The cannabinoid receptor interacting protein CRIP1 binds to glutamate receptors in the CNS: Molecular mechanisms and functional implications

谷氨酸是中枢神经系统中最重要的兴奋性神经递质，可激活g蛋白偶联（代谢性）谷氨酸受体（mGluR1-mGluR8）。此外，内源性大麻素调节调节神经元活动的g蛋白偶联受体（CB1和CB2），并对大麻的精神病效应负责。哺乳动物视网膜的光感受器在突触前共同表达mGluR8和CB1，它们都是调节光感受器末端谷氨酸释放的负反馈回路的一部分。通过这种方式，mGluR8和CB1参与调节视网膜在大范围照明下的敏感性的适应性机制。不同神经递质受体之间的串扰是协调神经元信号通路的重要机制。报道了CB1和mGluR类型之间的功能相互作用。此外，腺苷和5 -羟色胺受体与CB1或mGluR2形成异源二聚体，并以不同的组合方式形成由腺苷、多巴胺、谷氨酸和内源性大麻素受体组成的异源三聚体受体复合物。此外，细胞内支架蛋白可以物理连接功能连接的受体类型。最近，我们发现大麻素受体相互作用蛋白1a CRIP1a是mGluR8a异构体的新结合伙伴。因此，我们认为mGluR8a和CB1之间通过CRIP1存在功能/物理相互作用。这一假设是基于(i)发现CRIP1a是mGluR8a相互作用因子，（ii） CRIP1a、CB1和mGluR8a在突触前光受体末端共表达，（iii） CB1和mGluR8a具有相似的生理功能（抑制突触前谷氨酸释放），（iv） CB1 c端绘制的9个氨基酸长的CRIP1a结合序列与mGluR8a c端6个氨基酸的线性延伸之间具有高度的同源性；(v)最后根据已知的生物学原理，受体串扰可以通过细胞内蛋白质发生。我们计划描述介导已鉴定的mGluR8a/CRIP1a相互作用的分子机制，并阐明mGluR8a和CB1在中枢神经系统中可能存在的物理/功能串扰。我们的工作计划将首先分析mGluR8a， CB1和CRIP1a之间结合的分子机制，从而为后续的功能研究提供分子工具和机制知识。这些数据将通过解剖共定位实验加以补充。之后，我们计划研究新发现的蛋白质相互作用的功能后果，首先在HEK-293细胞中使用重组表达的蛋白质，然后在光感受器中使用。我们的研究旨在描述中枢神经系统中神经递质受体功能的新原理，并将具体阐明哺乳动物视网膜中第一视觉突触信号调节的分子机制。

Glutamate is the most important excitatory neurotransmitter in the central nervous system (CNS) and activates G-protein coupled (metabotropic) glutamate receptors (mGluR1-mGluR8). Also endocannabinoids regulate G-protein coupled receptors (CB1 and CB2) that regulate neuronal activity and are responsible for psychotic effects of marijuana. Photoreceptors of the mammalian retina co-express pre-synaptically mGluR8 and CB1 that are both part of negative feedback loops regulating glutamate release from photoreceptor terminals. In this way, mGluR8 and CB1 participate in adaptive mechanisms that regulate the sensitivity of the retina over a large range of illumination.Crosstalk between different neurotransmitter receptor classes represents a key mechanism to coordinate signal pathways in neurons. Functional interactions between CB1 and mGluR types were reported. In addition, receptors for adenosine and serotonin form heterodimers with CB1 or mGluR2, and the formation of heterotrimeric receptor complexes composed of adenosine, dopamine, glutamate and endocannabinoid receptors in different combinations was demonstrated. In addition, intracellular scaffold proteins can physically link functionally connected receptor types.Recently, we identified the cannabinoid receptor interacting protein 1a CRIP1a as a new binding partner of the mGluR8a isoform. Therefore, we suggest a functional/physical interaction between mGluR8a and CB1 via CRIP1. This hypothesis is based on (i) the identification of CRIP1a as a mGluR8a interactor, (ii) the co-expression of CRIP1a, CB1 and mGluR8a in pre-synaptic photoreceptor terminals, (iii) similar physiological functions of CB1 and mGluR8a (inhibition of the pre-synaptic glutamate release), (iv) a high homology between the mapped 9 amino acid long CRIP1a binding sequence in the CB1 C-terminus and a linear stretch of 6 amino acids in the mGluR8a C-terminus and (v) finally on the known biological principle that receptor crosstalk can occur via intracellular proteins.We plan to characterise molecular mechanisms that mediate the identified mGluR8a/CRIP1a interaction, as well as to elucidate a suggested physical/functional crosstalk between mGluR8a and CB1 in the CNS. Our working programme will first analyse molecular mechanisms of the binding between mGluR8a, CB1 and CRIP1a, thereby providing molecular tools and mechanistic knowledge for successive functional studies. These data will be complemented by anatomical co-localization experiments. Thereafter, we plan to investigate functional consequences of the newly identified protein interactions, first using recombinantly expressed proteins in HEK-293 cells and thereafter in photoreceptors.Our studies are designed to describe new principles of neurotransmitter receptor function in the CNS and will specifically elucidate molecular mechanisms that regulate signalling at the first visual synapse in the mammalian retina.