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CCR7 signalering en ligand bias: Een uitgebreide karakterisering met behulp van label-gebaseerde en label-vrije methodologieën

Year: 2024
Type: dissertation
Abstract: G protein-coupled receptors (GPCR) are intricate transmembrane proteins that relay signals from the extracellular to the intracellular environment using heterotrimeric G proteins. GPCRs mediate a vast array of physiological functions, but when deregulated, they also contribute to many pathological diseases such as diabetes, Alzheimer's disease, and cancer. This makes GPCRs an attractive therapeutic target. Indeed, approximately 35% of all Food and Drug Administration (FDA)-approved drugs on the market target GPCRs. The chemokine signaling system, a subfamily of GPCRs that coordinates the movement of immune cells throughout the body, remains challenging to drug. Substantial promiscuity exists within said system and many unique ligand-receptor interactions mediate specific downstream signalling pathways, a phenomenon referred to as biased signalling. This dissertation focuses on the CC type chemokine receptor (CCR) 7, which mediates the migration of a subset of lymphocytes and dendritic cells (DC) to the secondary lymphoid organs, specifically the lymph nodes. CCR7, often described as the prototypical example of biased signalling, is an interesting therapeutic target, particularly with regards to cancer metastasis. Unfortunately, no small molecules specifically targeting CCR7 are currently available. As such, the main aim of this work is to develop a better fundamental understanding of CCR7 signalling and its reported signalling bias. In the first part of this work, we generated a comprehensive fingerprint of the CCR7-mediated downstream signalling. Proximal to the receptor, we investigated the activation of 11 different G protein subunits using a novel Nanoluciferase (NLuc)-based bioluminescence resonance energy transfer (BRET) assay. We found that CC chemokine ligand (CCL) 19 and CCL21 had well-conserved G protein activation profiles, activating all cognate G proteins and some non-cognate couplings. Investigation of G protein-dependent second messengers such as 3',5'-cyclic adenosine monophosphate (cAMP) and calcium (Ca2+) revealed similar findings. Additionally, we found a discrepancy with regards to β-arrestin recruitment and receptor internalisation, where CCL19 was significantly more potent. The results outlined above suggested a potential ligand bias between G protein activation and β-arrestin recruitment. To quantify whether this was indeed the case, we performed an extensive ligand bias analysis. Overall, CCL19 and CCL21 showed largely conserved pathway preferences, but small differences were detected. However, these differences resulted in little to no ligand bias in human embryonic kidney (HEK) 293A cells. Interestingly, investigation of ligand bias in different cellular contexts showed that ligand bias at the chemokine receptor CCR7 was not conserved across cell types. Together, these data suggest that CCL19 and CCL21 share mostly overlapping, weakly biased, transducer profiles that can be influenced by the cellular system. The second aim of our work was to validate and use cellular electrical impedance (CEI) as a method to investigate CCR7 signalling. CEI is a label-free and non-invasive technique that reflects an integrated readout of all downstream signalling. We found that CCL19 and CCL21 induced distinct impedance profiles, which prominently differed in the desensitisation phase of the response. This discrepancy was mainly modulated by differential β-arrestin recruitment, which shaped the impedance profile but did not seem to contribute to it directly. These results correlated well with the label-based fingerprint established earlier. We further expanded this study by leveraging the label-free nature of CEI and investigated CCR7 signalling in primary immune cells. We demonstrated that CEI was capable of measuring CCR7-mediated impedance profiles in naïve T cells (nTC) and mature monocyte-derived dendritic cells (mMoDCs), which endogenously express CCR7. While impedance profiles within a unique cell type shared similar kinetics, pathway deconvolution revealed that ligand-specific impedance profiles were modulated by distinct underlying signalling cascades. Additionally, impedance profiles differed drastically between the two cell types, further validating that the cellular system provides additional mechanisms to control CCR7 signalling in an in vivo setting. While CCR7 is an attractive therapeutic target, it remains poorly druggable with conventional small molecules modalities. For the final aim of this work, we set out to develop and characterise CCR7-specific nanobodies (Nb). To this end, we developed eight Nbs that specifically bound CCR7 and were capable of inhibiting CCL19 binding as well as CCL19- and CCL21-induced Ca2+ mobilisation. In conclusion, this work demonstrates the complexity of CCR7 signalling and provides further insight into the ligand-receptor specific responses induced by the endogenous ligands CCL19 and CCL21. It also establishes CEI as a valid method to investigate receptor signalling in systems that are challenging to transfect, as well as a powerful technique to screen G protein-β-arrestin biased ligands. Finally, we generated and characterised eight CCR7 specific Nbs, which could serve as future therapeutics or research tools.
Source: Lirias
Language: English
Open Access status: green
OpenAlex ID: W6980439491
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