GPCR activation triggers MVB–PM fusion in HeLa cells via SNAP23-Ser110 phosphorylation. (a) Fusion activity of histamine-stimulated cells nontreated or treated with the Gα_q inhibitor UBO-QIC (1 µM). n ≥ 24 cells per condition. (b) Basal fusion activity in cells treated with PKC inhibitors GÖ6976 (1 µM) or GÖ6983 (1 µM). n ≥ 11 cells per condition. (c) Fusion activity of histamine-stimulated cells nontreated or preincubated with GÖ6983 (1 µM). n ≥ 11 cells per condition. (d) Schematic representation of SNAP23 with SNARE motifs, a membrane-anchoring domain (M), and all phosphosites with the posphosite targeted by histamine stimulation (Ser110) in bold. (e) Fusion activity of histamine-stimulated cells transfected with WT SNAP23, phosphomutant SNAP23-S110A, or phosphomimic SNAP23-S110D. n ≥ 16 cells per condition. (f) Left: fusion activity of CD63-, CD81-, and CD9-pHluorin HeLa cells cotransfected with SNAP23 WT or SNAP23-S110A. n ≥ 16 cells per condition. Western blot on exosomes isolated from SNAP23-WT and SNAP23-S110A HeLa cells labeled for CD63, CD9, CD81, flotillin-1, and syntenin-1. (g) Schematic representation of the histamine-stimulated pathway leading to exosome release as identified by phosphoproteomics and specific inhibitors. Blue-rimmed proteins represent the putative pathway implicated by both experiments. The IP3–Ca²⁺ pathway is represented in gray as a direct link with MVB–PM fusion is missing. **, P < 0.01; ****, P < 0.0001 using Student’s two-tailed two-sample t test. All t tests were paired except for b and f. Whiskers in the box plots in b and f represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter.