Not Just One Way In: Mapping Chemoattractant Receptor Trafficking Through Parallel CRISPR Screens

Tuning of receptor levels controls immune cell sensitivity 

Immune cells are critical for defending the body against infection, but their excessive activity can cause inflammatory disease. The sensitivity of these cells must be tuned to achieve protection while minimizing incidental harm to the body. As immune cells detect and respond to stimuli using specialized receptors, the levels of these receptors on the cell surface have big impacts on response sensitivity. A driving question for our recent paper in Communications Biology was: how do immune cells control the levels of receptors on their surface?

We wanted to address this question from a systems perspective to get insights into coordination between basal and stimulus-dependent pathways, including biogenesis, forward trafficking, endocytosis, and recycling.  As a model, we focused on the chemoattractant receptor formyl peptide receptor 1 (FPR1) in human neutrophils. FPR1 recognizes short formylated peptides, and it is critical for phagocytic cell function, innate immunity, and inflammatory responses^1,2. Furthermore, even though FPR1 has been heavily studied, the mechanisms controlling its trafficking are poorly understood.

Assay throughput enables phenotype quantification and genome-wide screening

Before we started this project, the picture of FPR1 endocytosis in the literature was based in large part on negative results that showed the non-requirement of classic endocytosis regulators. Knockout of β-arrestin 1 and 2, GRK2, or components of the best characterized endocytosis mechanisms (clathrin and flotillin) did not block FPR1 internalization^3,4, so FPR1 was suggested to follow a non-canonical, β-arrestin independent pathway for internalization. Some experiments were performed in non-immune cell models, and we wondered whether differences in the expression of components such as GRKs could affect the endocytic routes. Moreover, we found that gene editing can result in large changes in basal FPR1 surface expression through biological effects, but also sometimes through stochastic effects associated with clonal selection. Therefore, we felt that to understand internalization of FPR1, it was critical to measure and study its basal expression in parallel. 

To clarify roles of classic endocytosis regulators and gain a systems-level view of the regulation of FPR1 surface levels, we optimized a quantitative flow-cytometry based internalization assay and developed a four-sample statistical approach to adapt it for genome-scale detection of effects on different aspects of FPR1 surface regulation. 

FPR1 has multiple ways to enter the cell

A major conclusion of our study is that FPR1 is regulated by multiple complementary pathways. While our data confirmed the lack of a strict requirement for β-arrestins, we found that FPR1 does use an arrestin-dependent pathway, and that loss of this pathway cannot be entirely compensated for by other pathways. Moreover, we found that phosphorylation is crucial for efficient FPR1 internalization, but multiple kinases (GRK2, GRK3, and GRK6) play overlapping roles. 

It is not uncommon for receptors to utilize different endocytosis pathways based on cell types, ligands, and ligand concentrations, as internalization is a context dependent process that also diversifies downstream signaling events and receptor fate. Therefore, a natural next step will be to resolve how FPR1 is routed to different pathways and whether these pathways share components. GRK2/3 phosphorylation is closely associated with β-arrestin and clathrin dependent pathways, suggesting that GRK6 might be important for recruitment of the arrestin-independent pathway components. Consistent with recent evidence for GRK-specific regulation of receptors^5,6, it is possible that kinase specific regulation of FPR1 phosphorylation determines what components will be recruited to the receptor.

Genome-wide mapping of the regulators of FPR1 trafficking

Our screens for the regulators of FPR1 surface expression identified genes associated with FPR1 transcription, trafficking to the cell surface, internalization and recycling. The data set is large and multi-faceted, and we have gotten a lot of value from searching it in different ways. We feel that it will be similarly valuable for others working on formyl peptide-driven neutrophil behaviors.

To help others explore our screen results, we created a searchable database on our website (https://collinslab.ucdavis.edu/hl-60-crispr-screen-data/). As one application, researchers could use the database to quickly determine whether a gene of interest affects basal FPR1 surface levels, which could provide an unexpected explanation for observed phenotypes. For example, we found that multiple genes involved in cell migration (e.g., Moesin) also affect basal FPR1 levels, which is important to consider when interpreting results.

