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

描述（由申请人提供）：alpha2-肾上腺素能受体（ARs）属于g蛋白偶联受体（GPCR）超家族，介导多种生理/药理学反应，当alpha2-AR被用作治疗ADHD等疾病的治疗靶点时，可能会产生有利和不利的影响。我们研究的长期目标是了解α - 2ar功能在分子和细胞水平上的调控机制，从而为治疗策略提供新的见解。不同激动剂在刺激各种alpha2-AR介导的反应和诱导alpha2-AR内化和随后在天然细胞中分选方面的特性当然是alpha2-AR生理功能的基础。然而，迄今为止，对这些过程的清晰理解仍然难以捉摸。作为一种独特的工具，我已经生成了一个小鼠系，其中n端表位标记的HA-alpha2A-AR表达由内源性小鼠alpha2A-AR位点驱动（HA-alpha2A-AR敲入）。在这项研究中，我们将利用这条新线路来评估两种激动剂（可乐定和胍法辛）与内源性配体（去甲肾上腺素）在天然新皮质神经元中对α 2a - ar运输和信号传导的调节。虽然这两种激动剂都用于治疗多动症，并且可以通过激活α 2a - ar来改善认知，但可乐定和胍法辛在诱导镇静方面表现出不同的能力，这是一种不良的副作用。我们的假设是，这两种激动剂在诱导体内反应方面的差异是由于它们在诱导α 2a - ar转运和天然神经元信号反应方面的不同能力。为了验证这一假设，我们提出了两个特定的目的：1)我们将在HA-alpha2A-AR敲入小鼠的活皮层培养物中标记表面HA-alpha2A-AR，进行荧光和比色检测，并确定每种激动剂在诱导受体内化和受体内吞后分选方面的效力和功效；2)我们将评估每种激动剂刺激α 2a - ar诱导的两种信号通路的功效和效力；新皮质神经元电压门控钙电流的抑制和MAP激酶的激活。鉴于一种GPCR通常调节多种生理反应，鉴定和开发反应特异性激动剂代表了一种新的和有前途的治疗策略，可以治疗多种疾病状态，同时限制不良副作用。揭示不同激动剂对alpha2-AR调节的分子和细胞基础，对于指导ADHD治疗中alpha2-AR激动剂的开发和后续应用，以及在其他可能需要使用alpha2ar激动剂的临床环境中具有重要意义。

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.