Embargoed until 2020-01-14
- Doctoral Thesis
As part of our endocannabinoid system, human cannabinoid CB2 receptor is involved in regulation of many physiological processes. Due to its involvement in inflammation, CB2 is an interesting pharmacological target for treating conditions such as chronic pain, osteoporosis and Alzheimer’s disease. Although naturally existing cannabinoids have been used for millennia, and there are drugs targeting the endocannabinoid system, the mechanism of their action at molecular level is still not well understood. CB2 belongs to a large family of versatile membrane proteins, G protein‑coupled receptors (GPCRs). In response to extracellular stimuli, GPCRs undergo conformational changes, interact with intracellular signalling partners, such as G protein and arrestins, resulting in an appropriate intracellular response. GPCRs are known to be able to selectively activate distinct signalling pathways, in response to different biased ligands. Although it was shown that activation of only certain signalling pathways can be pharmacologically beneficial, it remains unknown how the signal is propagated within a GPCR, leading to biased signalling. Within my thesis, I studied the potential for biased signalling by cannabinoid CB1 and CB2 receptors, as well as the molecular basis for biased signalling by CB2, using biosensors based on bioluminescence resonance energy transfer (BRET). I screened a library of 35 structurally diverse ligands for the activation of CB1 and CB2. Furthermore, I tested signalling profiles of over 360 CB2 mutants, collecting over 7000 concentration‑response curves. As a result, I identified CB2 residues involved in selective activation of distinct signalling pathways and the maintenance of its constitutive activity. These residues important for signalling formed extensive allosteric networks connecting the ligand and the effector binding sites. Through a collaboration with a computational group performing molecular dynamics simulations on the same system, I was able to add a structural and dynamic information to the functional data. In addition, I compared my data with analogous data obtained for another GPCR, vasopressin V2 receptor, identifying conserved receptor regions of crucial importance for signal propagation, as well as unique ways of obtaining active conformations utilised by the two receptors. Taken together, my data allow determination of the molecular basis of biased signalling and constitutive activity in CB2 receptor, setting the foundation for the rational design of biased ligands with fewer side effects. Due to their high structural similarity, my results are likely to be transferrable to other GPCRs, improving our understanding of the mechanisms underlying their signalling. Show more
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ContributorsExaminer: Schertler, Gebhard F.X.
Examiner: Veprintsev, Dmitry
Examiner: Korkhov, Volodymyr
Examiner: Jaakola, Veli‑Pekka
SubjectCB2 cannabinoid receptor; GPCR; Biased signalling; Alanine scanning
Organisational unit03866 - Schertler, Gebhard F. / Schertler, Gebhard F.
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