Desensitization and downregulation of CB1 during cannabinoid tolerance

描述（由申请人提供）：本研究将确定c-Jun n -末端激酶（JNK）介导的对δ 9-四氢大麻酚耐受的分子机制。我们已经产生了突变小鼠（S426A/S430A），表达大麻素受体1 （CB1）的突变形式，导致对德尔塔9thc的耐受性延迟。然而，S426A/S430A突变体最终对δ 9- thc完全耐受。用抑制剂JNK SP600125预处理S426A/S430A突变体，可消除对δ 9- thc镇痛作用的耐受。然而，jnk介导的delta - 9- thc耐受性的机制尚不清楚。本研究将确定JNK介导的δ 9- thc耐受性是由于CB1脱敏、下调还是两者兼有。这个问题将通过hu -210刺激的[35S]GTPgS结合测定，[3H]SR141716A结合测定和腺苷酸环化酶活性测定来回答。由于我的实验室目前没有采用这些技术，因此本K01的一个核心组成部分是向Lakshmi Devi博士的团队学习这些检测方法，并在我的实验室中实施。目前，我的实验室设备齐全，可以测量大麻素对行为和生理影响的耐受性。然而，我们在R01资金方面的竞争力将需要这些检测的实施，这些检测代表了评估G蛋白偶联受体（GPCR）脱敏和下调的金标准。在这个奖项中，Dr. Devi和Dr. Richard Mailman是GPCR信号领域的两位专家，他们将提供杰出的共同指导，帮助我向完全独立的方向过渡，并建立一个资金充足的学术研究实验室。我获得了宾夕法尼亚州立大学的青年教师学者研究奖（JFRSA），我的部门也为我提供了优秀的启动资金和研究空间。作为JFRSA的一员，我还获得了包括dr。Kent Vrana, Richard Mailman， Andras Hajnal和Victor Ruiz-Velasco。K01奖的研究目标是：1)确定JNK介导的δ - 9- THC耐受性的分子机制，2)确定哪种形式的JNK负责CB1的脱敏和/或下调，以及3)将hu -210刺激的[S]GTPupsilonS结合，腺苷酸环化酶活性和[H]SR141716A 353结合分析纳入我的实验室的技术库。

DESCRIPTION (provided by applicant): This study will determine the molecular mechanism responsible for c-Jun N-terminal kinase (JNK)-mediated tolerance to delta9-THC. We have produced mutant mice (S426A/S430A) expressing a mutant form of the cannabinoid receptor 1 (CB1) that causes delayed tolerance for delta9THC. However, S426A/S430A mutants eventually become completely tolerant to delta9-THC. Tolerance to the analgesic effects of delta9-THC was eliminated by pre-treatment of S426A/S430A mutants with SP600125, an inhibitor JNK. However, the mechanism responsible for JNK-mediated delta9-THC tolerance is not known. This study will determine whether JNK- mediated delta9-THC tolerance is due to CB1 desensitization, down-regulation, or a combination of both. This question will be answered using HU-210-stimulated [35S]GTPgS binding assays, [3H]SR141716A binding assays, and adenylyl cyclase activity assays. Since my lab does not currently employ these techniques, a central component of this K01 is to learn these assays from Dr. Lakshmi Devi's group and implement them in my laboratory. Currently, my laboratory is well equipped to measure tolerance to the behavioral and physiological effects of cannabinoids. However, our competitiveness for R01 funding will require the implementation of these assays, which represent the gold standards for assessing G protein-coupled receptor (GPCR) desensitization and down-regulation. During this award, Dr. Devi and Dr. Richard Mailman, two experts in the field of GPCR signaling, will provide outstanding co-mentorship as I transition towards full independence and develop a fully funded academic research lab. I have been awarded a Junior Faculty Scholar Research Award (JFRSA) from Penn State and have also been provided with excellent start-up funding and research space from my department. As part of the JFRSA, I receive additional outstanding mentorship from an in-house team of mentors including Drs. Kent Vrana, Richard Mailman, Andras Hajnal, and Victor Ruiz-Velasco. The research objectives of this K01 award are to: 1) determine the molecular mechanisms responsible for JNK-mediated delta9- THC tolerance, 2) determine which form of JNK is responsible for desensitization and/or down-regulation of CB1, and 3) incorporate HU-210-stimulated [ S]GTPupsilonS binding, adenylyl cyclase activity, and [ H]SR141716A 35 3 binding assays into my laboratory's technological repertoire.