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(Received for publication, March 7, 1997)
From the ABSTRACT 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.
INTRODUCTION G protein-coupled receptors (GPCR)1
are involved in numerous biological processes ranging from peptide
hormone and neurotransmitter-regulated function to smell, taste, and
light receptors and to viral entry into cells. This very broad spectrum
of activities indicates that these receptors have a central role in
cell biology and in many cases may be attractive targets for drug
development. G protein-coupled receptor function is significantly
regulated by the mechanisms that determine receptor trafficking within
the cell. The molecular and cellular mechanisms involved in regulation
of translocation, sequestration, recycling, and degradation of G
protein-coupled receptors are not well understood, and the available
data are largely controversial. The study of the trafficking by means
of localization of the receptors with antibodies is laborious and does
not allow for the efficient observation of receptor translocation in
intracellular compartments. The green fluorescent protein (GFP) from
jelly fish is becoming widely used as a molecular reporter to monitor
gene expression, localization, and intracellular protein trafficking in
living cells (1-3). Many GFP-tagged proteins retain their biological
activity and have the same trafficking pattern as the native proteins
(4-8). However, the majority of the proteins that have been labeled
with GFP for microscopy studies are expressed at relatively high
levels, which makes the detection of the fusion proteins significantly
easier. Localization of GPCR is a technically challenging procedure
because most of the receptors are expressed at very low levels. The
approaches utilizing either cells that overexpress the receptors or the
use of GFP mutants with much higher fluorescence than that of the wild
type protein can be applied for the localization of labeled GPCR in
living cells. In the present paper we have explored the possibilities
of GFP labeling for the study of G protein-coupled receptor trafficking using the type A cholecystokinin receptor (CCKAR). Cholecystokinin, a
well studied bioactive peptide, was initially discovered in the gut and
subsequently in the central nervous system (9). It exerts numerous
effects through its action as a hormone and a neurotransmitter. The
CCKAR was well characterized with respect to the mechanisms of signal
transduction (10) and phosphorylation upon ligand binding (11). It was
shown to undergo sequestration upon binding of agonists (12-17) like
many other GPCR. It was suggested that internalization of the CCKAR and
many other GPCRs is ligand-dependent, but direct proof has
been missing. It is also unclear whether only binding of agonists
versus antagonists can trigger GPCR internalization, since
it has been shown that signal transduction is not necessary for
internalization (18). Furthermore, cell surface receptor trafficking
has not been directly observed previously in real time in living cells.
To address these questions, we have developed the tools that allowed
for the direct observation of the CCKAR and its ligand in living cells
in real time, and have applied them for the study of receptor and
ligand trafficking in four different cell types. Attachment of GFP did
not influence receptor expression, affinity, signal transduction, or
internalization. The fluorescence of the newly created mutant of
GFP2 was intense enough for observation of
the CCKAR by confocal microscopy in different cell types that expressed
the receptor at natural levels. Direct observation of the CCKAR-GFP
fusion receptor proved to be a powerful tool for studying
ligand-dependent receptor internalization and
recycling.
EXPERIMENTAL PROCEDURES Materials
Rhodamine Green trifluoroacetic acid, succinimidyl ester,
rhodamine red concanavalin A (ConA), tetramethylrhodamine-transferrin, hexyl ester of rhodamine 6G, MitoTracker Red CMXRos, and LysoTracker Red and Green dyes were purchased from Molecular Probes Inc. (Eugene, OR). Cholecystokinin-8 was purchased from Research Plus, Inc. (Bayonne,
NJ). Cy3.29 N-hydroxysuccimidyl esters was a kind gift from
Dr. Brigitte Schmidt (Center for Light Microscope Imaging and
Biotechnology, Carnegie Mellon University, Pittsburgh, PA).
NIH/3T3, CHO, HeLa, and COS-1 cells were obtained from the American
Type Culture Collection (Rockville, MD).
Methods
The full open reading frame of the rat CCKAR (nucleotides
199-1485; Ref. 20) either with CCKAR-GFP or with GFP-CCKAR was amplified using appropriate primers containing EcoRI and
XbaI (CCKAR-hGFP) or XbaI and BamHI
along with a stop codon (hGFP-CCKAR) restriction sites at the 5 Alternatively, to generate pF25-CCKAR-GFP, the coding region of the rat
CCKAR was amplified by PCR using appropriate primers containing
NheI restriction sites. The PCR product was digested with
NheI and cloned into the NheI-cut vector
pCMV-GFPsg25. Plasmid pCMV-GFPsg25 expresses a mutated GFP protein
under the control of the early cytomegalovirus
promoter.2
Two micrograms of pCDL-SR The wild type rat CCKAR and CCKAR-hGFP cDNAs were
stably transfected by electroporation (500 millifarads, 0.25 kV,
Bio-Rad Gene Pulser) of 2 × 107 CHO and
NIH/3T3-cell/ml in a volume of 0.25 ml with 20 µg of the linearized
recombinant pCDL-SR Stably transfected
CHO cells were plated at a density of 2.0 × 105
cells/well in 24-well tissue culture plates and assayed the following day for radioligand binding. For binding displacement studies, cells
were incubated for 90 min with 50 pM 125I-CCK-8
in the absence or presence of increasing concentrations of cold CCK-8
in 0.5 ml of DMEM, 0.1% BSA at 37 °C. Nonspecific binding was
defined as total binding in the presence of 1 µM cold CCK-8 and was always less than 10% of total binding. After termination of the binding reaction by washing the cells two times with PBS, 0.1%
BSA at 4 °C, cells were solubilized with 1 ml of 0.1 N
KOH and radioactivity was detected in a Packard/Autogamma counter. Binding parameters, Kd and
Bmax, were determined with the nonlinear least
squares curve fitting computer program, LIGAND (22).
