The study of signal transduction mechanism of angiotensin II receptor by mean of site-directed mutagenesis

定点突变研究血管紧张素II受体信号转导机制

基本信息

批准号：
06660107
负责人：
YAMANO Yoshiaki
金额：
$ 1.34万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06660107/
关键词：
Angiotensin II Receptor Site-directed mutagenesis cDNA 部位突然変異法

项目摘要

Ligand binding of rat angiotensin II receptor (AT1A)The molecular interaction involved in the ligand binding of rat angiotensin II receptor (AT1A) was studied by site-directed mutagenesis and receptor model building. Five conserved residues in AT1A,R167, K199, W253, F259 and D263 were substituted with alanine, individually. These amino acid residues appeared to provide binding sites for angiotensin II.Binding of a nonpeptide angiotensin II antagonist was also reduced for R167 mutant. R167 is concerned for receptor conformation.G-protein coupling of rat angiotensin II receptor (AT1A)G-protein coupling in carboxyl-terminal tail of rat angiotensin II receptor was studied by site-directed mutagenesis. Deletion mutants, which have stop codons at K310 (K310DELTA) or at K318 (K318DELTA), have angiotensin II binding affinity similar to wild type receptor. In case of K318DELTA, both the effects of GTPgammaS and the IP3 production was retained in response to angiotensin II,but K310DELTA had neither of the effects. It is concluded that G-protein couples to amino acid residues between K310 and K318.Expression of the chicken angiotensin II receptor in adrenalAves have a unique angiotensin II receptor, whose character is different from AT1 and AT2. The probe used for the hybridization to determine the expression of the receptor was synthesized using PCR.The receptor was expressed in adrenal but not in liver.

大鼠血管紧张素II受体（AT1A）的配体结合通过位置定向的诱变和受体模型构建研究了大鼠血管紧张素II受体（AT1A）配体结合（AT1A）的分子相互作用。单独将AT1A，R167，K199，W253，F259和D263的五个保守残留物分别用丙氨酸代替。这些氨基酸残基似乎为血管紧张素II提供了结合位点。对于R167突变体而言，非肽血管紧张素II拮抗剂的结合也减少了。 R167涉及受体构象。通过定位定向的诱变研究了大鼠血管紧张素II受体（AT1A）G蛋白II受体中G蛋白偶联的G蛋白偶联。删除突变体在K310（K310delta）或K318（K318DELTA）中具有类似于野生型受体的血管紧张素II结合亲和力。在K318delta的情况下，GTPGAMMAS和IP3产生的作用都是响应血管紧张素II的响应，但K310delta都没有效果。得出的结论是，G蛋白夫妇伴有K310和K318之间的氨基酸残基。肾上腺中鸡血管紧张素II受体的表达具有独特的血管紧张素II受体，其特征与AT1和AT2不同。使用PCR合成用于杂交来确定受体表达的探针。受体在肾上腺中表达，但在肝脏中不表达。