Endocytic regulation of chemokine receptor expression

The activity of stimulated cell surface signalling receptors is frequently regulated by endocytosis, which provides a mechanism to internalise and either degrade or reprogram the protein. Clathrin-mediated endocytosis (CME) is one of the principal mechanisms responsible for these events. Although clathrin and many of its associated proteins are relatively well characterised, many aspects of how CME operates are yet to be established. In particular, the early steps of internalisation, the formation of clathrin-coated pits (CCPs) and the mechanism by which receptors are recruited into CCPs remain controversial. This thesis investigates the early events of CME occurring at the plasma membrane using as a model various cell lines expressing CC Chemokine receptor 5 (CCR5), a G-protein coupled receptor (GPCR). Upon agonist binding, CCR5 undergoes rapid phosphorylation by a GPCR kinase (GRK) and protein kinase C (PKC) on four C-terminal serines, p-arrestin is subsequently recruited to the receptor, abrogates CCR5 interaction with the G- protein and links the receptor to the endocytic machinery by binding to AP-2 and clathrin. Using confocal immunofluorescence microscopy and electron microscopy, I show that, on agonist binding, CCR5 relocates in the plasma membrane and clusters into flat clathrin lattices. CCPs were observed to invaginate at the edge of these lattices and proteins from the endocytic machinery were identified by immunolabelling within these domains. As C-terminal serine phosphorylation has been found to be important for efficient agonist-induced internalisation of CCR5, the requirement for specific serines for plasma membrane relocation, association with clathrin lattices and endocytosis were analysed. A mutant lacking all serines failed to be recruited into flat clathrin lattices. Further analysis showed that specific GRK phosphorylation of CCR5 was sufficient for this recruitment, suggesting a distinct role for different kinases in receptor desensitisation and internalisation. This study brings new insights into the mechanism of recruitment of activated GPCRs into CCPs and reveals the importance of flat clathrin lattices in the formation of endocytic clathrin-coated vesicles and CME. Part of the results presented in this thesis have been published (Signoret et al., 2005) (see appendix)