Department of Biomedical Sciences, University of Teramo, Teramo, Italy European Center for Brain Research (CERC)/Santa Lucia Foundation I.R.C.C.S., Rome, Italy.
Br J Pharmacol. 2012 Apr;165(8):2635-51. doi: 10.1111/j.1476-5381.2011.01658.x.
The CB(1) cannabinoid receptor is regulated by its association with membrane microdomains such as lipid rafts. Here, we investigated the role of palmitoylation of the CB(1) receptor by analysing the functional consequences of site-specific mutation of Cys(415) , the likely site of palmitoylation at the end of helix 8, in terms of membrane association, raft targeting and signalling.
The palmitoylation state of CB(1) receptors in rat forebrain was assessed by depalmitoylation/repalmitoylation experiments. Cys(415) was replaced with alanine by site-directed mutagenesis. Green fluorescence protein chimeras of both wild-type and mutant receptors were transiently expressed and functionally characterized in SH-SY5Y cells and HEK-293 cells by means of confocal microscopy, cytofluorimetry and competitive binding assays. Confocal fluorescence recovery after photobleaching was used to assess receptor membrane dynamics, whereas signalling activity was assessed by [(35) S]GTPγS, cAMP and co-immunoprecipitation assays.
Endogenous CB(1) receptors in rat brain were palmitoylated. Mutation of Cys(415) prevented the palmitoylation of the receptor in transfected cells and reduced its recruitment to plasma membrane and lipid rafts; it also increased protein diffusional mobility. The same mutation markedly reduced the functional coupling of CB(1) receptors with G-proteins and adenylyl cyclase, whereas depalmitoylation abolished receptor association with a specific subset of G-proteins.
CB(1) receptors were post-translationally modified by palmitoylation. Mutation of Cys(415) provides a receptor that is functionally impaired in terms of membrane targeting and signalling.
This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.
大麻素 CB1 受体通过与细胞膜微区如脂筏的结合而受到调节。在此，我们通过分析 CB1 受体第 8 螺旋末端可能的棕榈酰化位点 Cys415 的定点突变，研究了该受体棕榈酰化对膜结合、筏定位和信号转导的功能影响。
通过去棕榈酰化/再棕榈酰化实验评估大鼠前脑 CB1 受体的棕榈酰化状态。通过定点突变将 Cys415 替换为丙氨酸。通过共聚焦显微镜、细胞荧光术和竞争结合实验，在 SH-SY5Y 细胞和 HEK-293 细胞中瞬时表达野生型和突变型受体的绿色荧光蛋白嵌合体，并进行功能表征。通过荧光漂白后荧光恢复实验评估受体膜动力学，通过 [(35) S]GTPγS、cAMP 和共免疫沉淀实验评估信号转导活性。
大鼠脑中的内源性 CB1 受体发生棕榈酰化。Cys415 突变阻止了转染细胞中受体的棕榈酰化，减少了其向质膜和脂筏的募集；还增加了蛋白质的扩散迁移率。同样的突变显著降低了 CB1 受体与 G 蛋白和腺苷酸环化酶的功能偶联，而去棕榈酰化则消除了受体与特定 G 蛋白亚群的结合。
CB1 受体通过棕榈酰化进行翻译后修饰。Cys415 突变使受体在膜靶向和信号转导方面的功能受损。
本文是关于大麻素在生物学和医学中的主题部分的一部分。要查看该部分中的其他文章，请访问 http://dx.doi.org/10.1111/bph.2012.165.issue-8。要查看大麻素在生物学和医学中的第一部分，请访问 http://dx.doi.org/10.1111/bph.2011.163.issue-7。