The wild type rat CCKAR and CCKAR-GFP
cDNAs were sequenced using the Dye Terminator kit and a model 377 DNA Sequencer (Applied Biosystems).
Stably transfected CHO cells were
plated at a density of 2.0 × 105 cells/well on
24-well culture plates with DMEM, 10% calf serum in the presence of
100 mCi/ml myo-2-[3H]inositol and incubated
overnight. The following day, the medium was aspirated and the cells
were incubated with PI buffer (20 mM HEPES, 2 mM CaCl2, 1.2 mM MgSO4,
10 mM LiCl, 11.1 mM, glucose, 0.5% BSA) and
exposed to the indicated concentrations of peptide. Total
[3H]inositol phosphates were measured by strong anion
exchange chromatography (Dowex AG 1-X8), using a modification of the
method described by Berridge et al. (23). Eluates were
assayed using a Beckman liquid scintillation counter.
Cholecystokinin octapeptide (6 mg, 5 µmol) was
dissolved in a mixture of acetonitrile (50 µl) and aqueous solution
of sodium bicarbonate (0.2 M, pH 9.0, 50 µl). A solution
of either Rhodamine Green trifluoroacetic acid, succinimidyl ester (5 mg, 7.5 µmol) or Cy3.29 succinimidyl ester (5 mg, 7.5 µmol) in
dimethylformamide (10 µl) was added under nitrogen in the dark. The
reaction mixtures were stirred at room temperature overnight. A fresh,
aqueous solution of hydroxylamine (25 µl, 1.5 M, pH = 7.8) was added. After 2 h the reaction mixtures were dissolved
in 30% aqueous acetonitrile (4 ml), and products were separated by
HPLC under reverse-phase conditions using a semipreparative YMC-Pack
ODS-AM column (10 mm × 300 mm), (eluent:
acetonitrile/water/0.05% trifluoroacetic acid, gradient 30-70% of
acetonitrile, 60 min). Yields were 30-50%. NMR spectroscopy was
performed on a Varian VXR-500 spectrometer (500 MHz for
1H). The mass spectra were generated on a Bruker Reflex II
time of flight mass spectrometer. For RG-CCK-8, mass spectra were as follows:
C70H73N12O20S3Na
calculated, 1521.6; found, m/z = 1521.9; C70H73N12O17S2Na
calculated, 1441.5; found, m/z = 1441.7;
C70H74N12O17S2 calculated, 1419.5; found, m/z = 1420.2. For Cy3.29-CCK-8,
mass spectra were as follows:
C76H90N12O23S5
calculated, 1699.9; found, m/z = 1696.8. Double quantum
tilted correlation NMR spectra of RG-CCK-8 and Cy3.29-CCK-8 (data not
shown) had all expected cross-peaks for NH- Cells were grown in Nunc
cover glass chamber slides in medium without phenol red and observed on
a Zeiss inverted LSM 410 laser scanning confocal microscope.
Fluorescence of rhodamine green, LysoTracker Green, and GFP was excited
using a 488-nm argon/krypton laser, and emitted fluorescence was
detected with 515-540-nm band pass filter. For LysoTracker Red,
rhodamine red, Cy3.29-CCK-8, and tetramethylrhodamine, a 568-nm
helium/neon laser was used for excitation and fluorescence was detected
with a 590-nm band pass filter.
The cells were incubated for 1 h with various
concentrations of the compound in phenol red-free medium in a
CO2 incubator. Cells were rinsed three times with medium
and observed under the microscope using identical parameters for all
concentrations. The images were stored on an optical disk and analyzed
with Zeiss LSM software. A minimum of 10 images for each concentration
were quantitated.
The cells were rinsed with cold PBS and
treated with rhodamine red concanavalin A (10 µg/ml in PBS) for 2 min
at 4 °C. After a final rinse with cold PBS, the cell were observed
with a confocal microscope using 63× oil immersion lens, a pinhole of
35, and an electronic zoom of 3 to yield a final magnification of
2016× of the stored images. Fluorescence pixels of the green
fluorescence and of the colocalization mask of green and red
fluorescence were counted with Zeiss LSM software. The percentage of
the receptor on the cell surface was determined as the ratio of the
colocalization mask pixels and total green fluorescence.
The cells were
preincubated for 30 min with 100 nM RG-CCK-8 or
Cy3.29-CCK-8 at 4 °C, rinsed with cold medium, and placed under the
microscope. Images of a selected group of cells were taken at 1-min
intervals at 20 °C.
The cells were treated with the
lysosomal markers at 37 °C in CO2 incubator for 15 min,
rinsed with medium and incubated with 100 nM RG-CCK-8 or
Cy3.29-CCK-8 in a CO2 incubator for various time periods,
rinsed, and observed with the confocal microscope.
CHO
cells, transfected with rat CCK-A receptor cDNA were grown almost
to confluence and pretreated with 25 µg/ml cycloheximide. After 30 min of incubation, 100 nM RG-CCK-8 was added in the medium without phenol red. The cells were incubated in a CO2
incubator for various time periods, rinsed with medium and observed
with the inverted confocal microscope. A minimum of 10 images for each incubation time point were recorded, and the fluorescence intensity was
quantitated by processing the data using Zeiss LSM software.
RESULTS Construction and Characterization of CCKAR and GFP Fusion
Proteins
To study the localization and trafficking of the CCKAR in live
cells, we constructed a chimeric cDNA of the coding region of the
rat CCKAR and fused its C terminus before the stop codon, to the full
coding region of the mutant GFP cDNA. The C terminus of GFP was
also fused to the N terminus of the receptor resulting in GFP-CCKAR.
