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.
We have found that tolerance to the analgesic effects of delta9-THC are blocked by pharmacological inhibition of c-Jun N-terminal kinase (JNK) in S426A/S430A mutant mice that are unable to undergo G protein-coupled receptor kinase and betaarrestin-mediated desensitization. This study will determine whether tolerance for delta9-THC is caused by JNK-mediated desensitization or down-regulation of CB1. This study will also determine the form of JNK that is responsible for CB1 desensitization and/or down-regulation.
