Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1994 Oct 1;303(Pt 1):177–186. doi: 10.1042/bj3030177

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

A M Garland 1, E F Grady 1, D G Payan 1, S R Vigna 1, N W Bunnett 1
PMCID: PMC1137573  PMID: 7524481

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.

Full text

PDF
177

Images in this article

180Figure 1
on p.180
180Figure 2
on p.180
181Figure 3
on p.181
182Figure 4
on p.182

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Feldman R. D., McArdle W., Lai C. Phenylarsine oxide inhibits agonist-induced changes in photolabeling but not agonist-induced desensitization of the beta-adrenergic receptor. Mol Pharmacol. 1986 Nov;30(5):459–462. [PubMed] [Google Scholar]
  2. Frost S. C., Lane M. D. Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes. J Biol Chem. 1985 Mar 10;260(5):2646–2652. [PubMed] [Google Scholar]
  3. Hausdorff W. P., Caron M. G., Lefkowitz R. J. Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J. 1990 Aug;4(11):2881–2889. [PubMed] [Google Scholar]
  4. Hershey A. D., Krause J. E. Molecular characterization of a functional cDNA encoding the rat substance P receptor. Science. 1990 Feb 23;247(4945):958–962. doi: 10.1126/science.2154852. [DOI] [PubMed] [Google Scholar]
  5. Heuser J. E., Anderson R. G. Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol. 1989 Feb;108(2):389–400. doi: 10.1083/jcb.108.2.389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hopkins C. R., Gibson A., Shipman M., Miller K. Movement of internalized ligand-receptor complexes along a continuous endosomal reticulum. Nature. 1990 Jul 26;346(6282):335–339. doi: 10.1038/346335a0. [DOI] [PubMed] [Google Scholar]
  7. Hoxie J. A., Ahuja M., Belmonte E., Pizarro S., Parton R., Brass L. F. Internalization and recycling of activated thrombin receptors. J Biol Chem. 1993 Jun 25;268(18):13756–13763. [PubMed] [Google Scholar]
  8. Kwatra M. M., Schwinn D. A., Schreurs J., Blank J. L., Kim C. M., Benovic J. L., Krause J. E., Caron M. G., Lefkowitz R. J. The substance P receptor, which couples to Gq/11, is a substrate of beta-adrenergic receptor kinase 1 and 2. J Biol Chem. 1993 May 5;268(13):9161–9164. [PubMed] [Google Scholar]
  9. Larsen P. J., Mikkelsen J. D., Mau S., Saermark T. Binding and internalization of a iodinated substance P analog by cultured anterior pituitary cells. Mol Cell Endocrinol. 1989 Aug;65(1-2):91–101. doi: 10.1016/0303-7207(89)90169-x. [DOI] [PubMed] [Google Scholar]
  10. Lee P. C., Jensen R. T., Gardner J. D. Bombesin-induced desensitization of enzyme secretion in dispersed acini from guinea pig pancreas. Am J Physiol. 1980 Mar;238(3):G213–G218. doi: 10.1152/ajpgi.1980.238.3.G213. [DOI] [PubMed] [Google Scholar]
  11. Lohse M. J., Benovic J. L., Caron M. G., Lefkowitz R. J. Multiple pathways of rapid beta 2-adrenergic receptor desensitization. Delineation with specific inhibitors. J Biol Chem. 1990 Feb 25;265(6):3202–3211. [PubMed] [Google Scholar]
  12. McMillian M. K., Soltoff S. P., Talamo B. R. Rapid desensitization of substance P- but not carbachol-induced increases in inositol trisphosphate and intracellular Ca++ in rat parotid acinar cells. Biochem Biophys Res Commun. 1987 Nov 13;148(3):1017–1024. doi: 10.1016/s0006-291x(87)80233-4. [DOI] [PubMed] [Google Scholar]
