Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Sedej, M.
Signaling and trafficking properties of the PGD2 receptors CRTH2 and DP in a recombinant HEK293 cell model and in human peripheral blood eosinophils
[ Dissertation ] Medical University of Graz; 2012. pp. 108
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- Autor*innen der Med Uni Graz:
- Sedej Miriam
- Betreuer*innen:
- Heinemann Akos
- Wadsack Christian
- Waldhoer Maria
- Altmetrics:
- Abstract:
- Prostaglandin (PG) D2 is a cardinal mediator released from activated immune cells during the allergic response and thus, is fundamentally involved in the initiation and perpetuation of allergic inflammation. The effects of PGD2 are mediated by the seven transmembrane spanning (7TM)/ G-protein coupled receptors (GPCRs) D-type prostanoid receptor (DP) and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). Both receptors are co-expressed in eosinophils, among other immune cells. PGD2 and its two receptors have raised considerable attention in the field of allergy research during the past years and have become attractive therapeutic targets. CRTH2 is well recognized to mediate proinflammatory effects, in vitro as well as in vivo, and a variety of CRTH2 antagonists are being developed. However, the particular role of the DP receptor in allergic inflammation is controversial and many of the observed DP-mediated effects both in vitro and in vivo are difficult to connect with the signaling pathways so far described for this receptor. During the last decades, the importance of 7TM/GPCR heteromerization has been recognized. A plethora of studies showed, that heteromerization of 7TM/GPCR can profoundly alter the pharmacological profile of a receptor and consequently the cellular response. In my PhD thesis I aimed to investigate individual and combinatorial signaling and trafficking properties of CRTH2 and DP receptors. I hypothesized that the divisive signaling properties of the DP receptor are based on a cross-talk with CRTH2. I studied both receptors in a HEK293 cell model and in human peripheral blood eosinophils and could thereby demonstrate that the cell model recombinantly expressing the PGD2 receptors resembles eosinophils endogenously expressing CRTH2 and DP. I show that CRTH2 and DP receptors essentially modulate the signaling properties of each other and form CRTH2/DP heteromers without altering their ligand binding capacities. The DP receptor amplifies the CRTH2-induced Ca2+ release from intracellular stores and, coincidentally, forfeits its own signaling potency. Moreover, desensitization or pharmacological blockade of the DP receptor hinders CRTH2-mediated signal transduction. In contrast, CRTH2 internalization occurs independently of the DP receptor. In cells that express both receptors pharmacological blockade of G¿q/11-proteins abolishes the Ca2+ response to both CRTH2 and DP agonists, while inhibition of G¿i-proteins selectively attenuates the CRTH2-mediated response but not the DP signal. Further, I could show that CRTH2 and DP receptors synergistically mediate actin polymerization and eosinophil adhesion to activated endothelial cells. In doing so, CRTH2 is the dominant receptor as revealed by application of selective antagonists. I also demonstrate the interactive involvement of the PGD2 receptors in activating transcription factors, which play essential roles in allergic inflammation: NFAT activation induced by CRTH2 relies on the presence of the DP receptor and can be modulated by both, CRTH2 and DP antagonists. SRE can be solely activated by DP, but underlies an inhibitory regulation by CRTH2. CREB is exclusively activated by the DP receptor. NF-¿B is not regulated by PGD2 receptors. In summary, my PhD thesis demonstrates outstanding cooperativity between the PGD2 receptors, CRTH2 and DP. They exist as a dynamic signaling unit, whose functionality might be altered by the G-protein pathway activated. Thus, the PGD2 receptor signaling unit represents a potential target for ...