Molecular Regulation of Metabotropic Glutamate Receptors in Striatal Neurons

描述(申请人提供)：蛋白质磷酸化是谷氨酸受体翻译后调节的重要机制。通过磷酸化细胞内域中的特定氨基酸，蛋白激酶调节特定谷氨酸受体的锚定、运输和信号传递。I组代谢性谷氨酸受体(mGluR1/5)在纹状体密集表达，纹状体是大脑中参与精神刺激剂成瘾特性的区域。长型mGluR1/5剪接变异体(1a、5a和5b)有一个很大的细胞内C末端尾巴，这为蛋白质-蛋白质的直接相互作用和磷酸化提供了基础。在我们最近的研究中，我们发现钙/钙调蛋白依赖的蛋白激酶II(CaMKII)直接与mGluR5a C末端的近端结合。这种结合将mGluR5a转化为生化底物，可能在选择性的丝氨酸位点进行磷酸化。这些发现提出了创新的问题，即CaMKII是否通过直接的蛋白质-蛋白质相互作用和磷酸化来调节mGluR1/5，以及这种调节在疾病模型中是否具有高度的临床相关性。在这个延续方案中，提出了一系列从分子到行为的连贯实验，以证实CaMKII在体外与mGluR1/5直接结合，并在体内证实天然CaMKII和mGluR1/5在纹状体神经元中相互作用。我们将在体外和体内研究钙离子是否以及如何调节CaMKII和mGluR1/5之间的相互作用。然后，我们将研究钙离子调节的CaMKII-mGluR1/5相互作用是否调节1)mGluR1/5的信号效应，2)受体的运输，以及3)mGluR1/5与纹状体神经元或异种细胞中关键支架Hmer蛋白的相互作用。最后，我们将进行神经行为实验，以确定CaMKII-mGluR1/5相互作用在精神刺激剂苯丙胺成瘾作用中的作用。我们的结果将为一种新的激酶调节的mGluRs突触模型及其与精神疾病(物质成瘾)的联系提供证据和见解。它们最终还将通过靶向mGluRs和CaMKII来帮助开发新的药物疗法，用于治疗包括成瘾在内的各种精神疾病。

DESCRIPTION (provided by applicant): Protein phosphorylation is an important mechanism for post-translational regulation of glutamate receptors. Through phosphorylating a specific amino acid in the intracellular domain, protein kinases regulate anchoring, trafficking, and signaling of a given glutamate receptor. Group I metabotropic glutamate receptors (mGluR1/5) are densely expressed in the striatum, a brain area involved in addictive properties of psychostimlants. The long-form mGluR1/5 splice variants (1a, 5a, and 5b) have a large intracellular C-terminal tail, which provides a basis for direct protein-protein interactions and phosphorylation. In our recent studies, we found that Ca2+/calmodulin-dependent protein kinase II (CaMKII) binds directly to the proximal region of mGluR5a C-terminus. This binding converts mGluR5a into a biochemical substrate for phosphorylation likely at a selective serine site. These findings raise innovative questions as to if CaMKII regulates mGluR1/5 via a direct protein-protein interaction and phosphorylation and if this regulation has a high clinical relevance in a disease model. In this continuation proposal, a series of coherent experiments from molecule to behavior was proposed to confirm the direct binding of CaMKII to mGluR1/5 in vitro and to establish that native CaMKII and mGluR1/5 interact with each other in striatal neurons in vivo. We will characterize if and how Ca2+ regulates the interaction between CaMKII and mGluR1/5 in vitro and in vivo. We will then investigate whether Ca2+-regulated CaMKII-mGluR1/5 interactions regulate 1) signaling efficacy of mGluR1/5, 2) trafficking of the receptors, and 3) interactions of mGluR1/5 with key scaffold Homer proteins, in striatal neurons or heterologous cells. Finally, we will carry out neurobehavioral experiments to define the role of CaMKII-mGluR1/5 interactions in the addictive action of the psychostimulant amphetamine. Our results will provide evidence and insights for a new synaptic model of kinase-regulated mGluRs and for its linkage to a mental illness (substance addiction). They will also ultimately contribute to the development of novel pharmacotherapies, by targeting mGluRs and CaMKII, for treating various mental illnesses, including addiction.