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Human Molecular Genetics Advance Access originally published online on July 15, 2003
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Human Molecular Genetics, 2003, Vol. 12, No. 17 2133-2144
DOI: 10.1093/hmg/ddg227
© 2003 Oxford University Press

Intrinsic susceptibility to misfolding of a hot-spot for Hirschsprung disease mutations in the ectodomain of RET

Svend Kjær and Carlos F. Ibáñez*

Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden

Received April 16, 2003; Accepted July 5, 2003

Loss-of-function mutations in RET cause abnormal development of the enteric nervous system, a congenital condition known as Hirschsprung disease. Hirschsprung mutations in the extracellular domain of RET (RETECD) affect processing in the endoplasmic reticulum (ER) and prevent RET expression at the cell surface. We have investigated the processing and function of a series of Hirschsprung disease mutations affecting different biochemical properties of the RETECD. All mutations examined prevented the maturation of RETECD in the ER and abolished its ability to interact with the GDNF/GFR{alpha}1 ligand complex, indicating defects in protein folding. Immature forms of RETECD accumulating intracellularly associated with the ER chaperone Grp78/BiP and showed different degrees of protein ubiquitination. Maturation of RETECD mutants, including those deficient in Ca2+ binding and disulfide bridge formation, could be rescued by allowing protein expression to proceed at 30°C, a condition known to facilitate protein folding. Several of the mutants produced at 30°C regained their ability to bind to the GDNF/GFR{alpha}1 complex comparable to wild-type, demonstrating that the mutations affected RETECD folding but not function. Analysis of autonomous folding subunits in the RETECD indicated an intrinsic propensity to misfolding in three N-terminal cadherin-like domains, CLD1–3, which also concentrate the majority of Hirschsprung mutations affecting the RETECD. In agreement with this, expression and maturation of these subdomains was specifically improved at 30°C, identifying them as temperature-sensitive determinants in RETECD. Intriguingly, while production of human and mouse RETECD was suboptimal at 37°C compared with 30°C, expression of Xenopus RETECD was higher at 37°C, a non-physiological temperature for amphibians. The intrinsic susceptibility to misfolding of mammalian RETECD may be the result of a trade-off that helps to avoid an increased incidence of tumors, at the expense of a greater vulnerability to Hirschsprung disease.

* To whom correspondence should be addressed. Email: carlos.ibanez{at}neuro.ki.se


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