Direct Interaction of GABA-A & Beta-Adrenergic Receptors

描述（由申请人提供）：γ -氨基丁酸A型受体（GABAARs）是中枢神经系统神经元抑制的主要效应器。大量证据表明，GABAARs和β -肾上腺素能受体（β -肾上腺素能受体）在体内表现出生理上的串扰和相互调节。考虑到这两种受体都位于神经元质膜附近，以及最近发现神经递质受体可以异二聚化，我们决定探索GABAARs和β aars之间可能存在直接相互作用的可能性。在这里，我们提出了初步的证据，证明GABAARs和β 1ars在HEK 293细胞和天然脑组织中异种表达后异二聚化。通过GST融合蛋白亲和纯化、诱变和进一步的免疫沉淀研究，我们提出鉴定GABAAR和β 1ar中介导相互作用的特定氨基酸。我们还将使用多模式方法来阐明GABAAR/beta1AR异源二聚化对两种受体的功能后果。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 - 1ar的药理学和调控的影响。了解伴随异源二聚化的功能变化，可能有助于了解迄今为止无法解释的GABAergic和β -肾上腺素ergic生理串扰在体内的观察结果。这些研究在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方面迈出了重要的一步。

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.