The receptor fused to the T65S mutant of GFP (24) could be visualized
by CLSM in transiently transfected COS-1 that overexpressed CCKAR-GFP,
but the intensity of T65S GFP fluorescence was not sufficient for
detection of receptor molecules in stably transfected NIH/3T3 cells
that expressed natural levels of the receptor. The use of the GFP
molecule with mutations that were found previously to increase the
fluorescence approximately 150-fold (F64L, T65C, and
I167T)2 allowed for direct observation of the fusion
receptor in stably transfected cells. GFP-CCKAR, when expressed in
COS-1 cells, showed no binding of 125I-CCK-8. Confocal
microscopy revealed that GFP-CCKAR had an intracellular localization,
suggesting that attachment of the GFP to the N terminus of the CCKAR
abrogates the transport of the receptor to the cell membrane.
To determine the effect of fusing GFP to the intracellular C terminus
of the CCKAR on receptor function, CHO cells expressing wild type CCKAR
were compared with CHO cells expressing CCKAR-GFP for cell surface
receptor density, CCK-8 affinity, and CCK-8-stimulated signal
transduction. The CHO cells expressed CCKAR-GFP on their surface with a
similar density (Bmax = 1.75 ± 0.63 × 105 receptors/cell) and affinity for CCK-8
(Kd = 5.93 ± 2.19 × 10
Fluorescent Derivatives of CCK-8
In a previous study we compared the properties of three
fluorescent derivatives of gastrin, rhodamine green, BODIPY, and Cy3.29 heptagastrin (25). The rhodamine green (RG) moiety provided the
strongest fluorescence intensity, chemical and light stability and
resistance to intracellular degradation within the cells necessary for
the kinetic studies of ligand uptake. Cy3.29 derivatives have emission
maximum at 570 nm and do not emit below 540 nm and thus allow for
simultaneous detection of the GFP fluorescence. Rhodamine green and
Cy3.29-CCK-8 were obtained by the method used earlier for the synthesis
of heptagastrin derivatives (25). The structures of the compounds were
confirmed by NMR and mass spectroscopy. Both RG and Cy3.29-CCK-8
retained a high affinity toward the gastrin receptor
(Kd = 8 ± 3.0 nM in displacement
of 125I-labeled cholecystokinin-8 by RG-CCK-8) and showed
specific binding to NIH/3T3, CHO, and HeLa cells stably transfected
with rat CCKAR cDNA, but not to untransfected cells. The
concentration dependence of binding of RG-CCK-8 and Cy3.29-CCK-8 to
NIH/3T3 cells transfected with CCK-A receptor cDNA (Fig.
3A) showed a typical saturation curve,
usually observed for radiolabeled hormones upon binding to their
receptors. The KD for the binding was calculated from these data to be 0.7 ± 0.2 nM for RG-CCK-8 and
2.5 ± 0.5 nM for Cy3.29-CCK-8. Preincubation of the
cells with 10 µM cholecystokinin for 10 min reduced
fluorescence of bound fluorescent derivatives of CCK to background
levels. Both compounds induced intracellular Ca2+ release
in NIH/3T3 cells transfected with CCKAR at concentrations as low as 10 nM (Fig. 3B), which was identical to that of the parent peptide, indicating that the derivatives retained the biological properties of CCK-8.
To estimate the time needed for internalization, the cells expressing
the CCKAR or CCKAR-GFP were saturated with Cy3.29-CCK-8 at 4 °C for
30 min, rinsed with medium, and observed by confocal microscopy at
20 °C at 1-min intervals. No internalization was observed at
4 °C, but at 20 °C the bound ligand was clustered into aggregates
on the cell surface with evidence of internalization as early as 4-7
min of incubation. The pattern of ligand binding and internalization
time was identical in the case of the cells expressing wild type CCKAR
and CCKAR-GFP (data not shown) and identical to cholecystokinin
receptor type B, which had been characterized earlier (26).
Intracellular stability of rhodamine green dye allowed us to develop a
technique for the study of accumulation of fluorescent peptides within
cells (26), which was applied to RG-CCK-8. The curve for the
accumulation of the fluorescent ligand with time (Fig.
4) revealed a periodic pattern, that was observed
earlier for the gastrin/cholecystokinin B receptor (26) and suggests that there is a periodicity for receptor recycling back to the cell
surface and subsequent uptake of RG-CCK-8. Assuming that the plateaus
on the curve (Fig. 4) correspond to the periods when nearly all
receptor molecules are sequestered, an average recycling time for the
CCKAR deduced from these data is approximately 60 min. The kinetics of
ligand accumulation in the cells expressing the fluorescent receptor
could not be studied because the fluorescence of RG-CCK-8 interferes
with the fluorescence of GFP and the Cy3.29 moiety was not sufficiently
stable within intracellular compartments.
Intracellular Localization of CCKAR-GFP in Different Cell Types
in the Absence of Ligands
The major part of the green fluorescence corresponding to
CCKAR-GFP was observed on the cell membrane in CHO and HeLa cells stably transfected with CCKAR-GFP (Fig. 5, A
and B). The receptor molecules appeared to be evenly
distributed on the plasma membrane. A significant part of the
fluorescence was on the cellular membrane in transiently transfected
COS-1 cells (Fig. 5D). However, COS-1 and HeLa cells also
had significant labeling of the endoplasmic reticulum (ER) (identified
with the help of the red fluorescent ER marker, the hexyl ester of
rhodamine 6G (27); Fig. 6, A and B, respectively). In CHO cells, a small part of the
fluorescence was diffusely distributed throughout the cytoplasm (Fig.
