Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Aghaei Bandbon Balenga, N.
The pharmacology and signaling of GPR55 in a recombinant system and in primary human blood neutrophils
[ Dissertation ] Medical University of Graz; 2010. pp. 205
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- Autor*innen der Med Uni Graz:
- Aghaei Bandbon Balenga Nariman
- Betreuer*innen:
- Heinemann Akos
- Waldhoer Maria
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- Abstract:
- Cannabis sativa is a widespread illegal drug and an old medicine, which contains more than 60 bioactive compounds that bind to cannabinoid receptors. Cannabinoid receptors and their endogenous ligands encompass the endocannabinoid system (ECS), which is known to be involved in many physiological and pathophysiological conditions. Cannabinoid receptors belong to the G Protein Couple Receptors (GPCRs) family of proteins. The cannabinoid 2 receptor (CB2R) is the main peripheral receptor that controls the trafficking of macrophages and neutrophils. Neutrophils represent the main granular innate immune cells recruited to inflammatory foci and there have been controversies about the underlying mechanisms of ECS action on these cells. Moreover, some of the cannabinoid effects were not attributable to either CB1R or CB2R. Recently, the G Protein-coupled Receptor 55 (GPR55) was suggested to be the third member of the cannabinoid receptor family. First, I explored the pharmacology and signaling pathways of GPR55 in a recombinant expression system, the Human Embryonic Kidney cells (HEK293). I over-expressed GPR55 in HEK293 cells (HEK-GPR55). I discovered that some of the CB1R antagonists/inverse agonists acted as agonists on GPR55 and induced the activation of the transcription factor Nuclear Factor of Activated T cells (NFAT). In addition, I found that L-alpha-LysoPhosphatidylInositol (LPI) was the most potent and efficacious agonist on GPR55. All of these ligands showed a functional selectivity for the activation of NFAT, NF-kB and cAMP Response Element Binding protein (CREB) transcription factors. Moreover, the activation of GPR55 triggered actin polymerization through the function of the small GTPase RhoA and its effector ROCK. In order to unravel the underpinning mechanisms of neutrophil migration towards endocannabinoids, I set out to investigate the putative function of GPR55 in neutrophils. Initially, I discovered that GPR55 was co expressed with CB2R in human peripheral blood neutrophils at high levels. Activation of GPR55 by a gradient of agonists induced the migration of neutrophils. However, this directional migration was augmented when CB2R was co-activated by its endogenous cannabinoid agonist, 2-arachidonoyl glycerol (2-AG). Moreover, the concomitant activation of GPR55 and CB2R resulted in a distinct polarized morphology of neutrophils: polarized neutrophils showed extending head and retracting tails. Next, I found that LPI significantly augmented the 2-AG-induced activity of Cdc42, a small GTPase which is typically involved in the rearrangement of the cytoskeleton. This finding may explain the polarization and directional migration of neutrophils. In line with these observations, I found that the co-activation of GPR55 and CB2R in HEK-CB2R/GPR55 cells led to a more intensive stress fiber formation and NFAT activation compared to treatment with each agonist alone. The bactericidal functions of neutrophils are mediated by the release of Reactive Oxygen Species (ROS) and myeloperoxidase (MPO) from intracellular granules. GPR55 activation inhibited the 2-AG- and complement factor 5a (C5a)-induced ROS production in neutrophils. In addition, the MPO release triggered by C5a was prevented by activation of GPR55. Moreover, ROS production and MPO release were regulated by the Rac2 small GTPase. In addition, LPI treatment i) suppressed the Rac2 activation induced by 2-AG and further ii) blocked the translocation of Rac2 to the plasma membrane. In summary, I provide evidence that GPR55 activation is linked to multiple signaling pathways. The Galpha13/PDZ-RhoGEF/RhoA/ROCK signaling axis governs most of the functions of GPR55. In addition, I discovered that GPR55 and CB2R crosstalk at the level of small GTPases; thereby GPR55 augments the migratory capacity of neutrophils, while it limits the bactericidal functions of neutrophils, such as ROS production and degranulation.