Assessing The Real-time Activation Of The Cannabinoid CB1 Receptor And Studying Its Biological Activities Based On FRET Biosensor

Cannabinoid receptor 1?CB1?is a member of the G protein-coupled receptors?GPCRs?family,it 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?i3c-loop?and the C-terminal tail.To monitor CB1 activation and its downstream signaling pathways,we constructed the first FRET molecular probes.The FRET sensor is named p5-VSV-CB1-eYFP at3-loop-M-eCFP?the following referred as CB1-M?.It is constructs with cyan fluorescent protein?eCFP?is fused in the C-terminal of the CB1 receptor and the yellow fluorescent protein?eYFP?is inserted directly into the i3c-loop.Then we study the biology activities of the sensor.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 Ca²⁺,inhibited cAMP accumulation,and increased phosphorylation of the ERK1/2 MAP kinases.And also,the activation kinetics?such as,Kon,Koff and Kd?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 CB1activation in real-time and can be used to monitor CB1 structure and function.In addition,the i3c-loop of GPCRs is a key domain of sensing receptor structure changes,and it is unclear whether the i3c-loop is too long to influence receptor activation and function.Therefore,in order to ensure the normal folding of the i3c-loop,we truncated the i3c-loop to 12 amino acids on both sides and inserted eYFP to construct a second FRET molecular probe?p5-VSV-CB1-eYFP at3-loop-M-eCFP,the following referred as CB1-12?.Results show that CB1-12 sensor also induced FRET signal change and downstream ERK1/2 phosphorylation.While,CB1-12 sensor-induced a weaker FRET signal change than CB1-M,indicating that the i3c-loop plays an important role in receptor activation.Therefore,the i3c-loop is associated with the activation of GPCRs,internalization and activation of ERK signaling.This study provides a method for direct monitoring of CB1 activation and systematically studies the effects of CB1 structural changes on receptor activation and biological activity.This study provides a new method for studying the biological activity of GPCRs.