BPS2026 – Comparative proximity biotinylation mapping reveals the role of endolysosomal retrofusion in intracellular trafficking of monocytic GPCRs

As drivers of immune cell trafficking into tumors, the G protein-coupled chemokine receptors are pursued for immuno-oncology. However, agents targeting the monocytic receptors CCR2 and CCR5 have shown mixed results and none reached approval. Cryptic endomembrane trafficking patterns contribute to variations in chemokine pharmacology for CCR5 and to unexpected side effects of CCR2 antagonists, complicating therapeutic development. GPCR trafficking can be explored using ascorbic-acid peroxidase (APEX) proximity biotinylation and mass spectrometry; however, multi-receptor experiments are challenging. Our innovative computational approach enables comparisons between separately acquired receptor-APEX data sets. By applying it to APEX experiments on CCR2 and CCR5, we revealed fundamental trafficking differences between these receptors when stimulated by their endogenous chemokines. Initial patterns (membrane departure and retromer recruitment) are similar between CCR2 stimulated with CCL2 and CCR5 stimulated with engineered superagonist [6P4]CCL5 but not with endogenous CCL5. Strikingly, CCR2, but not CCR5, exhibited strong basal association with lysosomal intralumenal vesicle (ILV) machinery and only transiently lost it upon agonist stimulation. Combined with CCR2’s constitutive recycling, this suggested endolysosomal retrofusion as a CCR2-specific trafficking mechanism. Immunofluorescence microscopy showed that agonist-activated CCR2, but not CCR5, localizes to endosomal lumen. BRET trafficking assays demonstrated that IFITM3, an inhibitor of ILV formation and retrofusion, prevents agonist-induced GPCR trafficking to ILVs, but only partially affects CCR2. Moreover, IFITM3 inhibited CCR2 recycling following agonist stimulation. Altogether, this study maximizes informative power of APEX proximity proteomics and illuminates an intriguing GPCR intracellular trafficking pathway, offering an alternative avenue for disrupting monocyte recruitment to tumor microenvironments.