The convergence of extracellular vesicle and G protein-coupled receptor biology

Intercellular communication enables individual cells to work together and is therefore crucial for the life of multicellular organisms. Cells communicate with each other via direct cell-to-cell contact or by releasing signaling molecules in the extracellular space that activate receptor proteins located on recipient cells. In the last decades, extracellular vesicles (EVs) have emerged as another means of intercellular communication. EVs are nanosized membrane vesicles that mediate the intercellular transport of a wide range of cargo molecules, including lipids, RNA and proteins. As such, they are involved in numerous physiological processes, ranging from the regulation of metabolism to the protection of neurons by oligodendrocytes. In addition, distorted EV communication has been found to contribute to a number of diseases. Especially in cancer, where EVs contribute to key tumorigenic processes such as metastasis, drug resistance and immune evasion. EVs are a heterogeneous group of membrane vesicles consisting of microvesicles that are generated by direct budding from the plasma membrane, and of exosomes that are released when late endosomal compartments called multivesicular bodies (MVBs) fuse with the plasma membrane and release their intraluminal vesicles (ILVs) in the extracellular space. Over the years, research into EV biogenesis has identified key molecules involved in microvesicle budding, as well as in MVB biogenesis, trafficking and exocytosis. However, the regulatory mechanisms governing EV secretion remain largely unknown. Furthermore, despite the clear clinical potential of modulating EV-mediated communication, there are currently only few pharmacological opportunities for inhibiting EV secretion. Research into mechanisms underlying EV biogenesis and the regulation of their secretion is hampered by the limitations of currently available EV isolation and quantification methods. In this thesis we describe the development of two novel and complementary approaches to quantify EV secretion. The first two studies describe the development of a live imaging approach using tetraspanin-based, pH-sensitive, optical reporters that can be used to visualize MVB exocytosis in single cells. Using this approach, we show that the fusion of MVBs with the plasma membrane is mediated by the SNARE proteins SNAP23 and Syntaxin-4. Furthermore, we report that stimulation of the Histamine H1 receptor, a G protein-coupled receptor (GPCR), promotes MVB-plasma membrane fusion by increasing Ser110 phosphorylation of SNAP23, demonstrating a regulatory role for GPCR signaling in exosome release. In our third study we used the live cell imaging approach to demonstrate that the GPCR US28, a viral chemokine receptor encoded by the human cytomegalovirus (HCMV), is secreted on exosomes. Exosomal US28 binds the chemokine CX3CL1, and US28-containing exosomes inhibit the CX3CL1-CX3CR1 signaling axis. These findings suggest that exosomal release of US28 contributes to chemokine scavenging and immune evasion by HCMV. The final study reports the development of HA-NanoLuc-CD63, a bioluminescent reporter that enables robust, fast and scalable quantification of EV secretion. We observed that under basal culture conditions, cells secrete this reporter through a SNAP23-independent mechanism, presumably via plasma membrane-derived microvesicles. Using the vATPase inhibitor bafilomycin to stimulate MVB-plasma membrane fusion, we performed a broad-spectrum kinase inhibitor screen and identified a role for the kinase PI4KIIIβ in exosome secretion. This study demonstrates the potential of HA-NanoLuc-CD63 as a tool for high-throughput screening for modulators of EV secretion. Overall, the two novel approaches to study EV secretion described in this thesis, combined with traditional EV quantification techniques, will enable us to unravel the mechanisms of EV secretion and will aid the development of pharmacological strategies targeting distorted EV-communication in disease. Furthermore, the research described in this thesis highlights two points of convergence between GPCR and EV biology.