5A). Surprisingly, in stably transfected NIH/3T3 cells, the
fluorescent receptor had a predominantly intracellular localization
(Fig. 5C). To determine the relative distribution of the
fluorescent CCKAR-GFP, the cell surface was labeled by a brief exposure
to rhodamine B concanavalin A. The green fluorescence of CCKAR-GFP that
appeared to colocalize with the red fluorescence of ConA was considered
to correspond to the receptor molecules residing on the plasma
membrane. Total CCKAR-GFP fluorescence and the fluorescent component
that colocalized with ConA were quantitated with the help of Zeiss LSM
software. In the absence of the ligand, CHO cells had 75.8 ± 9.4% of the receptors on the cell surface, HeLa cells had 64 ± 9.1%, and NIH/3T3 had 25.6 ± 6.3%. In transiently transfected
COS-1 cells that overexpressed CCKAR-GFP, part of the GFP fluorescence
colocalized with the mitochondrial marker, MitoTracker Red CMXRos (28),
while no receptor could be detected in mitochondria in stably
transfected cells (data not shown). Fluorescence could also be detected
in endosomes and lysosomes, indicating that overexpression of the
receptor may change the pattern of receptor localization.
Ligand-induced Translocation of CCKAR-GFP
In all three stably transfected cell
lines, addition of CCK-8 caused the green fluorescence of the receptor
to disappear almost completely from the cell surface and to move into
intracellular vesicles identified by the tetramethyl rhodamine
derivative of transferrin, used as a marker of endosomal compartments.
In the absence of the ligand, almost no receptor molecules could be
detected in endosomes in CHO and HeLa cells (Fig.
7A). Within minutes after addition of the
ligand, significant intracellular colocalization of the receptor with
transferrin could be observed indicating the occurrence of
ligand-induced endocytosis of the receptor (Fig. 7B). In
NIH/3T3 cells, CCKAR-GFP showed partial colocalization with transferrin
even in the absence of the ligand (Fig. 7C), suggesting that
spontaneous endocytosis may occur in this cell type. In transiently
transfected COS-1 cells even an excess of the ligand did not deplete
the receptor from the plasma membrane, suggesting that a fraction of
the receptor molecules in the cells that overexpress CCKAR-GFP either
lack the ability to be internalized or overwhelm the endocytic
machinery.
CCKAR antagonists L-374,718 and CCK
27-32 amide not only did not stimulate internalization of the receptor
but significantly inhibited the spontaneous endocytosis of CCKAR-GFP.
In NIH/3T3 cells, incubation with 100 nM L-364,718 and 10 µM CCK-27-32 amide for 2 h increased the amount of
the receptor on the cellular membrane to 74.6 ± 5.4% in case of
L-364,718 and to 68.4 ± 4.8% in case of CCK 27-32 amide,
compared with 25.6 ± 6.3% in the absence of the antagonists.
Intracellular Sorting of the Ligand and the Receptor
The optical properties of the receptor agonist Cy3.29-CCK-8
allowed for simultaneous observation of the receptor and the ligand during endocytosis in real time (Fig. 8). To observe the
changes in receptor and ligand colocalization with time, several
chamber slides with CHO cells expressing CCKAR-GFP were treated with
Cy3.29-CCK-8 at 37 °C for 10 min, rinsed, and incubated at 37 °C
in the presence of the protein synthesis inhibitor, cycloheximide. At
various time intervals, the slides were removed from the incubator, the images of the green and red fluorescence were recorded, and the pixels
corresponding to CCKAR-GFP and Cy3.29 fluorescence and to the
colocalization mask were counted with the help of Zeiss LSM software.
The degree of colocalization was determined as the ratio of green
fluorescence that colocalized with the red fluorescence to the total
green fluorescence of CCKAR-GFP. The degree of colocalization was found
to decrease rapidly with incubation time (Fig. 9). The half-time of dissociation of the receptor from the ligand in CHO cells
was approximately 25 min (Fig. 9). After 1 h of incubation at
37 °C, all ligand-associated red fluorescence was found in the
intracellular vesicles (presumably lysosomes), while the majority of
receptor molecules shuttled back to the cell surface (Fig. 8C). Cy3.29-CCK-8 and RG-CCK-8 showed significant
colocalization with the lysosomal markers LysoTracker Green and Red
(29) in CHO, HeLa, and NIH/3T3 cells stably transfected with wild type CCKAR (Fig. 10A), while there was very
little colocalization of the fluorescent receptor with LysoTracker Red
after 10, 20, 30, and 40 min of incubation with CCK-8 (Fig.
10B). Incubation of the cells expressing CCKAR-GFP with
CCK-8 in the presence of the protein synthesis inhibitor cycloheximide
for 5 h did not change the fluorescence of the cells, indicating
that there was no significant degradation of the receptor-GFP fusion
protein during ligand-induced trafficking.
Time Course of Receptor Recycling
The relative amount of CCKAR-GFP on the cell surface was
determined after exposure to the agonist CCK-8. CCKAR-GFP-expressing CHO cells were pretreated with cycloheximide to inhibit de
novo synthesis of receptor molecules, and incubated with CCK-8 for 10 min at 37 °C, a time period previously shown to be sufficient to
cause internalization of the receptor from the cell surface. The cells
were then rinsed with a medium containing cycloheximide and left in the
incubator for varying time intervals, and briefly exposed to ice-cold
rhodamine B ConA and observed by CLSM. The number of receptor molecules
on the cell surface increased gradually after 10 min exposure to CCK-8
and returned to the initial value of the untreated cells within 60 min
(Fig. 11), consistent with the data from Fig. 4.
