STRUCTURAL BASIS OF G-PROTEIN SPECIFICITY OF HUMAN ENDOTHELIN RECEPTORS - A STUDY WITH ENDOTHELIN(A/B) CHIMERAS
STRUCTURAL BASIS OF G-PROTEIN SPECIFICITY OF HUMAN ENDOTHELIN RECEPTORS - A STUDY WITH ENDOTHELIN(A/B) CHIMERAS
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DOI：
10.1074/jbc.270.17.10072
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发表时间：
1995-04-28
影响因子：
4.8
通讯作者：
MASAKI, T
中科院分区：
文献类型：
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作者：
TAKAGI, Y;NINOMIYA, H;MASAKI, T
内皮肽(ET)家族通过两种亚型的鸟核苷酸结合调节蛋白(G蛋白)偶联受体ET(A)和ET(B)发挥作用。ET-1在稳定表达人野生型ET(A)的中国仓鼠卵巢(CHO)细胞(CHO/HET(A)细胞)中刺激cAMP的形成，而在表达人野生型ET(B)的CHO细胞(CHO/HET(B)细胞)中抑制cAMP的形成，药理学证据表明，相反的作用是由于每个受体亚型与GαS/GαI选择性偶联。为了找到决定选择性偶联的受体结构域(S)，在CHO细胞上表达了一系列HET(A)和HET(B)之间的嵌合受体。检测ET-1对各细胞系cAMP形成的影响。HET(A)将细胞内第二和/或第三环(ICLII和/或-III)替换到HET(B)的相应区域(S)，但不能传递ET-1的刺激作用。HET(B)在HET(A)的相应区域替换ICLIII，不能传递ET-1的抑制作用。HET(B)的ICLII和HET(A)的ICLIII的嵌合受体不能传递这两种作用。在表达HET(A)的ICLII和HET(B)的ICLIII的嵌合受体的细胞中，经百日咳毒素处理后，ET-1抑制cAMP的形成，而刺激cAMP的形成。这些结果表明，HETR的ICLII和-III分别是HET(A)和HET(B)与GαS/GαI选择性偶联的主要决定因素。我们还证明，在同一细胞上表达的每个受体亚型都可以独立地发挥作用，即HET(A)激活GαS，HET(B)激活GαI，从而导致ET-1对cAMP形成的剂量依赖性双重作用。
The endothelin (ET) family of peptides acts via two subtypes of guanine nucleotide-binding regulatory protein (G protein) coupled receptors termed ET(A) and ET(B). ET-1 stimulated cAMP formation in Chinese hamster ovary (CHO) cells stably expressing human wild-type ET(A) (CHO/hET(A) cells) while it inhibited cAMP formation in CHO cells expressing human wild-type ET(B) (CHO/hET(B) cells), and pharmacological evidence indicated that the opposite effects were due to the selective coupling of each receptor subtype with G alpha s/G alpha i. To find out a receptor domain(s) that determined the selective coupling, a series of chimeric receptors between hET(A) and hET(B) was expressed on CHO cells, and the effect of ET-1 on cAMP formation in each cell line was tested. hET(A) with the replacement of second and/or third intracellular loop (ICLII and/or -III) to the corresponding region(s) of hET(B) failed to transmit the stimulatory effect of ET-1. hET(B) with the replacement of ICLIII to the corresponding region of hET(A) failed to transmit the inhibitory effect of ET-1. A chimeric receptor with ICLII of hET(B) and with ICLIII of hET(A) failed to transmit both effects. In cells expressing chimeric receptors with ICLII of hET(A) and with ICLIII of hET(B), ET-1 inhibited cAMP formation while it stimulated cAMP formation when cells were pretreated with pertussis toxin. These results indicated the roles of ICLII and -III of hETR as a major determinant of the selective coupling of hET(A) and hET(B) with G alpha s/G alpha i, respectively. We also demonstrated that each receptor subtype expressed on the same cell could work independently, i.e. for hET(A) to activate G alpha s and for hET(B) to activate G alpha i, resulting in dose dependent dual effects of ET-1 on cAMP formation.