  13. Okamoto A., Lovett M., Payan D. G., Bunnett N. W. Interactions between neutral endopeptidase (EC 3.4.24.11) and the substance P (NK1) receptor expressed in mammalian cells. Biochem J. 1994 May 1;299(Pt 3):683–693. doi: 10.1042/bj2990683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Otsuka M., Yoshioka K. Neurotransmitter functions of mammalian tachykinins. Physiol Rev. 1993 Apr;73(2):229–308. doi: 10.1152/physrev.1993.73.2.229. [DOI] [PubMed] [Google Scholar]
  15. Roth N. S., Campbell P. T., Caron M. G., Lefkowitz R. J., Lohse M. J. Comparative rates of desensitization of beta-adrenergic receptors by the beta-adrenergic receptor kinase and the cyclic AMP-dependent protein kinase. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6201–6204. doi: 10.1073/pnas.88.14.6201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sibley D. R., Strasser R. H., Benovic J. L., Daniel K., Lefkowitz R. J. Phosphorylation/dephosphorylation of the beta-adrenergic receptor regulates its functional coupling to adenylate cyclase and subcellular distribution. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9408–9412. doi: 10.1073/pnas.83.24.9408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sjödin L. Cholecystokinin-induced inhibition of endocytosis of receptor-bound substance P in pancreatic acinar cells. J Recept Res. 1992;12(3):323–350. doi: 10.3109/10799899209074799. [DOI] [PubMed] [Google Scholar]
  18. Vigna S. R., Bowden J. J., McDonald D. M., Fisher J., Okamoto A., McVey D. C., Payan D. G., Bunnett N. W. Characterization of antibodies to the rat substance P (NK-1) receptor and to a chimeric substance P receptor expressed in mammalian cells. J Neurosci. 1994 Feb;14(2):834–845. doi: 10.1523/JNEUROSCI.14-02-00834.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Wang L. H., Mantey S. A., Lin J. T., Frucht H., Jensen R. T. Ligand binding, internalization, degradation and regulation by guanine nucleotides of bombesin receptor subtypes: a comparative study. Biochim Biophys Acta. 1993 Jan 17;1175(2):232–242. doi: 10.1016/0167-4889(93)90028-n. [DOI] [PubMed] [Google Scholar]
  20. Wileman T., Harding C., Stahl P. Receptor-mediated endocytosis. Biochem J. 1985 Nov 15;232(1):1–14. doi: 10.1042/bj2320001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Yokota Y., Sasai Y., Tanaka K., Fujiwara T., Tsuchida K., Shigemoto R., Kakizuka A., Ohkubo H., Nakanishi S. Molecular characterization of a functional cDNA for rat substance P receptor. J Biol Chem. 1989 Oct 25;264(30):17649–17652. [PubMed] [Google Scholar]
  22. Yu S. S., Lefkowitz R. J., Hausdorff W. P. Beta-adrenergic receptor sequestration. A potential mechanism of receptor resensitization. J Biol Chem. 1993 Jan 5;268(1):337–341. [PubMed] [Google Scholar]
  23. Zachary I., Rozengurt E. Internalization and degradation of peptides of the bombesin family in Swiss 3T3 cells occurs without ligand-induced receptor down-regulation. EMBO J. 1987 Aug;6(8):2233–2239. doi: 10.1002/j.1460-2075.1987.tb02495.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Zhou X. M., Fishman P. H. Desensitization of the human beta 1-adrenergic receptor. Involvement of the cyclic AMP-dependent but not a receptor-specific protein kinase. J Biol Chem. 1991 Apr 25;266(12):7462–7468. [PubMed] [Google Scholar]
  25. Zhu W. Y., Göke B., Williams J. A. Binding, internalization, and processing of bombesin by rat pancreatic acini. Am J Physiol. 1991 Jul;261(1 Pt 1):G57–G64. doi: 10.1152/ajpgi.1991.261.1.G57. [DOI] [PubMed] [Google Scholar]
  26. von Zastrow M., Kobilka B. K. Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors. J Biol Chem. 1992 Feb 15;267(5):3530–3538. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES