Gαs signalling of the CB1 receptor and the influence of receptor number
Gαs signalling of the CB1 receptor and the influence of receptor number
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DOI：
10.1111/bph.13866
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发表时间：
2017-08-01
影响因子：
7.3
通讯作者：
Glass, Michelle
中科院分区：
文献类型：
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作者：
Finlay, David B.;Cawston, Erin E.;Glass, Michelle
背景和目的CB 1受体信号传导通常通过抑制性G α（i）蛋白介导，但在某些情况下通过其他G蛋白发生，G α（s）是最具特征的二级途径。迄今为止确定的这种信号传导开关的决定因素包括G α（i）阻断、CB 1/D-2受体共刺激、CB 1激动剂类别和细胞背景。实验方法CB 1受体在HEK细胞中以不同水平表达，并且通过实时BRET生物传感器-CAMYEL -表征cAMP的信号传导，通过AlphaScreen表征磷酸化ERK的信号传导。匀浆和全细胞放射性配体结合试验进行表征AM 6544，一种新的不可逆CB 1受体拮抗剂。关键结果在HEK细胞表达高水平的CB 1受体，激动剂治疗刺激cAMP，响应不知道是由受体数量介导的。δ（9）-THC和BAY 59 -3074仅在用百日咳毒素预处理的高表达细胞中增加cAMP，并且激动剂在刺激途径中比典型抑制途径中表现出更多样化的信号传导谱。药理学CB 1受体敲除和G alpha（i）1补充恢复了高表达细胞的经典Gai信号传导。在低和高表达的细胞组成的信号是G α（i）-mediated.CONCLUSION和IMPLICATIONS CB 1受体偶联到对立的G蛋白是由受体和G蛋白的表达水平，这支持了一个机制，非典型的信号在一个符合G α（S）信号的方式。CB 1受体介导相反的结果，如肿瘤的生存能力取决于表达水平的终点，我们的研究结果可能有助于解释这种影响在G蛋白偶联的水平。
BACKGROUND AND PURPOSE CB1 receptor signalling is canonically mediated through inhibitory G alpha(i) proteins, but occurs through other G proteins under some circumstances, G alpha(s) being the most characterized secondary pathway. Determinants of this signalling switch identified to date include G alpha(i) blockade, CB1/D-2 receptor co-stimulation, CB1 agonist class and cell background. Hence, we examined the effects of receptor number and different ligands on CB1 receptor signalling.EXPERIMENTAL APPROACH CB1 receptors were expressed in HEK cells at different levels, and signalling characterized for cAMP by real-time BRET biosensor -CAMYEL - and for phospho-ERK by AlphaScreen. Homogenate and whole cell radioligand binding assays were performed to characterize AM6544, a novel irreversible CB1 receptor antagonist.KEY RESULTS In HEK cells expressing high levels of CB1 receptors, agonist treatment stimulated cAMP, a response not known to be mediated by receptor number. Delta(9)-THC and BAY59-3074 increased cAMP only in high-expressing cells pretreated with pertussis toxin, and agonists demonstrated more diverse signalling profiles in the stimulatory pathway than the canonical inhibitory pathway. Pharmacological CB1 receptor knockdown and G alpha(i)1 supplementation restored canonical Gai signalling to high-expressing cells. Constitutive signalling in both low- and high-expressing cells was G alpha(i)-mediated.CONCLUSION AND IMPLICATIONS CB1 receptor coupling to opposing G proteins is determined by both receptor and G protein expression levels, which underpins a mechanism for non-canonical signalling in a fashion consistent with G alpha(s) signalling. CB1 receptorsmediate opposite consequences in endpoints such as tumour viability depending on expression levels; our results may help to explain such effects at the level of G protein coupling.