title: Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells.
abstract: Internalization of the NK1 receptor (NK1R) and substance P was observed in cells transfected with cDNA encoding the rat NK1R by using anti-receptor antibodies and cyanine 3-labelled substance P (cy3-substance P). After incubation at 4 degrees C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37 degrees C, NK1R immunoreactivity and cy3-substance P were internalized into small intracellular vesicles located beneath the plasma membrane. Fluorescein isothiocyanate-labelled transferrin and cy3-substance P were internalized into the same vesicles, identifying them as early endosomes. After 60 min at 37 degrees C, NK1R immunoreactivity was detected in larger, perinuclear vesicles. Internalization of 125I-labelled substance P was studied by using an acid wash to dissociate cell-surface label from that which has been internalized. Binding reached equilibrium after incubation for 60 min at 4 degrees C with no detectable internalization. After 10 min incubation at 37 degrees C, 83.5 +/- 1.0% of specifically bound counts were internalized. Hyperosmolar sucrose and phenylarsine oxide, which are inhibitors of endocytosis, prevented internalization of 125I-labelled substance P and accumulation of NK1R immunoreactivity into endosomes. Acidotropic agents caused retention of 125I-labelled substance P within the cell and inhibited degradation of the internalized peptide. Continuous incubation of cells with substance P at 37 degrees C reduced 125I-substance P binding at the cell surface. Therefore, substance P and its receptor are internalized into early endosomes within minutes of binding, and internalized substance P is degraded. Internalization depletes NK1Rs from the cell surface and may down-regulate the response of a cell to substance P.
authors.affiliation: Department of Surgery, University of California, San Francisco.
authors.forename: A M
authors.initials: AM
authors.lastname: Garland
authors.forename: E F
authors.initials: EF
authors.lastname: Grady
authors.forename: D G
authors.initials: DG
authors.lastname: Payan
authors.forename: S R
authors.initials: SR
authors.lastname: Vigna
authors.forename: N W
authors.initials: NW
authors.lastname: Bunnett
mesh_terms: D000595:Amino Acid Sequence; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology*; D002461:Cell Line, Transformed; D002738:Chloroquine / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D004705:Endocytosis / Q000187:drug effects; D004848:Epithelium / Q000378:metabolism; D006982:Hypertonic Solutions; D007106:Immune Sera; D007150:Immunohistochemistry; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D051381:Rats; D018040:Receptors, Neurokinin-1 / Q000819:agonists / Q000096:biosynthesis / Q000378:metabolism*; D011994:Recombinant Proteins / Q000819:agonists / Q000096:biosynthesis / Q000378:metabolism; D013373:Substance P / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D001152:Arsenicals; D006982:Hypertonic Solutions; D007106:Immune Sera; D007976:Leupeptins; D010455:Peptides; D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins; D000643:Ammonium Chloride; C029341:oxophenylarsine; D013373:Substance P; D002738:Chloroquine; D008985:Monensin; C032854:leupeptin; D003078:Colchicine