DISCUSSION CCK acting at CCKARs present in the gastrointestinal tract and
nervous systems regulates the physiological processes governing digestion and satiety (9, 30). At the cellular level, the CCKAR
mediates ligand-induced activation of phospholipase C, adenylyl cyclase, and phospholipase A2 (10, 31). For several
receptors, the cellular responses to effectors have been shown to be
regulated in part by the movement and compartmentalization of the
receptors and their effectors within the cell. Elucidation of the
mechanisms underlying the trafficking of receptors and their effectors
is therefore critical for understanding the physiological response to a
variety of hormones such as CCK. Earlier studies have demonstrated sequestration of the CCKAR upon binding of agonists (12, 13) and
internalization of CCK in pancreatic acinar cells and in CHO cells
transfected with CCKAR cDNA (14-17). The present work describes the study of CCKAR trafficking upon binding of agonists and antagonists that was observed in real time in four different cell types. The convenience of labeling of the receptor with the highly fluorescent marker, GFP, in combination with the recent advances in the techniques of laser scanning confocal microscopy and the development of image processing software, allowed for not only qualitative, but also quantitative characterization of spontaneous and ligand-induced receptor trafficking. Direct observation of fluorescent receptor allowed the characterization of CCKAR-GFP localization and distribution inside the cells avoiding the artifacts of fixation.
Our previous studies have demonstrated that the C terminus of the CCKAR
is totally dispensable for ligand binding, signal transduction, and
internalization of the CCKAR (32). Consistent with these studies, the
attachment of GFP to the C terminus of the receptor did not cause
noticeable changes in receptor function demonstrated by radioligand
displacement studies and CCK-stimulated increase in total
[3H]inositol phosphates in CHO cells stably expressing
wild type CCKAR and CCKAR-GFP, nor were there noticeable changes in the pattern and rate of receptor-mediated ligand internalization. Fusion of
the N terminus of the CCKAR to the C terminus of GFP (resulting in
GFP-CCKAR) disrupted the transport of the receptor to the plasma
membrane. In COS-1 cells expressing GFP-CCKAR, the fluorescent receptor
was distributed throughout the cytoplasm and nucleus, and no binding of
CCK could be detected. In contrast, when the C terminus of the receptor
was fused to the C terminus of GFP, the resulting fusion receptor was
correctly transported to the cell surface. The GFP-tagged receptor
molecules on the plasma membrane were able to bind CCK, transduce a
signal, and be internalized similar to the wild type receptor.
CCKAR-GFP was distributed evenly along the cell membrane in all studied
cell lines, suggesting that there is no preassociation of the receptor
molecules with certain parts of the membrane. In contrast, the
endothelin receptor, which also belongs to the GPCR family, was found
to reside in caveolae in COS-1 cells overexpressing the receptor, and
immunofluorescence microscopy revealed a distribution of the receptor
to many small micropatches in the periphery of the cells (33). Thus,
different GPCRs may differ in distribution along the membrane.
In all three cell lines stably expressing the fluorescent CCKAR, a
significant fraction of the receptor molecules was found in the
intracellular compartments even in the absence of the ligand. Intracellular receptor molecules were localized predominantly at the
site of their synthesis, the endoplasmic reticulum. The receptor was
found in the ER in all studied cell lines. CCKAR-GFP molecules in the
ER do not appear to be short-lived, since incubation of the cells with
the protein synthesis inhibitor cycloheximide did not lead to complete
disappearance of CCKAR-GFP from the ER. The amount of the receptor in
the ER differed from one cell line to another significantly, suggesting
that the time needed to transport CCKAR from the ER is different in
different cell types.
Overexpression of CCKAR-GFP led to saturation of all intracellular
membranes with the receptor. In COS-1 cells that overexpressed the
fluorescent receptor, fluorescence corresponding to CCKAR-GFP was
detected not only in the ER, endosomes, and plasma membrane (as in the
cells with natural levels of receptor expression), but also in
lysosomes and mitochondria. Although COS-1 cells that were transiently
transfected with CCKAR-GFP had normal binding of the radiolabeled
hormone, microscopy studies have revealed that the majority of the
receptor molecules on their cell surface was not able to be
internalized even in the presence of micromolar concentrations of the
ligand, suggesting that they were not functionally coupled for
internalization. Overexpression of GPCR and their mutants in COS-1
cells is frequently applied for the functional studies of the
receptors. Our results suggest that this strategy should be used with
caution, since unnaturally high levels of receptor expression may
titrate out cellular factors that normally participate in their
function, thus changing the properties of the system and creating
artifacts.
Receptors are frequently categorized into two types, based on their
pattern of internalization, receptors that internalize and recycle
constitutively and receptors that internalize upon binding of the
agonist (39, 40). Our results suggest that both ligand-induced and
spontaneous internalization of the same GPCR may exist and one pathway
can prevail over the other depending on the cell type. Constitutive
internalization of the CCKAR was negligible in HeLa and CHO cells, but
was very significant in NIH/3T3 cells expressing a similar number of
receptors, and it could be inhibited with the receptor antagonists
L-364,718 and CCK 27-32 amide in all cell lines. Inhibition led to an
increased number of receptor molecules on the cell surface. It was
suggested that arrestins (41) and possibly other molecules (42, 43) mediate the internalization of GPCR. Arrestins are able to interact with both the resting receptor molecules and ligand bound receptors, although the affinity of the arrestin toward ligand-induced
phosphorylated receptor is much higher (19). If the mediators of
internalization are present in certain cells at higher levels, some
spontaneous internalization may take place. We speculate that different
levels of arrestins or other cellular mediators of endocytosis in
different cell lines may be responsible for the observed differences in the degree of constitutive internalization of the CCKAR.
