Molecular-mechanism for nephrogenic diabetes insipidus

肾源性尿崩症（NDI）最常见的是一种X连锁疾病，其中尿液不浓缩，由于肾脏抵抗精氨酸加压素。我们最近在不相关的X连锁NDI家族中发现了4个加压素2型受体基因突变，包括R143 P、DELTA V278、R202 C和8 O 4 insG，所有这些突变都使配体结合活性降低到正常的10%以下，而不影响mRNA的积累。为了阐明受体是否在细胞表面上表达，我们使用两种针对不同末端的抗体分析了在中国仓鼠卵巢（CHO）细胞中稳定表达的标记或未标记受体的生物合成和定位。全细胞和表面标记研究表明，R202 C克隆具有表面定位和细胞内蛋白，类似于野生型AVPR 2克隆，而R143 P和DELTA V278克隆缺乏表面受体，尽管相对增加的细胞内成分。8 O 4 insG突变体细胞不产生蛋白质，尽管足够的mRNA水平。免疫荧光染色证实R202 C突变体到达细胞表面，而R143 P和AV 278保留在细胞质区室内。因此，R202 C、R143 P/DELTA V278和8 O 4 insG分别导致三种不同的表型，即细胞表面的简单结合损伤、细胞内转运受阻和受体的无效生物合成或/和加速降解，因此是NDI的原因。

Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type2 receptor gene mutations in unrelated X-linked NDI families, including R143P, DELTA V278, R202C, and 8O4insG.All these mutations reduced ligand binding activity to <10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized and intracellular proteins, similar to the wild-type AVPR2 clone, whereas the R143P and DELTA V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 8O4insG mutant cell produced no proteins despite adequate mRNA level. Immunofluorescence staining confirmed that R202C mutants reached cell surface, whereas the R143P and A V278 are retained within the cytoplasmic compartment. Thus, R202C, R143P/DELTA V278, and 8O4insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI.