Nickols Hilary Highfield, Shah Vikas N, Chazin Walter J, Limbird Lee E
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600, USA.
J Biol Chem. 2004 Nov 5;279(45):46969-80. doi: 10.1074/jbc.M407351200. Epub 2004 Aug 19.
To identify molecules that might contribute to V2 vasopressin receptor (V2R) trafficking or signaling, we searched for novel interacting proteins with this receptor. Preliminary data, using the V2R C terminus as bait in a yeast two-hybrid screen, revealed calmodulin as a binding partner. Because calmodulin interacts with other G protein-coupled receptors, we explored this interaction and its possible functional relevance in greater detail. A Ca2+ -dependent interaction occurs between calmodulin-linked agarose and the holo-V2R as well as the V2R C terminus. Truncation and site-directed mutagenesis of the V2R C terminus revealed an involvement of an RGR sequence in this interaction. NMR studies showed that a peptide fragment of the V2R C terminus containing the RGR sequence binds to calmodulin in a Ca2+ -dependent manner with a Kd < or =1.5 microm; concentration-dependent binding of the V2R C terminus to calmodulin-agarose was used to estimate a Kd value of approximately 200 nm for this entire C-terminal sequence as expressed in mammalian cells. Madin-Darby canine kidney II cells stably expressing either wild type or a mutant V2R, in which the RGR C-terminal sequence was mutated to alanines (AAA V2R), revealed that the steady-state localization and agonist-induced internalization of the AAA V2R resembled that of the wild type V2R in polarized Madin-Darby canine kidney II cells. V2R binding of agonist similarly was unchanged in the AAA V2R, as was the concentration response for arginine vasopressin (AVP)-stimulated cAMP accumulation. Most interestingly, AVP-induced increases in intracellular Ca2+ observed for the wild type V2R were virtually eliminated for the AAA V2R. Taken together, the data suggest that a C-terminal region of the V2R important for calmodulin interaction is also important in modulation of V2R elevation of intracellular Ca2+, a prerequisite for AVP-induced fusion of aquaporin-containing vesicles with the apical surface of renal epithelial cells.
为了鉴定可能参与血管升压素V2受体（V2R）转运或信号传导的分子，我们寻找了与该受体新的相互作用蛋白。利用V2R C末端作为酵母双杂交筛选中的诱饵，初步数据显示钙调蛋白是一种结合伴侣。由于钙调蛋白与其他G蛋白偶联受体相互作用，我们更详细地探究了这种相互作用及其可能存在功能相关性研究发现钙调蛋白连接的琼脂糖与完整的V2R以及V2R C末端之间存在Ca²⁺依赖性相互作用。V₂R C末端截短和定点诱变显示RGR序列参与了这种相互作用。核磁共振研究表明，V2R C末端含RGR序列的肽片段以Ca²⁺依赖性方式与钙调蛋白结合，解离常数Kd≤或 = 1.5 μmol；V2R C末端与钙调蛋白琼脂糖的浓度依赖性结合用于估计在哺乳动物细胞中表达的该完整C末端序列的Kd值约为200 nmol。稳定表达野生型或突变型V2R的Madin-Darby犬肾II细胞，其中RGR C末端序列突变为丙氨酸（AAA V2R），结果显示在极化的Madin-Darby犬肾II细胞中，AAA V2R的稳态定位和激动剂诱导的内化与野生型V2R相似。激动剂与AAA V2R的V2R结合同样未改变，精氨酸血管加压素（AVP）刺激的cAMP积累的浓度反应也是如此。最有趣的是，野生型V2R观察到的AVP诱导的细胞内Ca²⁺增加在AAA V2R中几乎完全消除。综上所述，数据表明V2R中对钙调蛋白相互作用重要的C末端区域在调节V2R升高细胞内Ca²⁺方面也很重要，这是AVP诱导含水通道蛋白的囊泡与肾上皮细胞顶端表面融合的前提条件。
