Neuropeptide signalling at the plasma membrane is terminated by neuropeptide degradation by cell-surface peptidases, and by β-arrestin-dependent receptor desensitization and endocytosis. However, receptors continue to signal from endosomes by β-arrestin-dependent processes, and endosomal sorting mediates recycling and resensitization of plasma membrane signalling. The mechanisms that control signalling and trafficking of receptors in endosomes are poorly defined. We report a major role for endothelin-converting enzyme-1 (ECE-1) in controlling substance P (SP) and the neurokinin 1 receptor (NK 1R) in endosomes of myenteric neurones. ECE-1 mRNA and protein were expressed by myenteric neurones of rat and mouse intestine. SP (10 nm, 10 min) induced interaction of NK 1R and β-arrestin at the plasma membrane, and the SP-NK 1R-β-arrestin signalosome complex trafficked by a dynamin-mediated mechanism to ECE-1-containing early endosomes, where ECE-1 can degrade SP. After 120 min, NK 1R recycled from endosomes to the plasma membrane. ECE-1 inhibitors (SM-19712, PD-069185) and the vacuolar H +ATPase inhibitor bafilomycin A 1, which prevent endosomal SP degradation, suppressed NK 1R recycling by >50%. Preincubation of neurones with SP (10 nm, 5 min) desensitized Ca 2+ transients to a second SP challenge after 10 min, and SP signals resensitized after 60 min. SM-19712 inhibited NK 1R resensitization by >90%. ECE-1 inhibitors also caused sustained SP-induced activation of extracellular signal-regulated kinases, consistent with stabilization of the SP-NK 1R-β-arrestin signalosome. By degrading SP and destabilizing endosomal signalosomes, ECE-1 has a dual role in controlling endocytic signalling and trafficking of the NK 1R: promoting resensitization of G protein-mediated plasma membrane signalling, and terminating β-arrestin-mediated endosomal signalling.
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