Functional and Pharmacological Implications of mGluR Heteromerization

描述（由申请人提供）：G蛋白偶联受体（GPCR）是临床治疗中最常见的靶蛋白家族。 GPCR 之间的分子相互作用可以对其功能以及针对它们的药物的效果产生深远的影响。我们研究了两种名为 mGluR1 和 mGluR5 的谷氨酸激活 GPCR 之间相互作用的功能和药理作用，已知这两种 GPCR 形成同源二聚体复合物（mGluR1-mGluR1 和 mGluR5-mGluR5 二聚体），参与许多病理生理过程，并在中枢神经系统中广泛分布。我们的数据表明这些受体可以在功能上相互作用，并且这种相互作用改变了它们的功能 对针对它们的选择性药物的敏感性。我们提出了一种新的假设来解释这种相互作用，其中 mGluR1 和 mGluR5 二聚体结合形成具有反式激活能力的四聚体（或可能更大）复合物，使得与一个受体结合的配体可以通过另一个受体激活 G 蛋白。此外，这些推定的四聚体受体似乎对某些药物的反应不同于其同二聚体成分。为了解决这一假设，我们将追求以下具体目标：1) 使用大鼠颈上神经节 (SCG) 神经元表达系统来表征 mGluR1/5 异聚体的独特药理学特性，2) 通过在 HeLa 细胞中表达并使用不同的效应器来测定受体信号传导来确认 mGluR1/5 异聚体的药理学和信号传导特性，3) 直接评估荧光标记的 mGluR1、mGluR5 的能力以及使用多光子荧光漂白后恢复 (FRAP) 和荧光相关光谱 (FCS) 将两种受体结合在一起形成二聚体、四聚体或更高级的多聚体受体，以及 4) 验证天然表达 mGluR1 和 mGluR5 的神经元中功能性 mGluR 相互作用的生理相关性。

DESCRIPTION (provided by applicant): G protein coupled receptors (GPCRs) are the most frequently targeted protein family in clinical therapeutics. The molecular interactions between GPCRs can have a profound impact on their function and on the effects of drugs that target them. We examined the functional and pharmacological effects of interactions between two glutamate-activated GPCRs called mGluR1 and mGluR5, which are known to form homodimeric complexes (mGluR1-mGluR1 and mGluR5-mGluR5 dimers), are involved in many patho-physiological processes, and have widespread distribution in the central nervous system. Our data suggest that these receptors can interact functionally, and that this interaction alters their sensitivity to selective pharmacological agents that target them. We suggest a novel hypothesis to explain this interaction in which mGluR1 and mGluR5 dimers combine to form tetrameric (or possibly larger) complexes with the ability to trans-activate, such that ligand binding to one receptor may activate G protein through the other. Further, these putative tetrameric receptors seem to respond to some pharmacological agents differently than their homodimeric constituents. To address this hypothesis, we will pursue the following Specific Aims: 1) To characterize the unique pharmacological properties of mGluR1/5 heteromers using the rat superior cervical ganglion (SCG) neuronal expression system, 2) To confirm the pharmacological and signaling properties of mGluR1/5 heteromers by expression in HeLa cells and using a different effectors to assay receptor signaling, 3) To directly assess the ability of fluorescently-tagged mGluR1, mGluR5 and both receptors together to form dimeric, tetrameric, or higher order multimeric receptors using multi-photon Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Correlation Spectroscopy (FCS), and 4) To verify the physiological relevance of functional mGluR interactions in neurons that natively express both mGluR1 and mGluR5.