Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells

A. M. Garland, E. F. Grady, D. G. Payan, S. R. Vigna, N. W. Bunnett

Research output: Contribution to journalArticle

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°C, NK1R immunoreactivity and cy3-substance P were confined to the plasma membrane. Within 3 min of incubation at 37°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°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°C with no detectable internalization. After 10 min incubation at 37°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°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.

Original languageEnglish (US)
Pages (from-to)177-186
Number of pages10
JournalBiochemical Journal
Volume303
Issue number1
DOIs
StatePublished - 1994

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Garland, A. M., Grady, E. F., Payan, D. G., Vigna, S. R., & Bunnett, N. W. (1994). Agonist-induced internalization of the substance P (NK1) receptor expressed in epithelial cells. Biochemical Journal, 303(1), 177-186. https://doi.org/10.1042/bj3030177