Visualization of G protein-coupled receptor trafficking with the aid of the green fluorescent protein. Endocytosis and recycling of cholecystokinin receptor type A.
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.
Tarasova
N I
NI
Molecular Aspects of Drug Design Section, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
Stauber
R H
RH
Choi
J K
JK
Hudson
E A
EA
Czerwinski
G
G
Miller
J L
JL
Pavlakis
G N
GN
Michejda
C J
CJ
Wank
S A
SA
D016475:3T3 Cells; D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D004705:Endocytosis; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007295:Inositol Phosphates / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000096:biosynthesis; D051379:Mice; D051381:Rats; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin / Q000096:biosynthesis / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012844:Sincalide / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
D007295:Inositol Phosphates; D008164:Luminescent Proteins; D043886:Receptor, Cholecystokinin A; D011949:Receptors, Cholecystokinin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012844:Sincalide