Direct Interaction of GABA-A & Beta-Adrenergic Receptors

描述（由申请方提供）：γ-氨基丁酸A型受体（GABAAR）是中枢神经系统中神经元抑制的主要效应物。大量证据表明，GABAARs和β-肾上腺素能受体（betaARs）在体内表现出生理性的相互作用和相互调节。鉴于这两种受体都位于神经元质膜中，并且最近发现神经递质受体可以异源二聚化，我们决定探索GABAAR和β AR之间可能存在直接相互作用的可能性。在这里，我们提出的初步证据表明，GABAAR和β 1 AR异源二聚体后，HEK 293细胞以及天然脑组织中的异源表达，通过使用免疫沉淀。使用GST融合蛋白的亲和纯化，诱变，和进一步的免疫沉淀研究，我们建议，以确定特定的氨基酸在GABAAR和β 1 AR介导的相互作用。我们还将使用多模式的方法来阐明两种受体的GABAAR/β 1AR异源二聚化的功能后果。我们将监测β 1AR的共表达和共刺激对GABAAR的生理、调节和磷酸化的影响。此外，我们将研究GABAAR的共表达和共刺激对β 1 AR的药理学和调节的影响。了解伴随异源二聚化的功能变化将可能提供对迄今为止无法解释的体内GABA能和β-肾上腺素能生理学串扰的观察的深入了解。这些研究代表了理解神经递质受体的蛋白质-蛋白质相互作用如何影响其内在药理学和行为的重要一步。

DESCRIPTION (provided by applicant): Gamma-aminobutyric acid type A receptors (GABAARs) are the main effectors of neuronal inhibition in the central nervous system. A large body of evidence has demonstrated that GABAARs and beta-adrenergic receptors (betaARs) exhibit physiological cross-talk and mutual regulation in vivo. Given that both receptors are proximately located in the neuronal plasma membrane, and recent findings that neurotransmitter receptors can heterodimerize, we decided to explore the possibility that there might be a direct interaction between GABAARs and betaARs. Here, we present preliminary evidence that GABAARs and beta1ARs heterodimerize following heterologous expression in HEK 293 cells as well as in native brain tissue by using immunoprecipitation. Using GST fusion protein affinity purifications, mutagenesis, and further immunoprecipitation studies, we propose to identify the specific amino acids in both the GABAAR and the beta1AR that mediate interaction. We will also use a multimodal approach to elucidate the functional consequences of GABAAR/beta1AR heterodimerization for both receptors. We will monitor the effect of coexpression and costimulation of the beta1AR on the physiology, regulation, and phosphorylation of the GABAAR. Additionally, we will investigate the effect of coexpression and costimulation of the GABAAR on the pharmacology and regulation of the beta1AR. Understanding the functional changes that accompany heterodimerization will likely provide insight into heretofore unexplained observations of GABAergic and beta-adrenergic physiological cross-talk in vivo. These studies represent an important step in understanding how the protein-protein interactions of a neurotransmitter receptor affect its intrinsic pharmacology and behavior.