Regulation of alpha2A-AR trafficking/signaling by different agonists in neurons

描述（由申请人提供）：α 2-肾上腺素能受体（AR）属于G蛋白偶联受体（GPCR）超家族，并介导许多生理/药理学反应，当α 2-AR被用作治疗ADHD等疾病的治疗靶点时，可导致有利和不利的影响。本研究的长期目标是在分子和细胞水平上了解α-2AR功能的调控机制，从而为治疗策略提供新的见解。不同激动剂在刺激各种α 2-AR介导的反应和诱导α 2-AR内化以及随后在天然细胞中分选中的性质肯定是α 2-AR生理功能的基础。然而，迄今为止，对这些过程的明确理解仍然难以捉摸。作为一个独特的工具，我已经产生了一个小鼠系，其中N-末端表位标记的HA-alpha 2A-AR表达是由内源性小鼠alpha 2A-AR基因座驱动的（HA-alpha 2A-AR敲入）。在这项研究中，我们将利用这种新的线，以评估调节α 2A-AR贩运和信号传导的两种激动剂，可乐定和胍法辛，与内源性配体，去甲肾上腺素，在本地新皮层神经元。尽管两种激动剂都用于治疗ADHD，并且可以通过激活α 2A-AR来改善认知，但可乐定和胍法辛表现出不同的引起镇静的能力，这是一种不希望的副作用。我们的假设是，这两种激动剂之间的差异，在引发体内反应是由于其不同的能力，在诱导α 2A-AR运输和信号转导反应的天然神经元。我们提出了两个具体的目的来验证这一假设：1）我们将在来自HA-α 2A-AR敲入小鼠的活的新皮层培养物中标记表面HA-α 2A-AR用于荧光和比色检测，并确定每种激动剂在诱导受体内化以及受体胞内后分选谱中的效力和功效; 2）我们将评估每种激动剂在刺激由α 2A-AR诱发的两种信号通路中的功效和效力：抑制新皮层神经元中的电压门控钙电流和激活MAP激酶。鉴于一种GPCR通常调节多种生理反应的事实，识别和开发反应特异性激动剂代表了用于治疗各种疾病状态同时限制不良副作用的新颖且有前景的治疗策略。揭示不同激动剂对α 2-AR调节的分子和细胞基础对于指导α 2-AR激动剂在ADHD治疗中的开发和后续利用以及在可能需要使用α 2-AR激动剂的其他临床环境中将是重要的。

DESCRIPTION (provided by applicant): The alpha2-adrenergic receptors (ARs) belong to the G-protein coupled receptor (GPCR) super-family and mediate a number of physiological/pharmacological responses, which can lead to both favorable and adverse effects when the alpha2-AR is exploited as a therapeutic target for treatment of diseases such as ADHD. The long-term goal of our studies is to understand the mechanisms by which alpha-2AR functions are regulated at the molecular and cellular levels, so as to provide new insights for therapeutic strategies. The properties of different agonists in stimulating various alpha2-AR-mediated responses and in inducing alpha2-AR internalization and subsequent sorting in native cells are certainly the bases for alpha2-AR physiological functions. However, to date, a clear understanding of these processes remains elusive. As a unique tool, I have generated a mouse line in which N-terminal epitope-tagged HA-alpha2A-AR expression is driven by the endogenous mouse alpha2A-AR locus (HA-alpha2A-AR knock-in). In this study, we will utilize this novel line to evaluate regulation of alpha2A-AR trafficking and signaling by two agonists, clonidine and guanfacine, in comparison with the endogenous ligand, norepinephrine, in native neocortical neurons. Although both agonists are used to treat ADHD and can improve cognition through activation of the alpha2A-AR, clonidine and guanfacine exhibit differential capacities to elicit sedation, an undesirable side-effect. Our hypothesis is that the difference between these two agonists in eliciting in vivo responses is due to their diverse capabilities in inducing alpha2A-AR trafficking and signaling responses in native neurons. Two Specific Aims are proposed to test this hypothesis: 1) We will label surface HA-alpha2A-AR in live neocortical cultures derived from the HA-alpha2A-AR knock-in mice for fluorescent and colorimetric detection, and determine the potency and efficacy of each agonist in inducing receptor internalization as well as receptor postendocytotic sorting profiles; 2) We will evaluate the efficacy and potency of each agonist in stimulating two signaling pathways evoked by the alpha2A-AR; inhibition of voltage-gated calcium current and activation of MAP kinase in neocortical neurons. Given the fact that one kind of GPCR often regulates multiple physiological responses, identification and development of response-specific agonists represents a novel and promising therapeutic strategy for treating a variety of disease states while restricting undesirable side effects. Revealing the molecular and cellular basis of alpha2-AR regulation by different agonists will be been important for guiding the development and subsequent utilization of alpha2-AR agonists in ADHD treatment as well as in other clinical settings where use of alpha2AR-agonists may be warranted.