A calcium-sensing receptor dileucine motif directs internalization to spatially distinct endosomal signaling pathways.

G protein-coupled receptors (GPCRs) can temporally alter signaling by generating secondary signals from internalized receptors. However, the motifs required to direct GPCRs to these pathways are ill defined. The GPCR, calcium-sensing receptor (CaSR), regulates parathyroid hormone release by promiscuous G protein coupling at cell surfaces and by selectively recruiting Gq/11 to early endosomal membranes. Here, we demonstrate that a dileucine endocytic motif is required for directing CaSR to dynamic spatiotemporal pathways by interacting with the adaptor protein-2 σ-subunit, which is mutated in hypercalcemic patients. From early endosomes, internalized CaSR is targeted to spatially distinct pathways involving early-to-recycling and late endosomes, to direct compartment-specific signals and regulate GPCR forward trafficking. The clinically available allosteric modulator cinacalcet biases signaling to preferentially enhance early endosomal-to-recycling pathways, demonstrating the veracity of targeting discrete endosomal pathways. These results reveal that single GPCRs can simultaneously activate spatially distinct endosomal pathways and demonstrate the structural motifs that facilitate such multidimensional signaling.