VAMP2 is not specifically colocalized with SpH-MOR–containing endosomes. (A) Representative confocal slices of cells expressing SpH-B2AR (light green) immunostained for VAMP2 (orange) and EEA1, Rab11, or Rab7 (purple) after 15 min of agonist treatment. (B) Representative confocal slices of cells expressing SpH-MOR (light green) immunostained for VAMP2 (orange) and EEA1, Rab11, or Rab7 (purple) after 15 min of agonist treatment. Scale bar = 6 µm. (C) Percent colocalization between the three labels calculated using an automated object-based method. Percent colocalization was first analyzed between SpH-B2AR (left) or SpH-MOR (right) and the endosomal markers (outer columns, ±SD), then the colocalized spots were further analyzed for the presence of VAMP2 (VAMP2+, inner dashed columns, ±SD, SpH-B2AR: EEA1: n = 10, Rab11: n = 11, Rab7: n = 10; SpH-MOR: EEA1: n = 12, Rab11: n = 10, Rab7: n = 10, samples of all conditions were from two independent coverslips). (D) Pie charts and stack columns showing the summary statistics of C show no enrichment of VAMP2 in SpH-MOR–containing endosomes. (E) Schematic of a proposed model of VAMP2’s specificity in GPCR recycling. VAMP2 is preferentially copackaged into vesicles from endosomal microdomains or tubules that contain a certain subset of GPCR cargos, such as MOR but not B2AR, allowing it to mediate fusion in a cargo-selective manner. TfR, on the other hand, can enter both microdomains or tubules (and other pathways if present), allowing redundant mechanisms of recycling.