Characterization of cell type-specific endocannabinoid signaling at biochemical level
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Abstract
The cannabinoid receptor type 1 (CB1) is one of the most abundant G protein-coupled receptors (GPCRs) in the brain and mediates a wide range of behavioral and physiological responses. It is differentially expressed in various neuronal subtypes and moreover, exerts distinct signaling in the respective cell types. Whereas its physiological functions have been well studied, the highly complex intracellular signaling at the molecular level, which leads to differential CB1 signaling, has not been well understood.
The present study aimed at the purification of cell type-specific CB1 protein complexes and identification of CB1-interacting proteins. By combining adeno-associated virus (AAV)-mediated transgene transfer, conditionally Cre-recombinase expressing mouse lines, and tandem affinity purification (TAP) of hippocampal synaptosome preparations, an experimental set up was established, which allowed the purification of CB1 multiprotein complexes in mouse hippocampal glutamatergic neurons and GABAergic interneurons under native conditions. The composition of these protein complexes was then revealed by deep-coverage mass spectrometry (MS).
In the MS approach, 951 proteins were identified in the TAP samples of glutamatergic and GABAergic CB1 protein complexes, with 41 being specific for glutamatergic neurons and 83 specific for GABAergic interneurons. The high number of co-purified proteins suggests that not only directly interacting proteins were purified, but higher order structures including cytoskeletal elements and scaffolding proteins. The whole dataset of purified proteins reflects known functions of CB1 and depicts the complex biosynthesis and trafficking of this membrane receptor, which acts in different subcellular compartments. Together with a number of identified already known CB1-interacting proteins, the dataset can be judged as coherent and meaningful, with a plethora of yet unknown distinctly interesting potential interactors. A number of cell type-specific co-purified proteins may hold the potential to contribute to differential signaling in the respective hippocampal neuronal subtype, but further functional studies need to be performed to reveal the functional relevance of interesting target proteins. Thus, an extensive basis for new hypotheses and further studies on differential CB1 signaling was generated.
To further test a yet unknown identified potential interactor of CB1, functional studies on G protein subunit alpha z (Gαz) were carried out. CB1 and Gαz are coexpressed in glutamatergic principal cells as well as GABAergic interneurons in the hippocampus of wildtype mice. A functional interaction of Gαz after activation of CB1 could be shown in an in vitro model using stably CB1 expressing HEK293 cells with transiently overexpressed inhibitory G proteins Gαz or Gαo. A kinetic assay to measure cyclic adenosine monophosphate (cAMP) turnover upon adenylyl cyclase (AC) stimulation with forskolin showed a CB1-mediated inhibition of cAMP synthesis upon CB1 agonist treatment of the cells. An additional treatment with pertussis toxin (PTX) led to an inhibition of cAMP synthesis only in the Gαz, but not in the Gαo overexpressing cells, since Gαz is the only PTX-insensitive inhibitory G protein. Thus, a functional interaction with one identified candidate interactor of CB1 could be verified.