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

大鼠血管紧张素II受体(AT1a)的配基结合用定点突变和受体模型建立的方法研究了大鼠血管紧张素II受体(AT1a)与配基结合的分子相互作用。AT1A、R167、K199、W253、F259和D263的5个保守残基分别被丙氨酸取代。这些氨基酸残基似乎为血管紧张素II提供了结合部位。对于R167突变体，非肽血管紧张素II拮抗剂的结合也减少了。R167与受体构象有关。用定点突变的方法研究了大鼠血管紧张素II受体(AT1A)羧基末端尾部的G蛋白偶联。K310(K310DELTA)或K318(K318DELTA)终止密码子的缺失突变体具有与野生型受体相似的血管紧张素II结合亲和力。K318DELTA对血管紧张素Ⅱ的反应保留了GTP-GammaS的作用和IP3的产生，而K310DELTA则没有这两种作用。结论：鸡血管紧张素II受体在肾上腺Aves中的表达与K310和K318之间的氨基酸残基偶联，具有不同于AT1和AT2的特性。用于杂交检测受体表达的探针是用PCR合成的，受体在肾上腺表达，而在肝脏不表达。

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.