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(Received for publication, March 7, 1997)
From the A chimeric protein consisting of the
cholecystokinin receptor type A (CCKAR) and the green fluorescent
protein (GFP) was used for studying receptor localization,
internalization, and recycling in live cells in real time in four
different cell lines. Fusion of the C terminus of the CCKAR to the N
terminus of the GFP did not alter receptor ligand binding affinity,
signal transduction, or the pattern of receptor surface expression and
receptor-mediated cholecystokinin (CCK) internalization. The use of a
new GFP mutant with increased fluorescence allowed the continuous
observation of CCKAR-GFP in stably expressing cell lines. Newly
obtained biologically active fluorescent derivatives of CCK were used
for simultaneous observation of receptor and ligand trafficking in CHO,
NIH/3T3, and HeLa cells stably expressing the fluorescent CCKAR and in transiently transfected COS-1 cells. Receptor internalization was
predominantly ligand dependent in HeLa, COS-1, and CHO cells, but was
mostly constitutive in NIH/3T3 cells, suggesting the existence of
cell-specific regulation of receptor internalization. The CCKAR antagonists, L-364,718 and CCK 27-32 amide potently inhibited spontaneous internalization of the receptor. The average sorting time
of CCK and the receptor in the endosomes was about 25 min. The receptor
recycled back to the cell membrane with an average time of 60 min.
While the ligands sorted to lysosomes, no receptor molecules could be
detected there, and no receptor degradation was observed during
recycling. These results demonstrate the usefulness of GFP tagging
for real time imaging of G protein-coupled receptor trafficking
in living cells and suggest that this technique may be
successfully applied to the study of the regulation and trafficking mechanisms of other receptors.
Volume 272, Number 23,
Issue of June 6, 1997
pp. 14817-14824
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
ENDOCYTOSIS AND RECYCLING OF CHOLECYSTOKININ RECEPTOR TYPE
A
,
,
,
and
Molecular Aspects of Drug Design Section,
§ Human Retroviruses Section, 
Confocal Microscopy,
ABL-Basic Research Program, NCI-Frederick Cancer Research and
Development Center, Frederick, Maryland 21702 and the
** Laboratory of Chemical Biology,
¶ Digestive Disease Branch, NIDDK, National Institutes
of Health, Bethesda, Maryland 20892
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