Antagonists may stabilize receptor molecules in an inactive
conformation that has lower affinity for an interaction with an internalization factor, thus inhibiting spontaneous internalization. This property may not be universal for all antagonists. Antagonists that behave as inverse agonists stabilize the inactive conformation of
the receptor and inhibit spontaneous signal transduction. The inverse
agonism of L-364,718 and CCK 27-32 amide was not tested in signal
transduction previously, but in internalization of the CCKAR they have
demonstrated the properties of inverse agonists. Further studies of
different types of antagonists are needed to understand whether it is
possible to activate internalization of the receptor without activating
signal transduction, and GFP-tagged receptors can provide the tools for
such studies.
The recycling time of the CCKAR estimated from the direct observation
of CCKAR-GFP after binding of the ligand (Fig. 11) correlates with the
kinetics of fluorescent ligand uptake (Fig. 4). The recycling time of
CCKAR-GFP was found to be within the range of recycling times for the
other GPCR, which are between 20 and 60 min (26, 35-38). The time
needed for intracellular sorting of the ligand and receptor has not
been previously estimated. It is remarkable that the sorting of CCK-8
and the CCKAR is very efficient, since no receptor molecules could be
detected in the lysosomes and no degradation of the receptor could be
observed by measurement of CCKAR-GFP fluorescence after prolonged
exposure to CCK in the presence of the protein synthesis inhibitor,
cycloheximide. Studying the time dependence of RG-CCK-8 accumulation
inside the cells confirmed the stability of the receptor molecules
during recycling. As demonstrated in Fig. 4, the uptake of the ligand
during the second and the third cycles is not much different from that
of the first cycle. However, such efficient sorting may not be a common
property of all GPCR. The hCG/LH receptor, for example, is degraded in
lysosomes after internalization and only a very small portion of
receptor molecules recycle back to the cell surface (35). Most of the
thrombin receptor molecules are degraded after endocytosis, with only
approximately 25% returning to the cell surface (36).
Thyrotropin-releasing hormone receptor is recycled, but a fraction of
the internalized receptor is targeted to a degradative pathway and
results in down-regulation of the receptor following prolonged exposure
to the ligand (37). Application of GFP tagging may allow for a direct
visualization of the fate of other GPCRs after ligand binding and
internalization, thus contributing to a better understanding of
receptor function.
CONCLUSIONS Fusion of the C terminus of GPCR to the N terminus of the GFP
mutant was found to be a valuable tool in studying receptor localization and trafficking. CCKAR-GFP allowed for the direct observation of spontaneous and ligand-induced internalization of the
receptor. Application of two-color microscopy to the receptor-GFP fusion protein and fluorescent ligands allowed for observation of
receptor and ligand sorting inside the endosomes in living cells in
real time. GPCR fusion to GFP has a great potential in studying the
structural requirements to GPCRs and their agonists and antagonists in
evoking receptor trafficking. It can also help elucidate the cellular
mechanisms of receptor translocation to the plasma membrane after
biosynthesis, molecular mechanisms of receptor sequestration upon
binding of the ligand, characterization of cellular factors involved in
receptor trafficking, and identification of structural elements of
GPCRs that define receptor localization.
FOOTNOTES REFERENCES
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
and 3
ends, respectively, using PCR. The humanized S65T GFP, phGFP-S65T
(CLONTECH), was mutated to phGFP-F64L,T65C,I167T by
site-directed mutagenesis (Muta-Gene® phagemid in
vitro mutagenesis kit, Bio-Rad).2 Either
XbaI and BamHI along with a stop codon
(CCKAR-hGFP) or EcoRI and XbaI (hGFP-CCKAR)
restriction sites were added to the 5 and 3 ends of the mutated hGFP
using PCR. Either the 5 or 3 end of CKKAR was ligated to the 3 or
the 5 end of the mutant hGFP, respectively, with T4 DNA ligase (Life
Technologies, Inc.) and the fused product subcloned into either
pCDL-SR
or pCDL-SR/Neo at the EcoRI and
BamHI sites.
containing
either the wild type rat CCKAR, CCKAR-hGFP, or hGFP-CCKAR cDNA
insert subcloned at the EcoRI and BamHI sites in
the sense orientation were transfected into near confluent COS-1 cells
using the DEAE/dextran method as described (21). Approximately 24 h after transfection, cells were trypsinized and replated on glass
coverslips for examination by confocal laser scanning microscopy at
48 h.
/Neo containing either the wild type rat CCKAR or
CCKAR-hGFP cDNA subcloned at the EcoRI and
BamHI sites in the sense orientation in the presence of 500 µg/ml salmon sperm DNA as a carrier. Cell clones stably expressing the receptors were then selected for G-418 resistance (250 µg of
G-418/ml) and by fluorescence-activated cell sorting (Coulter, ECP-
Elite, Miami, FL). NIH/3T3 cells were maintained in DMEM, 10% calf
serum, 250 µg/ml G-418, and CHO cells were maintained in Ham's F-12
medium, 10% fetal bovine serum, 250 µg/ml G-418 at 37 °C in a 6%
CO2 atmosphere. To generate CCKAR-GFPsg25-expressing HeLa cell lines, 5 × 105 HeLa cells were transfected
with 10 µg of pF25CCKAR-GFP by the calcium phosphate method. Two days
after transfection, single cell fluorescence-activated cell sorting was
performed into 96-well plates without prior selection. Sorting was
performed on a FACStar Plus platform (Becton Dickinson). An argon ion
laser at a wavelength of 488 nm was used to excite GFPsg25 (run at 200 milliwatts with a 500-nm long pass emission filter). Positive,
CCKAR-GFPsg25-expressing single cell colonies were identified using an
inverted fluorescence microscope and further expanded in DMEM, 10%
calf serum, 500 µg/ml G-418).
