Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to Gs and Gi
Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to Gs and Gi
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DOI：
10.1111/j.1476-5381.2010.01006.x
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发表时间：
2010-12-01
影响因子：
7.3
通讯作者：
Zhou, N. M.
中科院分区：
文献类型：
--
作者：
Chen, X. P.;Yang, W.;Zhou, N. M.
大麻素CB 1受体主要被认为在功能上与G蛋白的G（i）形式偶联，通过它负调节cAMP积累。本文研究了CB 1受体的双重偶联特性，并对介导与G（s）和G（i）选择性偶联的结构决定簇进行了表征。CB 1/CB 2受体嵌合体和定点突变结合功能分析和计算机模拟来确定介导G（s）和G（i）选择性偶联的结构决定因素。关键词TSCB 1受体可以偶联G（s）介导的cAMP积累和G（i）诱导的ERK 1/2激活和Ca 2+动员，而CB 2受体选择性偶联G（i）并抑制cAMP产生。使用CB 1/CB 2嵌合受体，CB 1受体的第二胞内环（ICL 2）被鉴定为主要负责介导G（s）和G（i）偶联特异性。此外，ICL 2中Leu-222突变为Ala或Pro可将G蛋白偶联从G（s）转换为G（i），而突变为Ile或瓦尔可导致突变受体与G（s）和G（i）的平衡偶联。在G（s）和G（i）蛋白偶联和特异性中起关键作用。我们的研究为CB 1受体与G蛋白偶联和大麻素诱导的耐受性的机制提供了新的见解。
BACKGROUND AND PURPOSEThe cannabinoid CB1 receptor is primarily thought to be functionally coupled to the G(i) form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB1 receptors and characterized the structural determinants that mediate selective coupling to G(s) and G(i).EXPERIMENTAL APPROACHA cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB1/CB2 receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to G(s) and G(i).KEY RESULTSCB1 receptors could couple to both G(s)-mediated cAMP accumulation and G(i)-induced activation of ERK1/2 and Ca2+ mobilization, whereas CB2 receptors selectively coupled to G(i) and inhibited cAMP production. Using CB1/CB2 chimeric receptors, the second intracellular loop (ICL2) of the CB1 receptor was identified as primarily responsible for mediating G(s) and G(i) coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from G(s) to G(i), while to Ile or Val led to balanced coupling of the mutant receptor with G(s) and G(i).CONCLUSIONS AND IMPLICATIONSThe ICL2 of CB1 receptors and in particular Leu-222, which resides within a highly conserved DRY(X)(5)PL motif, played a critical role in G(s) and G(i) protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB1 receptors to G proteins and cannabinoid-induced tolerance.