Biochemical basis of partial nephrogenic diabetes insipidus phenotypes.
Biochemical basis of partial nephrogenic diabetes insipidus phenotypes.
复制标题
DOI：
10.1210/mend.11.12.0017
复制
发表时间：
1997-11
影响因子：
--
通讯作者：
Heydar Sadeghi;Gary L. Robertson;D. Bichet;Giulio Innamorati;Mariel Birnbaumer
中科院分区：
文献类型：
--
作者：
Heydar Sadeghi;Gary L. Robertson;D. Bichet;Giulio Innamorati;Mariel Birnbaumer
在瞬时转染的 HEK 293 细胞中研究了导致肾性尿崩症部分表型的突变 2 型加压素受体 (V2R) 的生化特性。通过饱和结合测定确定，用 Asn 替代第二跨膜区中的 Asp85 不会改变 V2R 的细胞表面表达。尽管突变体 V2R 对精氨酸加压素 (AVP) 的亲和力仅降低了 6 倍，但腺苷酸环化酶活性对 AVP 的响应显示 EC50 右移 50 倍，并且突变体 V2R 的最大响应降低。这些数据表明，用 Asn 替代 Asp85 会影响受体与 Gs 的偶联，该受体的计算偶联效率降低 20 倍证实了这一结论。第二个细胞外环中的 Gly201Asp 突变也与 NDI 部分表型相关，降低了 V2R 的细胞表面表达，配体结合亲和力和与 Gs 的偶联效率略有降低。对于该突变体 V2R，AVP 和 V2 加压素受体激动剂脱氨基[Cys1,D-Arg8]-加压素促进的腺苷酸环化酶活性刺激之间观察到显着差异，表明 Gly201 参与受体对不同配体的选择性。这些数据表明，虽然配体结合亲和力降低和与 Gs 的偶联减少是 Asp85Asn 突变体 V2R 中配体反应减弱的原因，但 V2R 的细胞表面表达是减少细胞对 Gly201Asp 突变体 V2R 配体反应的主要因素。
Biochemical properties of mutant type 2 vasopressin receptors (V2Rs) causing a partial phenotype of nephrogenic diabetes insipidus were investigated in transiently transfected HEK 293 cells. Cell surface expression of the V2R was not altered by substituting Asp85 in the second transmembrane region by Asn as determined by saturation binding assays. Although the affinity of the mutant V2R for arginine vasopressin (AVP) was reduced only 6-fold, the response of adenylyl cyclase activity to AVP revealed a 50-fold right shift in EC50 and a decreased maximum response for the mutant V2R. These data indicated that replacement of Asp85 by Asn affected coupling of the receptor to Gs, a conclusion substantiated by a 20-fold decrease in the calculated coupling efficiency of this receptor. The Gly201Asp mutation in the second extracellular loop, also found associated with an NDI partial phenotype, decreased cell surface expression of the V2R with minor reduction in ligand-binding affinity and coupling efficiency to Gs. A pronounced difference was observed for this mutant V2R between the stimulation of adenylyl cyclase activity promoted by AVP and the V2 vasopressin receptor agonist deamino[Cys1,D-Arg8]-vasopressin, suggesting an involvement of Gly201 in the selectivity of the receptor for different ligands. These data demonstrated that while decreased ligand-binding affinity and decreased coupling to Gs are responsible for the attenuation of response to ligand in the Asp85Asn mutant V2R, cell surface expression of the V2R is the major factor reducing cellular responses to ligand for the Gly201Asp mutant V2R.