H-
H systems, and
aliphatic and aromatic parts of the molecules.
9)
compared with CHO cells expressing wild type CCKAR
(Bmax = 1.70 ± 0.29 × 105 receptors/cell and Kd = 3.78 ± 0.71 × 109) (Fig. 1). Consistent
with the receptor density and CCK-8 affinity results, the CHO cells
expressing wild type CCKAR responded to CCK-8 stimulation of total
inositol phosphates in a concentration-dependent manner
similar to the CHO cells expressing the CCKAR-GFP. CCK-8 stimulated
total inositol phosphates 5.4 ± 1.0-fold in the CHO cells
expressing wild type CCKAR, with an EC50 of 1.73 ± 0.28 nM compared with 7.36 ± 3.3-fold increase and an
EC50 of 4.38 ± 3.3 nM for the CHO cells
expressing the CCKAR-GFP (Fig. 2).
Fig. 1.
Displacement of 125I-CCK-8
binding to CHO cells stably expressing wild type CCKAR and
CCKAR-GFP. CHO cells stably expressing either the wild type CCKAR
or CCKAR fused to the GFP were incubated with 125I-CCK-8
(50 pM) either alone or with the indicated concentrations of CCK-8. Data are presented as percent of saturable binding in the
absence of unlabeled ligand. Each value represents the mean ± the
standard error from at least three experiments performed in
duplicate.
[View Larger Version of this Image (23K GIF file)]
Fig. 2.
Ability of CCK-8 to increase total inositol
phosphates in CHO cells stably expressing wild type CCKAR and
CCKAR-GFP. Data are shown as the CCK-8-stimulated increase in
total inositol phosphates divided by the unstimulated basal total
inositol phosphates and represent the mean ± the standard error
from at least three experiments performed in duplicate.
[View Larger Version of this Image (22K GIF file)]
Fig. 3.
A, concentration dependence of RG-CCK-8
and Cy3.29-CCK-8 binding to NIH/3T3 cells transfected with human CCK-A
receptor cDNA. B, change in intracellular concentration
of Ca2+ in NIH/3T3 cells stably expressing CCKAR upon
addition of 10 nM Cy3.29-CCK-8, RG-CCK-8, and CCK-8.
[View Larger Version of this Image (23K GIF file)]
Fig. 4.
Time dependence of RG-CCK-8 accumulation in
CHO cells, stably transfected with human CCKAR cDNA, in the
presence of protein synthesis inhibitor, cycloheximide. Cells were
exposed to 100 nM RG-CCK-8 for various length of time and,
bound fluorescence was measured by LSCM. Data are mean ± S.E. of
10 measurements.
[View Larger Version of this Image (16K GIF file)]
Fig. 5.
CLSM images of the cells expressing CCKAR-GFP
(in green). Cell surface marker, rhodamine B ConA, is
in red; colocalization of the two appears as
yellow. A, CHO-cells; B, HeLa cells;
C, NIH/3T3 cells; D, COS-1 cells.
[View Larger Version of this Image (157K GIF file)]
Fig. 6.
Colocalization of CCKAR-GFP (in
green) with the ER marker hexyl ester of rhodamine 6G
(red) in COS-1 cells, transiently transfected with the
receptor (A), and in HeLa cells stably expressing the
receptor (B).
[View Larger Version of this Image (86K GIF file)]
Fig. 7.
Endocytosis of CCKAR-GFP (green)
in the cells stably expressing the receptor. Endosomal marker
tetramethyl rhodamine transferrin is in red. A,
CHO cells without the ligand; B, CHO cells 10 min after
addition of 10 nM CCK-8. C, NIH/3T3 cells
expressing CCKAR-GFP in the absence of the ligands.
[View Larger Version of this Image (61K GIF file)]
Fig. 8.
Colocalization of CCKAR-GFP
(green) with Cy3.29-CCK-8 (red) in CHO cells.
A, 3 min after addition of the ligand; B, 15 min
after addition of the ligand; C, 60 min after removal of Cy3.29-CCK-8.
[View Larger Version of this Image (63K GIF file)]
Fig. 9.
Time dependence of Cy3.29-CCK-8 and CCKAR-GFP
colocalization in CHO cells stably expressing the receptor. The
cells were exposed to the ligand for 10 min at 37 °C. Colocalization was monitored by CLSM and quantitated with Zeiss LSM software.
[View Larger Version of this Image (19K GIF file)]
Fig. 10.
A, CHO cells, stably transfected with
CCK-A receptor cDNA, after 20 min of incubation with RG-CCK-8 (in
green), and LysoMark Red (in red). Partial
colocalization of cholecystokinin and LysoMark Red can be observed; it
appears as orange. B, CHO cells stably transfected with CCKAR-GFP (green) after 20 min of
incubation with CCK-8 and LysoMark Red (red).
[View Larger Version of this Image (99K GIF file)]
Fig. 11.
Recycling of CCKAR-GFP in CHO cells.
The relative amount of receptor molecules on the cell surface was
determined by colocalization with the cell surface marker rhodamine B
ConA, with the aid of Zeiss LSM software at different incubation time after 10 min of exposure to CCK-8.
