IN VIVO RELEVANCE OF ALPHA2AR TRAFFICKING ITINERARIES

我们实验室研究的总体目标是足够详细地了解α-2肾上腺素能受体（alpha 2AR）信号传导的机制，以便能够在各种病理生理状态下进行巧妙干预。 本提案旨在建立以下激动剂占用的几个部分反应的体内功能相关性：受体磷酸化，受体结合抑制蛋白，和受体内吞作用。 此外，我们希望了解不同的贩运路线的三个α 2 AR亚型和α 2 AR与14-3-3蛋白和与spinophilin在体内的相互作用的功能相关性。这些相关的目标最终将通过使用基于Cre-loxP的同源重组策略将具有修饰的运输特性或改变的部分反应的alpha 2AR结构引入小鼠的alpha 2AR基因座来解决。然而，为了优先考虑应开发哪些表达突变体α 2 AR的小鼠品系以及获得关于α 2 AR在天然靶细胞背景下的结构-功能关系的前所未有的见解，我们将利用立体定向程序将编码这些各种α 2 AR结构的腺病毒构建体递送到α 2 AAR“敲除”小鼠脑的第四脑室中。 然后，我们将评估这些受体结构在蓝斑中引起的运输特性和细胞功能。 对于成功开发的纯合子小鼠细胞系，我们将评估α 2 AR对蓝斑和上级颈神经节神经元中Ca 2+电流的抑制和K+电流的激活。 我们还将评估一些生理参数（包括镇静、镇痛、降低血压和抑制癫痫发生）和行为参数，包括抗癫痫药物疗效的测量和预先存在的“抑郁状态”指数。 这些拟议的研究代表了本研究的共同研究者Lee Limbird和Brian Kobilka实验室之间的合作，代表了在天然靶细胞和体内背景下探索α 2受体信号传导和α 2受体运输的部分反应的影响的首次努力。 我们预计，我们所获得的见解将为α 2AR调节的许多心血管，神经和行为障碍的新型治疗策略的开发提供信息。

The overall goal of the research in our laboratories is to understand the mechanisms of alpha-2 adrenergic receptor (alpha2AR) signaling in enough detail to be able to intervene with ingenuity in a variety of pathophysiological states. The present proposal seeks to establish the in vivo functional relevance of several partial reactions that follow agonist occupancy: receptor phosphorylation, receptor binding to arrestin, and receptor endocytosis. In addition, we wish to understand the functional relevance of differing trafficking itineraries for the three alpha2AR subtypes and of alpha2AR interactions with 14-3-3 proteins and with spinophilin in vivo. These linked goals ultimately will be addressed by introducing alpha2AR structures with modified trafficking properties or altered partial reactions into the alpha2AR locus of the mouse, using Cre-loxP based homologous recombination strategies. However, to prioritize which mouse lines expressing mutant alpha2AR should be developed as well as to gain unprecedented insights concerning the structure-function relationships of alpha2AR in the context of native target cells, we will utilize stereotactic procedures to deliver adenoviral constructs encoding these various alpha2AR structures into the fourth ventricle of the brain of alpha2AAR "knockout" mice. We will then evaluate the trafficking properties and the cellular functions elicited by these receptor structures in the locus ceruleus. For subsequently-developed homozygous mouse cell lines, we will evaluate alpha2AR suppression of Ca2+ currents and activation of K+ currents in the locus ceruleus and in superior cervical ganglion neurons. We will also evaluate a number of physiological parameters (including sedation, analgesia, lowering of blood pressure and suppression of epileptogenesis) and behavioral parameters, including measures of the efficacy of anti-depressant agents and indices for pre-existing "depressive states". These proposed studies, representing a collaboration between the laboratories of Lee Limbird and Brian Kobilka, co- investigators in this study, represent the first effort to explore, in the context of native target cells and in vivo, the impact of partial reactions of alpha2 receptor signaling and of alpha2 receptor trafficking. We anticipate that the insights we obtain will inform the development of novel therapeutic strategies for a number of cardiovascular, neurological and behavioral disorders regulated by alpha2AR.