Direct observation of endocytosis of gastrin releasing peptide and its receptor

E. F. Grady, L. W. Slice, W. O. Brant, J. H. Walsh, D. G. Payan, N. W. Bunnett

Research output: Contribution to journalArticlepeer-review

Abstract

Endocytosis of the gastrin releasing peptide receptor (GRP-R) may regulate cellular responses to GRP. We observed endocytosis in transfected epithelial cells by confocal microscopy using cyanine 3-GRP (cyanine 3.18-labeled gastrin releasing peptide) and GRP-R antibodies. At 4 °C, cy3-GRP and GRP-R were confined to the plasma membrane. After 5 min at 37 °C, ligand and receptor were internalized into early endosomes with fluorescein isothiocyanate-transferrin. After 10 min, cy3-GRP and GRP-R were in perinuclear vesicles, and at 60 min cy3-GRP was in large, central vesicles, while GRP-R was at the cell surface. We quantified surface GRP-R using an antibody to an extracellular epitope and an 125I-labeled secondary antibody. After exposure to GRP, there was a loss and subsequent recovery of surface GRP-R. Recovery was unaffected by cycloheximide, and thus independent of new protein synthesis, but was attenuated by acidotropic agents, and therefore required endosomal acidification. Internalization of 125I-GRP, assessed using an acid wash, was maximal after 10-20 min, and was clathrin- mediated since it was inhibited by hyperosmolar sucrose and phenylarsine oxide. Thus, GRP and its receptor are rapidly internalized into early endosomes and then dissociate in an acidified compartment. GRP is probably degraded whereas the GRP-R recycles.

Original languageEnglish (US)
Pages (from-to)4603-4611
Number of pages9
JournalJournal of Biological Chemistry
Volume270
Issue number9
DOIs
StatePublished - 1995

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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