Assessing the real-time activation of the cannabinoid CB1 receptor and the associated structural changes using a FRET biosensor. (June 2018)

• Record Type: Journal Article
• Authors: Liu, Ying; Chen, Lu-Yao; Zeng, Hong; Ward, Richard; Wu, Nan; Ma, Li; Mu, Xi; Li, Qiu-Lan; Yang, Yang; An, Su; Guo, Xiao-Xi; Hao, Qian; Xu, Tian-Rui
• Is Part Of: International journal of biochemistry & cell biology. Volume 99(2018)
• Page Start: 114
• Page End: 124
• Publication Date: 2018-06
• Subjects: CB1 cannabinoid type 1 receptor -- FRET fluorescence resonance energy transfer -- GPCRs G protein-coupled receptors -- HD Huntington's disease -- TM transmembrane -- G-proteins GTP binding-proteins -- cAMP cyclic adenosine monophosphate -- ERK1/2 extracellular signal-regulated kinases 1 and 2 -- PVDF polyvinylidene difluoride -- Kon the association rate constant -- Koff the dissociation rate constant -- Kd the equilibrium binding constant -- CREB cAMP-response element-binding protein -- i3 loop the third intracellular loop; Cannabinoid receptor -- ERK1/2 MAP kinase -- FRET sensors -- Receptor activation -- G protein-coupled receptor; Biochemistry -- Periodicals; Cytology -- Periodicals; Biochemistry -- Periodicals; Cell Biology -- Periodicals; Biochimie -- Périodiques; Cytologie -- Périodiques; Biochimie; Cytologie; Biochemistry; Cytology; Ressource Internet (Descripteur de forme); Périodique électronique (Descripteur de forme); Periodicals; 572.05
• DOI: 10.1016/j.biocel.2018.04.003
• Languages: English
• ISSNs: 1357-2725

Abstract

Abstract: The cannabinoid receptor 1 (CB1) is mainly expressed in the nervous system and regulates learning, memory processes, pain and energy metabolism. However, there is no way to directly measure its activation. In this study, we constructed a CB1 intramolecular fluorescence resonance energy transfer (FRET) sensor, which could measure CB1 activation by monitoring structural changes between the third intracellular loop and the C-terminal tail. CB1 agonists induced a time- and concentration-dependent increase in the FRET signal, corresponding to a reduction in the distance between the third intracellular loop and the C-terminal tail. This, in turn, mobilized intracellular Ca 2+, inhibited cAMP accumulation, and increased phosphorylation of the ERK1/2 MAP kinases. The activation kinetics detected using this method were consistent with those from previous reports. Moreover, the increased FRET signal was markedly inhibited by the CB1 antagonist rimonabant, which also reduced phosphorylation of the ERK1/2 MAP kinases. We mutated a single cysteine residue in the sensor (at position 257 or 264) to alanine. Both mutation reduced the agonist-induced increase in FRET signal and structural changes in the CB1 receptor, which attenuated phosphorylation of the ERK1/2 MAP kinases. In summary, our sensor directly assesses the kinetics of CB1 activation in real-time and can be used to monitor CB1 structure and function.