The endothelin subtype A receptor undergoes agonist- and antagonist-mediated internalization in the absence of signaling
The endothelin subtype A receptor undergoes agonist- and antagonist-mediated internalization in the absence of signaling
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DOI：
10.1210/en.139.7.3185
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发表时间：
1998-07-01
期刊：
影响因子：
4.8
通讯作者：
Puett, D
中科院分区：
文献类型：
--
作者：
Bhowmick, N;Narayan, P;Puett, D
平滑肌细胞的强效血管收缩剂和丝裂原内皮素-1（ET-1）通过两种不同的G蛋白偶联受体，亚型A （ETAR）和亚型B起作用，这两种受体主要偶联于G(q)-磷脂酶C信号通路。已知ET-1与ETAR结合促进内化，随后至少部分结合配体降解。为了研究内吞作用是否需要信号传导，我们建立了稳定转染的人胚胎肾293细胞系，表达野生型ETAR和受体嵌合体（ETARC），其中ETAR的c端细胞质尾部被另一种G蛋白偶联受体lutropin受体尾部取代，但该受体通过G(s)-腺苷酸环化酶途径发出信号。ETARC像ETAR一样结合ET-1，但缺乏信号传导。使用联合的豆豆蛋白a /蔗糖梯度离心技术将质膜与其他细胞膜分离，我们发现[I-125] ET-1在37℃（t(1/2)）下快速内化到表达etar的细胞中，内化= 5分钟；内吞速率常数= 0.1 min(-1)；表达etarc的细胞也会内化[I-125]ET-1，尽管速度比野生型受体慢一些(t（1/2）内化= 15分钟；内吞速率常数= 0.03 min(-1))。使用免疫荧光共聚焦显微镜和开发到ETAR n端区域的抗体，获得了定性相似的结果。此外，使用共聚焦显微镜发现，ETAR选择性拮抗剂BQ123也促进了表达ETAR的细胞的快速内化。这些结果表明，肌醇1,4,5-三磷酸信号不是配体介导的ETAR内化所必需的，并且表明受体构象改变是必要的。此外，BQ123促进ETAR内化的发现是新颖的，对其临床应用具有潜在的重要意义。
The potent vasoconstrictor and mitogen to smooth muscle cells, endothelin-1(ET-1), acts via two distinct G protein-coupled receptors, subtype A (ETAR) and subtype B, that are coupled primarily to the G(q)-phospholipase C signaling pathway. It is known that ET-1 binding to ETAR promotes internalization, with subsequent degradation of at least a portion of the bound ligand. To investigate whether signaling is required for endocytosis, we developed stably transfected lines of human embryonic kidney 293 cells expressing wild-type ETAR and a receptor chimera (ETARC) in which the C-terminal cytoplasmic tail to ETAR was replaced with that of the lutropin receptor, another G protein-coupled receptor, but one which signals through the G(s)-adenylyl cyclase pathway. ETARC binds ET-1 like ETAR, but is deficient in signaling. Using a combined concanavalin A/sucrose gradient centrifugation technique to separate plasma membranes from other cellular membranes, we found that [I-125] ET-1 is rapidly internalized into ETAR-expressing cells at 37 C (t(1/2) for internalization = 5 min; endocytic rate constant = 0.1 min(-1)); ETARC-expressing cells also internalize [I-125]ET-1, albeit at a somewhat slower rate than wild-type receptor (t(1/2) for internalization = 15 min; endocytic rate constant = 0.03 min(-1)). Using immunofluorescence confocal microscopy and an antibody developed to the N-terminal region of ETAR, qualitatively similar results were obtained. In addition, it was found using confocal microscopy that the ETAR-selective antagonist, BQ123, also promoted rapid internalization in cells expressing ETAR. These results establish that inositol 1,4,5-trisphosphate signaling is not required for ligand-mediated internalization of ETAR and suggest that a receptor conformational change is necessary. Moreover, the finding that BQ123 promotes ETAR internalization is novel and has potentially important implications in its clinical use.