title: Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor.
abstract: Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there 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 protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.
authors.affiliation: Department of Surgery, University of California, San Francisco 94143, USA.
authors.forename: E F
authors.initials: EF
authors.lastname: Grady
authors.forename: A M
authors.initials: AM
authors.lastname: Garland
authors.forename: P D
authors.initials: PD
authors.lastname: Gamp
authors.forename: M
authors.initials: M
authors.lastname: Lovett
authors.forename: D G
authors.initials: DG
authors.lastname: Payan
authors.forename: N W
authors.initials: NW
authors.lastname: Bunnett
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000502:physiology; D018699:Coated Vesicles / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D011992:Endosomes / Q000737:chemistry / Q000378:metabolism; D000939:Epitopes; D006863:Hydrogen-Ion Concentration; D009842:Oligopeptides; D010455:Peptides; D011188:Potassium / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000032:analysis; D018040:Receptors, Neurokinin-1 / Q000378:metabolism*; D011990:Receptors, Transferrin / Q000032:analysis; D013373:Substance P / Q000378:metabolism* / Q000494:pharmacology; D013395:Sucrose / Q000494:pharmacology
chemical_list: D002966:Clathrin; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D017527:Receptor, IGF Type 2; D018040:Receptors, Neurokinin-1; D011990:Receptors, Transferrin; D013373:Substance P; D013395:Sucrose; C085706:FLAG peptide; D006180:Proton-Translocating ATPases; D011188:Potassium