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控的重要机制。通过磷酸化细胞内特定氨基酸，蛋白激酶调节特定谷氨酸受体的锚定、运输和信号传导。I组代谢性谷氨酸受体（mGluR1/5）在纹状体中密集表达，纹状体是一个涉及精神兴奋剂成瘾特性的大脑区域。长形mGluR1/5剪接变异体（1a、5a和5b）具有较大的细胞内c端尾部，这为蛋白-蛋白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Ca2+/钙调素依赖性蛋白激酶II （CaMKII）直接结合到mGluR5a c端近端区域。这种结合将mGluR5a转化为生化底物，可能在选择性丝氨酸位点磷酸化。这些发现提出了一些创新的问题，如CaMKII是否通过直接的蛋白质相互作用和磷酸化来调节mGluR1/5，以及这种调节是否在疾病模型中具有很高的临床相关性。在这个延续的提议中，我们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的直接结合，并在体内建立天然CaMKII与mGluR1/5在纹状体神经元中相互作用。我们将描述Ca2+在体外和体内是否以及如何调节CaMKII和mGluR1/5之间的相互作用。然后，我们将研究Ca2+调节的CaMKII-mGluR1/5相互作用是否调节1)mGluR1/5的信号传导效果，2)受体的运输，以及3)mGluR1/5与关键支架Homer蛋白的相互作用，在纹状体神经元或异种细胞中。最后，我们将进行神经行为实验来确定CaMKII-mGluR1/5相互作用在精神兴奋剂安非他明成瘾作用中的作用。我们的研究结果将为激酶调节的mGluRs的新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。他们还将最终通过靶向mGluRs和CaMKII来促进新型药物疗法的发展，用于治疗各种精神疾病，包括成瘾。

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. PUBLIC HEALTH RELEVANCE: This research project is aimed to elucidate molecular mechanisms underlying the regulation of metabotropic glutamate receptors and roles of the receptor in drugs of abuse. The information obtained through this project is valuable for the development of new pharmacotherapies for mental illnesses stemming from dysfunctional glutamatergic transmission in the central nervous system.