DELINEATION OF THE ENDOCYTIC PATHWAY OF SUBSTANCE-P AND ITS 7-TRANSMEMBRANE DOMAIN NK1 RECEPTOR

Many of the actions of the neuropeptide substance P (SP) that are medi ated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We deli neated this endocytic pathway in transfected cells by confocal microsc opy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were int ernalized into the same clathrin immunoreactive vesicles, and then sor ted into different compartments. The NK1-R was colocalized with a mark er of early endosomes, but not with markers of late endosomes or lysos omes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, th ere was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protei n synthesis because it was' unaffected by cycloheximide. Recovery requ ired endosomal acidification because it was prevented by an H+-ATPase inhibitor. The fate of internalized I-125-Sp was examined by chromatog raphy. SP was intact at the cell surface and in early endosomes, but s lowly degraded in perinuclear vesicles. We conclude that SP induces cl athrin-dependent internalization of the NK1-R. The SP/NK1-R complex di ssociates in acidified endosomes. SP is degraded, whereas the NK1-R re cycles to the cell surface.