Direct Observation of Endocytosis of Gastrin Releasing Peptide and Its Receptor (*)
- Eileen F. Grady(1),
- Lee W. Slice(4),
- William O. Brant(1),
- John H. Walsh(4),
- Donald G. Payan(3) and
- Nigel W. Bunnett(1)(2)(§)
- From the (1)Departments of Surgery,
- (2)Physiology and
- (3)Medicine, University of California, San Francisco, California 94143 and
- (4)CURE:VA/UCLA Gastroenteric Biology Center, West Los Angeles Veterans Administration Center, Los Angeles, California 90073
- § To whom correspondence should be addressed: University of California, San Francisco, 521 Parnassus Ave., San Francisco, CA 94143-0660. Tel.: 415-476-0489; Fax: 415-476-0936.
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 
I-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 
I-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.
Footnotes
-
↵* This work was supported by National Institutes of Health Grants DK 39957 (to N. W. B. and D. G. P.), DK 43207 (to N. W. B.), NS 21710 (to N. W. B. and D. G. P.), DK 42341 (to J. H. W.), and DK 35740 (to J. H. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- GRP
-
gastrin releasing peptide
- GRP-R
-
gastrin releasing peptide receptor
- cy3-GRP
-
cyanine 3.18-labeled gastrin releasing peptide
- KNRK cells
-
Kirsten murine sarcoma virus transformed rat kidney cells
- DMEM
-
Dulbecco's modified Eagle's medium
- BSA
-
bovine serum albumin
- PBS
-
phosphate-buffered saline
- HPLC
-
high pressure liquid chromatography
- FITC
-
fluorescein isothiocyanate
- BES
-
2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid
- CMV
-
cytometalovirus.
-
↵2E. F. Grady and N. W. Bunnett, unpublished observations.
-
↵3E. F. Grady, A. M. Garland, P. D. Gamp, M. Lovett, D. G. Payan, and N. W. Bunnett, manuscript submitted.
-
- Received September 12, 1994.
