Rab23 GTPase and IFT43 regulate the trafficking of prostaglandin E receptor 4 to primary cilia.

Primary cilia are crucial organelles involved in various cellular signaling pathways. Prostaglandin E receptor 4 (EP4), a G protein-coupled receptor (GPCR), localizes to both cilia and cellular membranes, playing pivotal roles in inflammation, immunomodulation, and cancer progression. However, the mechanisms underlying EP4 trafficking to cilia remain poorly understood, hindering further exploration of its ciliary pathophysiological functions. In this study, we unveil EP4 expression in primary cilia across zebrafish embryos, mouse tissues, and human cells. We identify the third intracellular (IC3) loop and C-terminal (CT) region in EP4 as essential for its ciliary translocation, with the LPG motif serving as a key ciliary localization sequence. A single amino acid substitution in the LPG motif, introduced by CRISPR C-to-G base editing (CGBE1), blocks EP4 trafficking to cilia and reduces cAMP-PKA signaling. High-content siRNA screening identifies the small GTPase Rab23 and intraflagellar transport protein 43 (IFT43) as crucial regulators of EP4 ciliary trafficking in zebrafish and mammalian cells. Furthermore, EP4 physically interacts with IFT43 and Rab23 through its specific domains in the IC3 loop and CT region. These findings provide important insights into the conserved Rab23/IFT43-mediated ciliary trafficking mechanism, with significant implications for understanding the pathophysiological roles mediated by EP4 signaling in cilia.