Olivier LahunaFrance, France
Title: Molecular Intrication of the melatonergic pathway and the endocannabinoid system (ECS) through the Melatonin / Cannabinoid Type 1 Receptors (MT1/CB1) Heteromers
Complexity is the master word in biology. Human beings live in constant interaction with its environment, which shapes it in depth. In this environment there are natural causes such as the alternation of Day and Night and Human causes such as the regular consumption of cannabis. Melatonin is a neurohormone synthetized during the night with a circadian rhythm by the pineal gland. Among many functions, it regulates the periodicity of the circadian clocks found in every organs and thus participate on the homeostasis of the main functions in the body. If cannabis is well-known for its psychotropic effects, one should remind that the main psychotropic molecule Δ9-tetrahydrocannabinol (THC) highjacks the function of a family of molecules called endocannabinoids that are synthesized by the body. The endocannabinoid system (ECS) play important roles centrally and peripherally and as such is involved in the regulation of synapses functions or energy expenditure. Melatonin and cannabinoids act through membrane receptors belonging to the G proteins coupled receptors (GPCRs). GPCRs form the largest family of proteins in the human genome with more than 800 members and represent the main targets for medical treatments with about 40% of modern drugs targeting them. When GPCRs were discovered, the dogma was that GPCR functions were taking place at the plasma membrane upon binding of the hormone to a receptor and that one hormone acted through one type of receptor only. This scheme was complicated after identification of cross-talks between GPCRs upon physical interaction between them (heteromerization) and identification of functional GPCRs within intracellular organelles such as nucleus, endosomes or mitochondria. Among a few others receptors, functional melatonin receptor type 1 (MT1) and cannabinoid receptor type 1 (CB1) were found in neuronal mitochondria, suggesting a cross-talk between them. Here, we show that the melatonin type 1 receptor (MT1) and the cannabinoid type 1 receptor (CB1) can specifically interact one with each other to form an MT1/CB1 heteromer. This new complex enables a cross-talk between the melatonergic pathway and the endocannabinoid system.
To experimentally demonstrate interaction between the MT1 and CB1 receptors, we used cells in culture in which we expressed modified MT1 and CB1 receptors to allow their detection by antibodies. We studied the cellular distribution of the two receptors when they are coexpressed within the same cell by immunodetection conjugated to confocal microscopy and found that both are expressed in the same cellular intracellular compartments and at the plasma membrane. We prepare cellular homogenates and used it to perform an immunoprecipitation to fish one receptor with a specific antibody and showed that the other one was trapped with it. Thus we concluded that MT1/CB1 heteromers are formed in absence of melatonin or cannabinoids. We also identified this interaction in intact cells using PLA (Proximity Ligation Assay). PLA technique is based on a combination of immunodetection of proteins and PCR (Polymerase chain reaction) detection if proteins are close in a range of 10 to 40 nm. We detected MT1/CB1 heteromers both at the cell surface and intracellularly. We also performed ultrastructural studies with Transmission Electronic Microscopy (TEM) in cells coexpressing MT1 and CB1 receptors. Specific identification of each receptor was made with antibodies and gold particles of different sizes (6-nm and 15-nm) to enable the identification of each other. MT1/CB1 heteromers were identified at the cell surface. Penetrating interfering peptides are small synthetic oligopeptides able to insert into the plasma membrane and to dissociate complexes by competing with the interface of dimerization. Using PLA experiments we identified a penetrating interfering peptide including a CB1 transmembrane sequence, which specifically dissociates the MT1/CB1 heteromer and thus opens the door to studies of the MT1/CB1 heteromer properties. We were able to follow the melatonin-dependent internalization and trafficking of the MT1/CB1 heteromer from the plasma membrane to intracellular organelles using energy transfer techniques. Quantification of the signaling pathway induced by melatonin in presence of the interfering peptide pinpoints a specific MT1/CB1 dependent effect meaning that the complex shows specific properties. Those results will be discussed in the general context of the cross-talk between melatonergic pathways and the endocannabinoid system.
To be updated soon...