A new approach for studying GPCR dimers: drug-induced inactivation and reactivation to reveal GPCR dimer function in vitro, in primary culture, and in vivo.
A new approach for studying GPCR dimers: drug-induced inactivation and reactivation to reveal GPCR dimer function in vitro, in primary culture, and in vivo.
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DOI：
10.1016/j.pharmthera.2011.10.007
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发表时间：
2012-02
影响因子：
13.5
通讯作者：
Klein, Michael T.
中科院分区：
文献类型：
--
作者：
Teitler, Milt;Klein, Michael T.
GPCR是同源蛋白的主要家族，并且是许多内源性神经递质、激素、细胞因子、治疗药物和滥用药物的作用的关键介质。尽管对GPCR的药理学和生物化学性质进行了大量的研究，但关于它们是否以单体、二聚体或更高级结构存在于体内的问题尚未得到回答。GPCR二聚体领域一直由涉及表达突变受体的重组细胞系的技术主导，通常涉及受体的溶解。这些技术不能应用于体内，甚至不能应用于原代细胞培养。本文将重点介绍一种新的方法来探索同源二聚体的功能特性。对5-HT 7和5-HT 2A血清素受体的研究表明，假不可逆拮抗剂（“灭活剂”）与同二聚体的正构位点之一的结合消除了所有受体活性，随后竞争性拮抗剂与第二原聚体的正构位点的结合释放灭活剂，使受体恢复到活性状态。这种方法证明了天然GPCR同源二聚体的原聚体之间的变构串扰，表明GPCR在重组细胞和大鼠原代星形胶质细胞中确实存在并作为同源二聚体起作用。该技术可以普遍应用于使用完整的重组或原代细胞培养，膜匀浆制备，并可能在体内。使用5-HT 7和5-HT 2A受体获得的数据强烈支持GPCR同二聚体结构，几乎没有证据表明单体参与这些受体的功能。
GPCRs are a major family of homologous proteins and are key mediators of the effects of numerous endogenous neurotransmitters, hormones, cytokines, therapeutic drugs, and drugs-of-abuse. Despite the enormous amount of research on the pharmacological and biochemical properties of GPCRs, the question as to whether they exist as monomers, dimers, or higher order structures in the body is unanswered. The GPCR dimer field has been dominated by techniques involving recombinant cell lines expressing mutant receptors, often involving the solubilization of the receptors. These techniques cannot be applied in vivo or even to primary cell cultures. This review will focus on a novel approach to exploring the functional properties of homodimers. Studies of the 5-HT7 and 5-HT2A serotonin receptors have revealed that binding of a pseudo-irreversible antagonist (“inactivator”) to one of the orthosteric sites of a homodimer abolishes all receptor activity, and subsequent binding of a competitive antagonist to the orthosteric site of the second protomer releases the inactivator, allowing the receptor to return to an active state. This approach demonstrates allosteric crosstalk between protomers of native GPCR homodimers, indicating that GPCRs do exist and function as homodimers in both recombinant cells and rat primary astrocytes. This technique can be applied universally using intact recombinant or primary cells in culture, membrane homogenate preparations and, potentially, in vivo. The data obtained using the 5-HT7 and 5-HT2A receptors are strongly supportive of a GPCR homodimer structure, with little evidence of monomer involvement in the function of these receptors.
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影响因子：
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作者：
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通讯作者：
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DOI：
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发表时间：
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影响因子：
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作者：
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DOI：
10.1124/jpet.110.172098
发表时间：
2010-12-01
影响因子：
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作者：
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通讯作者：
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