Direct observation of endocytosis of gastrin releasing peptide and its receptor.
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 degrees C, cy3-GRP and GRP-R were confined to the plasma membrane. After 5 min at 37 degrees 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.
Grady
E F
EF
Department of Surgery, University of California, San Francisco 94143.
Slice
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Brant
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Walsh
J H
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Payan
D G
DG
Bunnett
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NW
D000595:Amino Acid Sequence; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D004705:Endocytosis; D019886:Gastrin-Releasing Peptide; D005755:Gastrins / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D018004:Receptors, Bombesin / Q000378:metabolism*; D013395:Sucrose / Q000494:pharmacology; D014168:Transferrin / Q000378:metabolism
D001152:Arsenicals; D005755:Gastrins; D010455:Peptides; D018004:Receptors, Bombesin; D014168:Transferrin; C029341:oxophenylarsine; D013395:Sucrose; D019886:Gastrin-Releasing Peptide