[View Larger Version of this Image (16K GIF file)]
To whom correspondence should be addressed.
1
The abbreviations used are: GPCR, G
protein-coupled receptor; CCK, cholecystokinin; CCK-8, CCK octapeptide;
RG-CCK-8, rhodamine green cholecystokinin octapeptide; CCKAR,
cholecystokinin receptor type A; GFP, green fluorescent protein; DMEM,
Dulbecco's modified Eagle's medium; CLSM, confocal laser scanning
microscopy; BSA, bovine serum albumin; PBS, phosphate-buffered saline;
ER, endoplasmic reticulum; CHO, Chinese hamster ovary; PCR, polymerase
chain reaction; ConA, concanavalin A.
2
R. H. Stauber, P. Carney, G. A. Gaitanaris, K. Horie, N. I. Tarasova, E. A. Hudson, and G. N. Pavlakis, submitted for
publication.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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Y. Cheng and R. Lotan Molecular Cloning and Characterization of a Novel Retinoic Acid-inducible Gene That Encodes a Putative G Protein-coupled Receptor J. Biol. Chem., December 25, 1998; 273(52): 35008 - 35015. [Abstract] [Full Text] [PDF]
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 G. Cai, T. Michigami, T. Yamamoto, N. Yasui, K. Satomura, M. Yamagata, M. Shima, S. Nakajima, S. Mushiake, S. Okada, et al. Analysis of Localization of Mutated Tissue-Nonspecific Alkaline Phosphatase Proteins Associated with Neonatal Hypophosphatasia Using Green Fluorescent Protein Chimeras J. Clin. Endocrinol. Metab., November 1, 1998; 83(11): 3936 - 3942. [Abstract] [Full Text]
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M. J. Shapiro and S. R. Coughlin Separate Signals for Agonist-independent and Agonist-triggered Trafficking of Protease-activated Receptor 1 J. Biol. Chem., October 30, 1998; 273(44): 29009 - 29014. [Abstract] [Full Text] [PDF]
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G. Czerwinski, N. I. Tarasova, and C. J. Michejda Cytotoxic agents directed to peptide hormone receptors: Defining the requirements for a successful drug PNAS, September 29, 1998; 95(20): 11520 - 11525. [Abstract] [Full Text] [PDF]
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T. Drmota, G. W. Gould, and G. Milligan Real Time Visualization of Agonist-mediated Redistribution and Internalization of a Green Fluorescent Protein-tagged Form of the Thyrotropin-releasing Hormone Receptor J. Biol. Chem., September 11, 1998; 273(37): 24000 - 24008. [Abstract] [Full Text] [PDF]
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R. Schülein, R. Hermosilla, A. Oksche, M. Dehe, B. Wiesner, G. Krause, and W. Rosenthal A Dileucine Sequence and an Upstream Glutamate Residue in the Intracellular Carboxyl Terminus of the Vasopressin V2 Receptor Are Essential for Cell Surface Transport in COS.M6 Cells Mol. Pharmacol., September 1, 1998; 54(3): 525 - 535. [Abstract] [Full Text]
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N. I. Tarasova, R. H. Stauber, and C. J. Michejda Spontaneous and Ligand-induced Trafficking of CXC-Chemokine Receptor 4 J. Biol. Chem., June 26, 1998; 273(26): 15883 - 15886. [Abstract] [Full Text] [PDF]
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A. R. Burt, M. Sautel, M. A. Wilson, S. Rees, A. Wise, and G. Milligan Agonist Occupation of an alpha 2A-Adrenoreceptor-Gi1alpha Fusion Protein Results in Activation of Both Receptor-linked and Endogenous Gi Proteins. COMPARISONS OF THEIR CONTRIBUTIONS TO GTPase ACTIVITY AND SIGNAL TRANSDUCTION AND ANALYSIS OF RECEPTOR-G PROTEIN ACTIVATION STOICHIOMETRY J. Biol. Chem., April 24, 1998; 273(17): 10367 - 10375. [Abstract] [Full Text] [PDF]
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S. A. Wank I. CCK receptors: an exemplary family Am J Physiol Gastrointest Liver Physiol, April 1, 1998; 274(4): G607 - G613. [Abstract] [Full Text] [PDF]
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A. W. Gagnon, L. Kallal, and J. L. Benovic Role of Clathrin-mediated Endocytosis in Agonist-induced Down-regulation of the beta 2-Adrenergic Receptor J. Biol. Chem., March 20, 1998; 273(12): 6976 - 6981. [Abstract] [Full Text] [PDF]
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J. G. Richman and J. W. Regan alpha 2-Adrenergic receptors increase cell migration and decrease F-actin labeling in rat aortic smooth muscle cells Am J Physiol Cell Physiol, March 1, 1998; 274(3): C654 - C662. [Abstract] [Full Text] [PDF]
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K. Berrada, C. L. Plesnicher, X. Luo, and M. Thibonnier Dynamic Interaction of Human Vasopressin/Oxytocin Receptor Subtypes with G Protein-coupled Receptor Kinases and Protein Kinase C after Agonist Stimulation J. Biol. Chem., August 25, 2000; 275(35): 27229 - 27237. [Abstract] [Full Text] [PDF]
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K. Kuwasako, Y. Shimekake, M. Masuda, K. Nakahara, T. Yoshida, M. Kitaura, K. Kitamura, T. Eto, and T. Sakata Visualization of the Calcitonin Receptor-like Receptor and Its Receptor Activity-modifying Proteins during Internalization and Recycling J. Biol. Chem., September 15, 2000; 275(38): 29602 - 29609. [Abstract] [Full Text] [PDF]
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