Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis.
Identification of amino acid residues of rat angiotensin II receptor for ligand binding by site directed mutagenesis.
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通过定点诱变鉴定大鼠血管紧张素 II 受体配体结合的氨基酸残基。
DOI：
10.1016/0006-291x(92)90461-s
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发表时间：
1992
影响因子：
3.1
通讯作者：
Inagami,T
中科院分区：
文献类型：
--
作者：
Yamano,Y;Ohyama,K;Chaki,S;Guo,DF;Inagami,T
为了确定配体与血管紧张素 (Ang II) 受体 AT1 结合的具体机制，在 COS-7 细胞中瞬时表达诱变的大鼠受体 cDNA，并通过斯卡查德图分析突变对与肽和非肽配体结合的影响。膜内结构域中 Lys199 突变为 Gln 强烈降低了与 [125I] Ang II 和 [125I]-1Sar,8Ile-Ang II 的亲和力，而其他两个 Lys 的突变几乎没有影响，表明 Lys199in 结合配体的参与。胞外域中四个半胱氨酸的每一个的替换都显着降低了结合亲和力，表明两个假定的二硫桥在活性受体构象形成中的重要性。 N-糖基化中用 Asp 替代 Asn 对配体结合或受体表达没有影响。这些研究表明突变的受体在质膜中表达，并且适合进一步的详细研究。
To determine the specific mechanism of ligand binding to angiotensin (Ang II) receptor AT1, mutagenized rat receptor cDNAs were expressed transiently in COS-7 cells and the effect of the mutations on the binding to peptidic and non-peptidic ligands was analyzed by Scatchard plots. Mutation of Lys199to Gln in the intramembrane domain strongly reduced the affinity to both [125I] Ang II and [125I]-1Sar,8Ile-Ang II whereas mutation of two other Lys had little effect, indicating involvement of Lys199in binding ligands. Replacement of each of four Cys in the extracellular domain markedly reduced binding affinity, indicating the importance of two putative disulfide bridges in the formation of active receptor conformation. Substitution of Asp for Asn in N-glycosylation had no effect on ligand binding or expression of the receptor. These studies indicate mutated receptors are expressed in the plasma membrane and are amenable for further detailed studies.