and lower vertebrate RET CLD regions. We identify two unpaired cysteines that predispose human RET to maturation impediments in the endoplasmic reticulum and establish a quantitative cell-based RET maturation assay that offers a biochemical correlate of HSCR disease severity. Our findings provide a key conceptual framework and means of testing and predicting genotype-phenotype correlations in HSCR.

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Figure 1: A clam-shell RET CLD(1-2) arrangement reveals mammalian clade-specific structural elements.
Figure 2: Mammalian RET-specific elements define a CLD1-mediated dimer interface.
Figure 3: Segregation of kinetic and thermodynamically compromised HSCR RET mutations by quantitative cell-based assays.
Figure 4: Discrete classes of HSCR mutations and their disease phenotypes.

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Acknowledgements

We thank C.F. Ibáñez (Karolinska Institute) for encouragement, support and the RET51 cDNA, A. Purkiss-Trew for help with phasing, S. Mouilleron for help with data collection, J. Murray-Rust for help with RET modeling, M. Santoro (Univ. of Naples) for the kind gift of mAb 1D9, J. Garcia-Fernandez (Univ. of Barcelona) for the unpublished amphioxus RET sequence, the LRI Peptide Synthesis Laboratory for the disulfide-linked xRET(1-2) peptide, the European Synchrotron Radiation Facility beamline staff on ID29 and Amgen for supplies of recombinant GDNF, in particular J. Treanor. This work was supported by funding from Cancer Research UK, the EU FP6 # LSHB-CT-2004-503467 Prokinase Network and a Marie Curie Intra-European Fellowship (#010656) to S.K.

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Authors and Affiliations

  1. Structural Biology Laboratory, the London Research Institute, Cancer Research UK, London, UK

    Svend Kjær & Neil Q McDonald

  2. Protein Analysis Laboratory, the London Research Institute, Cancer Research UK, London, UK

    Sarah Hanrahan & Nick Totty

  3. School of Crystallography, Birkbeck College, London, UK

    Neil Q McDonald

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  1. Svend Kjær
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  2. Sarah Hanrahan
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Contributions

S.K. performed the experiments, with the exception of the MS analysis, which was performed by S.H. and N.T.; N.Q.M. and S.K. designed the study, interpreted the results and wrote the paper.

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Correspondence to Neil Q McDonald.

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Supplementary Figures 1–7, Supplementary Discussion and Supplementary Methods (PDF 1675 kb)

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Kjær, S., Hanrahan, S., Totty, N. et al. Mammal-restricted elements predispose human RET to folding impairment by HSCR mutations. Nat Struct Mol Biol 17, 726–731 (2010). https://doi.org/10.1038/nsmb.1808

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