Parallel CRISPR screens reveal pathways controlling the cell surface levels of the attractant receptor FPR1.

Chemoattractants generate strong chemotactic and cytotoxic responses in immune cells by activating cognate receptors. Cell surface receptor levels control sensitivity, which is critical for achieving effective responses without excessive inflammation. The surface levels of the attractant receptor FPR1 are controlled through a balance of delivery and removal, which responds to receptor activation and other stimuli. While this regulation is critical for appropriate responses, the underlying mechanisms remain unclear, including the roles of classic endocytosis regulators. We address these questions using both focused and genome-scale approaches. We find that the receptor kinase GRK6 acts in parallel with GRK2 and GRK3 to trigger internalization, and that internalization uses a β-arrestin-independent pathway, as well as pathways involving β-arrestin1 and 2. Moreover, we use an integrated analysis of two parallel CRISPR/Cas9 screens to classify regulators of FPR1 biogenesis, surface expression, recycling, and endocytosis. We identify the formin mDia1 and the small GTPase ARF6 as specific regulators of FPR1 internalization, which we confirm using chemical inhibitors in primary human neutrophils. Finally, we find that ARF6 contributes to the β-arrestin-independent pathway. Together, our results provide a systems overview of the control of FPR1 surface levels and offer insights into alternative endocytosis mechanisms used by chemoattractant receptors.