BACKGROUND It has been hypothesized that heteromers of adenosine A2A receptors (A2AR) and cannabinoid CB1 receptors (CB1R) localized in glutamatergic nerve terminals mediate the integration of adenosine and endocannabinoid signaling involved in the modulation of striatal excitatory neurotransmission. Previous studies have demonstrated the existence of A2AR-CB1R heteromers in artificial cell systems. A dependence of A2AR signaling for the Gi protein-mediated CB1R signaling was described as one of its main biochemical characteristics. However, recent studies have questioned the localization of functionally significant A2AR-CB1R heteromers in striatal glutamatergic terminals. RESULTS Using a peptide-interfering approach combined with biophysical and biochemical techniques in mammalian transfected cells and computational modeling, we could establish a tetrameric quaternary structure of the A2AR-CB1R heterotetramer. This quaternary structure was different to the also tetrameric structure of heteromers of A2AR with adenosine A1 receptors or dopamine D2 receptors, with different heteromeric or homomeric interfaces. The specific quaternary structure of the A2A-CB1R, which depended on intermolecular interactions involving the long C-terminus of the A2AR, determined a significant A2AR and Gs protein-mediated constitutive activation of adenylyl cyclase. Using heteromer-interfering peptides in experiments with striatal glutamatergic terminals, we could then demonstrate the presence of functionally significant A2AR-CB1R heteromers with the same biochemical characteristics of those studied in mammalian transfected cells. First, either an A2AR agonist or an A2AR antagonist allosterically counteracted Gi-mediated CB1R agonist-induced inhibition of depolarization-induced glutamate release. Second, co-application of both an A2AR agonist and an antagonist cancelled each other effects. Finally, a CB1R agonist inhibited glutamate release dependent on a constitutive activation of A2AR by a canonical Gs-Gi antagonistic interaction at the adenylyl cyclase level. CONCLUSIONS We demonstrate that the well-established cannabinoid-induced inhibition of striatal glutamate release can mostly be explained by a CB1R-mediated counteraction of the A2AR-mediated constitutive activation of adenylyl cyclase in the A2AR-CB1R heteromer.

中文翻译：

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腺苷和大麻素受体复合物控制谷氨酸释放。

背景技术已经假设，位于谷氨酸能神经末梢的腺苷A2A受体（A2AR）和大麻素CB1受体（CB1R）的异聚体介导参与调节纹状体兴奋性神经传递的腺苷和内源性大麻素信号的整合。先前的研究表明，人工细胞系统中存在A2AR-CB1R异聚体。A2AR信号转导对Gi蛋白介导的CB1R信号转导的依赖性被描述为其主要生化特征之一。但是，最近的研究对纹状体谷氨酸能末端中功能上重要的A2AR-CB1R异聚体的定位提出了质疑。结果在哺乳动物转染细胞中，采用肽干扰法与生物物理和生化技术相结合并进行了计算建模，我们可以建立A2AR-CB1R异四聚体的四聚体四级结构。该四级结构不同于具有腺苷A1受体或多巴胺D2受体的A2AR异聚体的四聚体结构，其具有不同的异聚体或同聚体界面。A2A-CB1R的特定四级结构取决于涉及A2AR的长C端的分子间相互作用，决定了A2AR和Gs蛋白介导的腺苷酸环化酶的显着组成性活化。在纹状体谷氨酸能末端的实验中使用异源干扰物干扰肽，我们可以证明存在功能上重要的A2AR-CB1R异源异构体，其生化特性与在哺乳动物转染细胞中研究的相同。第一，A2AR激动剂或A2AR拮抗剂变构抵消了Gi介导的CB1R激动剂诱导的去极化诱导的谷氨酸释放抑制。第二，同时使用A2AR激动剂和拮抗剂可以互相抵消作用。最后，CB1R激动剂通过在腺苷酸环化酶水平上的典型Gs-Gi拮抗作用抑制A2AR的组成型活化，从而抑制谷氨酸释放。结论我们证明，建立良好的大麻素诱导的纹状体谷氨酸释放的抑制作用主要可以由A2AR-CB1R异源体中A2AR介导的腺苷酸环化酶的CB1R介导的反作用来解释。A2AR激动剂和拮抗剂的共同应用抵消了彼此的作用。最后，CB1R激动剂通过在腺苷酸环化酶水平上的典型Gs-Gi拮抗作用抑制A2AR的组成型活化，从而抑制谷氨酸释放。结论我们证明，建立良好的大麻素诱导的纹状体谷氨酸释放的抑制作用主要可以由A2AR-CB1R异源体中A2AR介导的腺苷酸环化酶的CB1R介导的反作用来解释。A2AR激动剂和拮抗剂的共同应用抵消了彼此的作用。最后，CB1R激动剂通过在腺苷酸环化酶水平上的典型Gs-Gi拮抗作用抑制A2AR的组成型活化，从而抑制谷氨酸释放。结论我们证明，建立良好的大麻素诱导的纹状体谷氨酸释放的抑制作用主要可以由A2AR-CB1R异源体中A2AR介导的腺苷酸环化酶的CB1R介导的反作用来解释。