Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides -: Endogenous 5-HT2C receptors in choroid plexus epithelial cells
Dissecting G protein-coupled receptor signaling pathways with membrane-permeable blocking peptides -: Endogenous 5-HT2C receptors in choroid plexus epithelial cells
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DOI：
10.1074/jbc.275.10.7021
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发表时间：
2000-03-10
影响因子：
4.8
通讯作者：
Sanders-Bush, E
中科院分区：
文献类型：
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作者：
Chang, M;Zhang, LS;Sanders-Bush, E
为了确定脉络丛上皮细胞中内源性表达的5-HT 2C受体的细胞内信号传导机制，我们实施了靶向破坏蛋白质-蛋白质相互作用的策略。该策略需要递送缀合的膜渗透性肽，其在信号级联中的特定步骤破坏结构域相互作用。作为概念的证明，仅检查了针对受体-G蛋白相互作用结构域的两种肽。只有靶向受体-G（q）蛋白相互作用结构域的G（q）CT破坏5-HT 2C受体介导的磷脂酰肌醇水解。G（s）CT靶向受体-G（s）蛋白，破坏β 2肾上腺素能受体介导的cAMCP激活，但不破坏5-HT 2C受体介导的磷脂酰肌醇水解。肽MPS-PLC β 1 M模拟磷脂酶C β 1（PLC β 1）与活性G α（q）相互作用的结构域，也阻断5-HT 2C受体激活。相比之下，肽PLC β 2 M和Phos结合60和螯合游离G β γ亚基在阻断5-HT 2C受体介导的磷酸肌醇周转方面无效。然而，这两种肽都破坏了G β γ介导的促分裂原活化蛋白激酶的α（2A）肾上腺素能受体活化。这些结果首次直接证明了活性G α（q）亚基介导PLC β的内源性5-HT 2C受体活化，而G α（q）异源三聚体蛋白释放的G β γ亚基不参与其中。在星形胶质细胞中表达的代谢型谷氨酸受体5获得了类似的结果。因此，coqiugated，膜渗透肽是有效的工具，用于解剖细胞内信号。
To determine the intracellular signaling mechanism of the 5-HT2C receptor endogenously expressed in choroid plexus epithelial cells, we implemented a strategy of targeted disruption of protein-protein interactions. This strategy entails the delivery of conjugated membrane-permeable peptides that disrupt domain interaction at specific steps in the signaling cascade. As proof of concept, two peptides targeted against receptor-G protein interaction domains mere examined. Only G(q)CT, which targets the receptor-G(q) protein interacting domain, disrupted 5-HT2C receptor-mediated phosphatidylinositide hydrolysis. G(s)CT, targeting the receptor-G(s) protein, disrupted beta 2 adrenergic receptor-mediated activation of cAMCP but not 5-HT2C receptor-mediated phosphatidylinositide hydrolysis, The peptide MPS-PLC beta 1M, mimicking the domain of phospholipase C beta 1 (PLC beta 1) interacting with active G alpha(q), also blocked 5-HT2C receptor activation. In contrast, peptides PLC beta 2M and Phos that bind 60 and sequester free G beta gamma subunits were ineffective at blocking 5-HT2C receptor-mediated phosphoinositol turnover. However, both peptides disrupted G beta gamma-mediated alpha(2A) adrenergic receptor activation of mitogen-activated protein kinase, These results provide the first direct demonstration that active G alpha(q) subunits mediate endogenous 5-HT2C receptor activation of PLC beta and that G beta gamma subunits released from G alpha(q) heterotrimeric proteins are not involved. Comparable results were obtained with metabotropic glutamate receptor 5 expressed in astrocytes. Thus, coqiugated, membrane-permeable peptides are effective tools for the dissection of intracellular signals.