Kjaer Svend, Ibáñez Carlos F
Division of Molecular Neurobiology, Department of Neuroscience, Karolinska Institute, 171 77 Stockholm, Sweden.
Hum Mol Genet. 2003 Sep 1;12(17):2133-44. doi: 10.1093/hmg/ddg227. Epub 2003 Jul 15.
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/GFRalpha1 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 degrees C, a condition known to facilitate protein folding. Several of the mutants produced at 30 degrees C regained their ability to bind to the GDNF/GFRalpha1 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 degrees C, identifying them as temperature-sensitive determinants in RETECD. Intriguingly, while production of human and mouse RETECD was suboptimal at 37 degrees C compared with 30 degrees C, expression of Xenopus RETECD was higher at 37 degrees 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.
RET功能丧失性突变会导致肠神经系统发育异常，这是一种被称为先天性巨结肠症的先天性疾病。RET细胞外结构域（RETECD）中的先天性巨结肠症突变会影响内质网（ER）中的加工过程，并阻止RET在细胞表面表达。我们研究了一系列影响RETECD不同生化特性的先天性巨结肠症突变的加工过程和功能。所有检测的突变都阻止了RETECD在ER中的成熟，并消除了其与GDNF/GFRα1配体复合物相互作用的能力，表明存在蛋白质折叠缺陷。在细胞内积累的未成熟形式的RETECD与ER伴侣蛋白Grp78/BiP相关，并表现出不同程度的蛋白质泛素化。通过在30℃下进行蛋白质表达，可以挽救RETECD突变体的成熟，30℃是已知有助于蛋白质折叠的条件。在30℃下产生的几个突变体恢复了与野生型相当的结合GDNF/GFRα1复合物的能力，表明这些突变影响RETECD的折叠但不影响其功能。对RETECD中自主折叠亚基的分析表明，在三个N端钙黏蛋白样结构域CLD1-3中存在内在的错误折叠倾向，这些结构域也集中了影响RETECD的大多数先天性巨结肠症突变。与此一致的是，这些亚结构域在30℃下的表达和成熟得到了特异性改善，将它们确定为RETECD中的温度敏感决定因素。有趣的是，虽然与30℃相比，人源和鼠源RETECD在37℃下的产生不理想，但非洲爪蟾RETECD在37℃下的表达更高，37℃对两栖动物来说是非生理温度。哺乳动物RETECD内在的错误折叠易感性可能是一种权衡的结果，这种权衡有助于避免肿瘤发病率的增加，但代价是对先天性巨结肠症的易感性增加。
