Cannabinoid Receptor Interacting Protein1a Structure-Function

描述(由申请人提供)：这项NRSA博士前奖学金将在维克森林大学健康科学系的Allyn C.Howlett博士的指导下为Lawrence C.Blume提供研究培训。大麻目前是美国使用最广泛的非法药物，主要通过中枢神经系统中的CB1大麻素受体产生精神作用。CB1受体可以通过蛋白质-蛋白质与多种分子的相互作用来调节，从而导致亚细胞定位、受体激活和信号转导的改变。CRIP1a是一种与CB1远端C末端结合的蛋白质，它的发现与这些受体的结构性(不受激动剂刺激)活性的调节有关。我第一次在实验室轮换的研究结果表明，CRIP1a在大鼠纹状体内的过度表达受到CB1和D2受体水平的影响，并且CRIP1a显著调节阿片肽及其受体的表达(提交发表)。因为CRIP1a似乎是神经元中CB1功能的一个完整的调节因子，所以我论文的剩余部分将集中在CRIP1a-CB1相互作用的生化和细胞机制上。目前，CRIP1a稳定过表达和下调的神经细胞系已经建立起来。在神经细胞中，我的工作表明CRIP1a的过度表达调节了CB1受体的定位、G蛋白的激活以及细胞外信号调节激酶(ERK)下游信号的构成活性。这些发现为研究这一应用中的三个特定目的奠定了基础，这将阐明CRIP1a调节CB1受体功能的机制。目的1利用生化技术确定CRIP1a在CB1在激动剂和拮抗剂条件下向质膜转位和内化中的作用。目的2通过[35S]GTP-γ-S结合、cAMP积聚和ERK磷酸化来确定CRIP1a如何影响Cb1-G蛋白介导的信号转导。目的3将通过下拉和免疫共沉淀实验评估CB1-CRIP1a复合体与含有PDZ结构域的支架蛋白相互作用的可能性。这些研究将提供有关CRIP1a调节CB1功能的能力的有价值的信息，并可能导致基于选择性微调CB1受体功能的新治疗策略的开发。因为CRIP1a逆转了神经细胞中CB1受体的构成活性，所以开发出阻断这种相互作用并进而增强CB1活性的药物是有可能的。为解决这些重点研究工作而提出的实验方法的多样性将为申请人提供出色的博士前培训经验，以支持他作为一名独立科学家的职业目标。

DESCRIPTION (provided by applicant): This NRSA Predoctoral Fellowship will provide Lawrence C. Blume with research training under the guidance of Dr. Allyn C. Howlett in the Department of Physiology and Pharmacology at Wake Forest University Health Sciences. Cannabis is currently the most widely used illicit drug in the United Sates, and produces its psychotropic effects primarily through CB1 cannabinoid receptors in the central nervous system. CB1 receptors can be regulated by protein-protein interactions with a variety of molecules, which can lead to alterations in sub-cellular localization, receptor activation and signal transduction. The discovery of CRIP1a, a protein that binds to the distal C-terminus of CB1, has been implicated in the regulation of constitutive (unstimulated by agonists) activity of these receptors. Research findings from my first laboratory rotation indicated that CRIP1a over-expression in rat striatum is influenced by CB1 and D2 receptor levels, and CRIP1a significantly modulates expression of opioid peptides and their receptors (submitted for publication). Because it appears that CRIP1a is an integral regulator of CB1 function in neurons, the remainder of my dissertation will focus on the biochemical and cellular mechanisms of the CRIP1a-CB1 interaction using cultured neuronal cells. Currently, CRIP1a stably over-expressing and knock-down neuronal cell lines have been developed. In neuronal cells, my work demonstrates that CRIP1a over-expression modulates CB1 receptor localization, G protein activation, and constitutive activity in downstream signaling to extra-cellular signal-regulated kinase (ERK). These findings form the basis for the investigation of three specific aims in this application that will elucidate the mechanisms by which CRIP1a modulates CB1 receptor functioning. Aim 1 will use biochemical techniques to establish the role of CRIP1a in CB1 translocation to the plasma membrane and internalization during agonist and antagonist conditions. Aim 2 will determine how CRIP1a affects CB1-G protein-mediated signal transduction using [35S]GTP-gamma-S binding, cAMP accumulation, and ERK phosphorylation in CRIP1a over- expressing and knock-down cells. Aim 3 will assess the potential for the CB1-CRIP1a complex to interact with PDZ domain-containing scaffolding proteins using pull-down and co-immunoprecipitation experiments. These studies will provide valuable information regarding CRIP1a's ability to modulate CB1 functioning and potentially lead to the development of novel therapeutic strategies based on selectively fine-tuning CB1 receptor function. Because CRIP1a reverses the constitutive activity of CB1 receptors in neuronal cells, it is plausible to develop drugs that block this interaction, and in turn enhance CB1 activity. The diversity of experimental approaches proposed to address these focused research efforts will provide an outstanding pre-doctoral training experience to the applicant in support of his career goals as an independent scientist.