| Summary: | The atypical chemokine receptor 3 (ACKR3) and the C-X-C chemokine receptor type 4 (CXCR4) are known to share the chemokine ligand CXCL12. CXCR4 and ACKR3 involvement in various types of cancer and their tumour environment has also been well-documented. Previous research indicated that, while CXCR4 canonically signals through G proteins, ACKR3 does not couple to G protein and suggested that this atypical receptor functions as a chemokine ‘scavenger’. Understanding the dynamic organisation of these receptors at the plasma membrane is crucial because this profoundly influences their signalling capabilities and receptor desensitisation. This thesis explored the membrane dynamics and organisation of CXCR4 and ACKR3 at the plasma membrane using a variety of advanced imaging and spectroscopic techniques. In addition, it provided insight into the roles of G protein-receptor kinases (GRKs) in ACKR3 trafficking.
To begin with, Surface-Alexa Flour 488-labelled SNAP-CXCR4 was used to assess receptor localisation and receptor diffusion parameters by Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Recovery After Photobleaching (FRAP). CXCR4 oligomeric state was investigated with Photon Counting Histogram (PCH) analysis. Membrane diffusion and oligomerisation parameters were compared in basal, agonist (CXCL12) and antagonist (IT1t) treated conditions. CXCR4 diffusion in the plasma membrane did not change upon ligand stimulation but showed a CXCL12-induced increase in oligomerisation and ligand-induced reduction in mobility, suggesting cluster formation.
ACKR3 dynamics and organisation were assessed using labelled SNAP-ACKR3. Confocal imaging already revealed a partial membrane but mainly the intracellular location of ACKR3 in both basal and CXCL12-stimulated conditions. ACKR3 dynamics were studied using FCS, FRAP and Raster Image Correlation Spectroscopy (RICS) which provide diffusion characteristics at different scales. The nanoscale (FCS) and microscale (FRAP) diffusion coefficients of ACKR3 showed significant reduction upon CXCL12 addition, whilst there was no significant change on the macro scale (RICS). The oligomeric state of the receptor was determined with Photon Counting Histogram (PCH) and Number and Brightness (N&B) analysis showing the presence of distinct oligomeric states of ACKR3, indicating cluster formation in both basal and CXCL12 conditions. Moreover, CXCL12 stimulation led to the reduction of ACKR3 mobility and a decrease in the number of particles on macro scale, suggesting internalisation or higher order oligomerisation and cluster formation.
Furthermore, this study investigated the role of G protein-coupled receptor kinases (GRKs) in the internalisation and trafficking of ACKR3 using CRISPR-Cas9 cells edited to lack GRK expression. Our data suggest that ACKR3 undergoes basal internalisation even in the absence of GRKs. However, GRKs appear to influence post-internalization trafficking of the receptor, as the absence of GRKs shifts ACKR3 towards a degradation (LAMP1-positive) pathway.
In conclusion, the data in this thesis provide insights into the distinct dynamics and organisation of CXCR4 and ACKR3 at the plasma membrane at various spatial scales. Additionally, our findings obtained with GRK depletion cell lines demonstrated a potential GRK role in ACKR3 localisation and trafficking post-internalisation.
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