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(Alpha2AR)信号的机制，以便能够在各种病理生理状态下巧妙地进行干预。本提案试图建立激动剂占据后的几个部分反应在体内的功能相关性：受体磷酸化，受体与arrestin结合，以及受体内吞。此外，我们希望了解三种α2AR亚型的不同转运路线以及体内α2AR与14-3-3蛋白和刺激性蛋白相互作用的功能相关性。这些相互关联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改的运输属性或改变的部分反应的alpha2AR结构引入到小鼠的alpha2AR基因座中来解决。然而，为了确定应该开发哪些表达突变的alpha2AR的小鼠系，以及在天然靶细胞的背景下获得关于alpha2AR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不同的alpha2AR结构的腺病毒载体运送到alpha2AAR“敲除”小鼠的第四脑室。然后，我们将评估这些受体结构在蓝斑中的运输特性和细胞功能。对于随后发展的纯合子小鼠细胞系，我们将评估Alpha2AR对蓝斑和颈上神经节神经元钙电流的抑制和K+电流的激活。我们还将评估一些生理参数(包括镇静、止痛、降低血压和抑制癫痫发生)和行为参数，包括抗抑郁剂疗效的测量和预先存在的“抑郁状态”的指数。这些拟议的研究是李·林伯德和布莱恩·科比尔卡的实验室之间的合作，他们是这项研究的共同研究者，代表了在天然靶细胞和体内探索α2受体信号转导的部分反应和α2受体转运的影响的第一次努力。我们预计，我们获得的见解将为开发新的治疗策略提供信息，以治疗由alpha2AR调节的一些心血管、神经和行为障碍。

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.