UC Davis

Neutrophils are key mediators of immune responses and inflammation in the body. Various neutrophil functions including chemotaxis, the rapid, directed migration of neutrophils towards chemical signals, are controlled by the recognition of chemoattractants by a specialized subfamily of G-protein-coupled receptors (GPCRs) including the widely studied formyl-peptide receptor 1 (FPR1) in humans. Receptor internalization prevents excessive activation of downstream ligand-induced signaling pathways, which allows cells to tune their sensitivity and limit inflammatory responses. Moreover, mutants deficient in FPR1-endocytosis migrate longer distances compared to their wild-type counterparts, suggesting that receptor internalization might act as a termination signal for neutrophil chemotaxis. Although precise regulation of chemotactic receptor internalization is critical for proper migration and function of neutrophils, the mechanism of FPR1 internalization and the role of internalization in chemotactic responses remain unclear.Taking genetic and cell biological approaches, we investigated the roles of both classic and unknown endocytosis regulators. Although their roles had been unclear, we found that multiple GPCR kinases (GRKs) and both β-arrestin 1 and 2 are important for FPR1 internalization using a flow-cytometry based endocytosis assay (Chapter 2). Moreover, we performed two parallel genome-wide CRISPR/Cas9 knock-out screens for the regulators of FPR1 surface expression and internalization (Chapter 3). Our results suggested multiple endocytic routes that FPR1 may follow, and this complexity may help explain conflicting data in the field. This screen identified potential new regulators of FPR1 endocytosis, including multiple subunits of endosome-associated protein complexes, small GTPases involved in membrane trafficking, and polarity signaling components. Through our ongoing investigation of some of these hits, we aim to validate and fully understand their involvement in chemoattractant receptor internalization and neutrophil chemotaxis. Better understanding of the roles of these regulators in FPR1 endocytosis and neutrophil function will unravel how the regulation of FPR1 endocytosis contributes to receptor-linked signaling events. Moreover, in Chapter 4, I summarize our findings on how ligand-dependent phosphorylation of another chemoattractant receptor, the LTB4 receptor, regulate its internalization. In conclusion, our efforts to gain insight into the mechanisms and biological consequences of chemoattractant receptor internalization broaden the current knowledge of regulation of these receptors and neutrophil chemotaxis. Accumulating evidence highlights the significance of neutrophil activity in the development of various ailments including inflammatory, autoimmune, and cardiovascular diseases. Gaining mechanistic insight into neutrophil function can help us develop novel strategies to modulate cell state and behavior during migration to control excessive or prolonged inflammation and thus prevent tissue damage.