Our results on the roles of multiprotein trafficking complexes in FPR1 surface expression could also contribute to the broader systems-level understanding of endocytosis. At a first glance, it could be surprising that deletion of many components of intracellular trafficking pathways cause an internalization phenotype in addition to expected changes in basal surface FPR1 levels. However, growing evidence supports a distributed control system used by cells to assess endocytosis kinetics based on endolysosome responses, raising the question: Are there active feedback mechanisms to stall the internalization process when there is a defect in endosomal sorting?

New regulators of FPR1 internalization: Arf6 and mDia1

We identified two new components controlling FPR1 endocytosis, with gene knockouts that specifically affect FPR1 surface levels after stimulation: the small GTPase Arf6 and the formin mDia1. Although a direct interaction between FPR1 and Arf6 seems unlikely, our data, in line with the literature⁷, suggests that fMLF induces Arf6 recruitment to the plasma membrane. 

Both Arf6 and mDia1 coordinate actin filament polymerization: Arf6 promotes branched actin assembly through effectors, while mDia1 drives linear filament assembly. Future efforts directly monitoring these proteins will clarify the step-by-step mechanisms of FPR1 internalization and determine which internalization pathways they contribute to. 

Looking ahead: What is the biological relevance of FPR1 internalization? 

FPR1 directs neutrophils to the precise locations of infections, and it initiates numerous inflammatory functions. These processes are central to immune defense but may also exacerbate inflammation in disease. Antagonizing FPR1 activity has already shown therapeutic potential in numerous preclinical models by dampening inflammatory reactions^8,9. Our current efforts focus on how receptor endocytosis shapes directed cell movement and optimizes signaling. By exploring the molecular details and understanding how FPR1 endocytosis affects neutrophil function, we might uncover new ways to modulate FPR1 signaling for future approaches. 

References

1. Metzemaekers, M., Gouwy, M. & Proost, P. Neutrophil chemoattractant receptors in health and disease: double-edged swords. Cell. Mol. Immunol. 17, 433–450 (2020). 
2. He, H.-Q. & Ye, R. D. The Formyl Peptide Receptors: Diversity of Ligands and Mechanism for Recognition. Mol. J. Synth. Chem. Nat. Prod. Chem. 22, 455 (2017). 
3. Subramanian, B. C., Moissoglu, K. & Parent, C. A. The LTB4–BLT1 axis regulates the polarized trafficking of chemoattractant GPCRs during neutrophil chemotaxis. J. Cell Sci. 131, jcs217422 (2018). 
4. Vines, C. M. et al. N-Formyl Peptide Receptors Internalize but Do Not Recycle in the Absence of Arrestins. J. Biol. Chem. 278, 41581–41584 (2003). 
5. Drube, J. et al. GPCR kinase knockout cells reveal the impact of individual GRKs on arrestin binding and GPCR regulation. Nat. Commun. 13, 540 (2022). 
6. Nakai, A. et al. The COMMD3/8 complex determines GRK6 specificity for chemoattractant receptors. J. Exp. Med. 216, 1630–1647 (2019). 
7. Dana, R. R., Eigsti, C., Holmes, K. L. & Leto, T. L. A Regulatory Role for ADP-ribosylation Factor 6 (ARF6) in Activation of the Phagocyte NADPH Oxidase. J. Biol. Chem. 275, 32566–32571 (2000). 
8. Li, Y. et al. Targeting formyl peptide receptor 1 reduces brain inflammation and neurodegeneration. Science 390, eadq1177 (2025). 
9. Honda, M. et al. Intravital Imaging of Neutrophil Recruitment Reveals the Efficacy of FPR1 Blockade in Hepatic Ischemia-Reperfusion Injury. J. Immunol. 198, 1718–1728 (2017).