Cannabinoid Receptor Interacting Protein1a Structure-Function

描述（由申请人提供）：这个NRSA博士前奖学金将提供劳伦斯C。Blume在Allyn C博士的指导下接受研究培训。维克森林大学健康科学生理学和药理学系的Howlett博士说。大麻是目前美国使用最广泛的非法药物，主要通过中枢神经系统中的CB 1大麻素受体产生精神作用。CB 1受体可通过与多种分子的蛋白质-蛋白质相互作用来调节，这可导致亚细胞定位、受体活化和信号转导的改变。CRIP 1a是一种与CB 1远端C末端结合的蛋白质，其发现与这些受体的组成性（未受激动剂刺激）活性的调节有关。我第一次实验室轮换的研究结果表明，大鼠纹状体中CRIP 1a的过度表达受到CB 1和D2受体水平的影响，CRIP 1a显著调节阿片肽及其受体的表达。由于CRIP 1a似乎是神经元中CB 1功能的不可或缺的调节剂，因此我的论文的其余部分将集中在使用培养的神经元细胞的CRIP 1a-CB 1相互作用的生化和细胞机制上。目前，已经开发了CRIP 1a稳定过表达和敲低的神经元细胞系。在神经元细胞中，我的工作表明，CRIP 1a过表达调节CB 1受体定位，G蛋白激活，并在下游信号转导细胞外信号调节激酶（ERK）的组成性活动。这些发现形成了本申请中三个具体目标的研究基础，这些目标将阐明CRIP 1a调节CB 1受体功能的机制。目的1将使用生物化学技术来确定CRIP 1a在激动剂和拮抗剂条件下CB 1转运至质膜和内化中的作用。目的2将确定CRIP 1a如何影响CB 1-G蛋白介导的信号转导，使用CRIP 1a过表达和敲低细胞中的[35 S] GTP-γ-S结合、cAMP积累和ERK磷酸化。目的3将使用下拉和免疫共沉淀实验评估CB 1-CRIP 1a复合物与含PDZ结构域的支架蛋白相互作用的潜力。这些研究将提供有关CRIP 1a调节CB 1功能的能力的有价值的信息，并可能导致基于选择性微调CB 1受体功能的新型治疗策略的开发。由于CRIP 1a逆转了神经元细胞中CB 1受体的组成性活性，因此开发阻断这种相互作用的药物并反过来增强CB 1活性是合理的。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.