Regulation of lateral mobility and cellular trafficking of the
CCK receptor by a partial agonist
Belinda F.
Roettger,
Delia I.
Pinon,
Thomas P.
Burghardt, and
Laurence J.
Miller
Center for Basic Research in Digestive Diseases and Department of
Biochemistry and Molecular Biology, Mayo Clinic and Foundation,
Rochester, Minnesota 55905
 |
ABSTRACT |
Partial agonists are effective tools for advancing development
of highly selective drugs and providing insights into molecular regulation of cellular functions. Here, we explore the impact of a
partial agonist on key aspects of cholecystokinin (CCK) receptor regulation, its lateral mobility and cellular trafficking, in native
pancreatic acinar cells and Chinese hamster ovary cells expressing CCK
receptor (CHO-CCKR). We developed and characterized a novel fluorescent
partial agonist,
rhodamine-Gly-[(Nle28,31)CCK-26-32]-phenethyl
ester, that binds specifically and with high affinity to CCK receptors.
Such analogs are fully efficacious pancreatic acinar cell secretagogues
without supramaximal inhibition that mobilize intracellular calcium
with little or no increase in phospholipase C (PLC) activity. Despite
minimal phosphorylation of CCK receptors in response to this partial
agonist, receptor trafficking was the same as that observed with full
agonist (CCK). This included normal internalization via
clathrin-dependent endocytosis in CHO-CCKR cells and insulation on the
surface of pancreatic acinar cells. Also, as with CCK-occupied
receptor, fluorescence recovery after photobleaching of partial
agonist-occupied receptor on the acinar cell surface demonstrated a
marked temperature-dependent slowing of its rate of diffusion. This was
similarly associated with resistance to acid-induced dissociation of
ligand. Thus some key molecular regulatory mechanisms for CCK receptor
internalization and insulation may be initiated by cellular signaling
cascades that are not dependent on PLC activation or receptor phosphorylation.
G protein-coupled receptor; receptor mobility; receptor
internalization
 |
INTRODUCTION |
PARTIAL AGONISTS HAVE BEEN extremely useful
pharmacologic tools, contributing key insights toward the development
of highly selective drugs and providing better understanding of
molecular events that control specific cellular functions (3). Analogs of cholecystokinin (CCK) in which the carboxy-terminal
phenylalanine-amide has been replaced with a phenethyl ester have been
demonstrated to act as partial agonists of the CCK receptor,
stimulating a subset of the biological responses that are stimulated by
occupation of the same receptor by natural CCK (6, 9, 15, 18, 34). This
was first recognized by net effects on amylase secretion by the
pancreatic acinar cell, where CCK stimulates a biphasic concentration-response curve and these compounds {JMV-180 and Gly-[(Nle28,31)CCK-26-32]-phenethyl
ester (OPE)} are fully efficacious secretagogues without the supramaximal inhibitory regions of their
concentration-response curves. They have also subsequently been
recognized to possess differences from the natural hormone in their
effects on desensitization and induction of experimental models of
pancreatitis (19, 33).
Although these compounds have been proposed to act as "agonists at
the high-affinity CCK receptor and antagonists at the low-affinity CCK
receptor" (36), a more cohesive molecular understanding of their
action is emerging. The high-affinity state of the CCK receptor is
known to be the ternary complex of agonist, receptor, and G protein
(21). Although natural CCK stabilizes that complex, as demonstrated by
the sensitivity of its binding to GTP (6), OPE binding does not have
this effect: its binding is unaffected by GTP analogs (6). As expected
from this observation, CCK binds with two distinct affinity states,
whereas OPE binds with a single apparent affinity state (6). OPE is
able to fully compete for CCK binding, but its affinity is intermediate
between the affinities for binding CCK, such that OPE has a lower
affinity than CCK for the "high-affinity state" of the receptor
and a higher affinity than CCK for the "low-affinity state" of
the receptor. Measurement of second messenger responses has
demonstrated little or no measurable inositol 1,4,5-trisphosphate
(IP3) response to phenethyl
ester analogs (15, 18, 32), likely reflecting their ineffective
stabilization of this ternary complex. This is consistent with the
IP3 response resulting from the
activation of phospholipase C (PLC) by
G
q-GTP after it dissociates
from such a complex. In contrast, the phenethyl ester analogs have normal phospholipase A2 responses
and induce clear increases in intracellular calcium (18,
38), which presumably emanate from an intact signaling cascade that is
distinct from the Gq pathway. This
secondary signaling pathway may be initiated either by receptor association with a G protein distinct from
Gq and having different kinetics
of receptor association or by a G protein-independent mechanism.
Interestingly, in the presence of minimal activation of protein kinase
C (PKC) achieved by preincubation with a low dose of phorbol ester, the
OPE concentration-response curve is transformed from a monophasic into
a fully biphasic concentration-response curve (7, 8). These data are
consistent with the interpretation that the phenethyl ester analogs are
partial agonists that are deficient in their stimulation of the PLC-PKC
signaling cascade.
Regulation of G protein-coupled receptors has been an area of
considerable interest. The CCK receptor has been demonstrated to be
regulated by phosphorylation, uncoupling from its G protein, "insulation" within the plasma membrane, and internalization into the cell interior (5, 14, 23, 24, 28-31). All of these events
occur in response to agonist occupation, with the details of these
processes varying based on the specific cell being studied (24, 28, 30,
31). We previously reported that stimulation with the partial agonist
OPE results in minimal phosphorylation of the CCK receptor (with a
stoichiometry of <1 mol phosphate/mol receptor) (10). This is
consistent with its activation of only a subset of postreceptor
signaling events and with the relative importance of PKC in CCK
receptor phosphorylation (8, 10, 23). In this work, we have studied the
effect of partial agonist stimulation of both CCK receptor-bearing
Chinese hamster ovary cells (CHO-CCKR) (11) and rat pancreatic acinar
cells on receptor mobility, internalization, and insulation. These
mechanisms of CCK receptor regulation are fully intact after
stimulation by OPE, demonstrating that they are regulated independently
of signaling events in the PLC cascade.
 |
METHODS |
Materials.
Synthetic CCK octapeptide (CCK amino acids 26-33; CCK-8) was
purchased from Peninsula Laboratories (Belmont, CA).
5(6)-Carboxytetramethylrhodamine succinimide ester was purchased from
Molecular Probes (Eugene, OR). The nonpeptidyl antagonist L-364718 was
provided by Dr. R. Freidinger (Merck, Sharp, and Dohme Laboratories,
West Point, PA). Tissue culture supplies were from GIBCO BRL
(Gaithersburg, MD), except for Falcon plasticware, which was purchased
from Becton Dickinson (Oxnard, CA). Electron microscopy grade
paraformaldehyde was from Electron Microscopy Sciences (Ft. Washington,
PA). BSA Cohn fraction V was purchased from Intergen (Purchase, NY),
and soybean trypsin inhibitor and collagenase were from Worthington Biochemical (Freehold, NJ). All other chemicals were analytical grade.
CCK analogs.
The fluorescent CCK analog
rhodamine-Gly-[(Nle28,31)CCK-26-33]
(Rho-CCK) was synthesized as described previously (31). The partial agonist CCK analog OPE was synthesized as described previously (6) and
was used as the base peptide for the synthesis of the fluorescent
partial agonist probe. OPE was dissolved in dimethyl formamide,
neutralized with
N,N-diisopropylethylamine,
and reacted with a twofold molar excess of
5(6)-carboxytetramethylrhodamine succinimide ester for 2 h at room
temperature. Once the reaction was complete, as confirmed by
reverse-phase HPLC, excess unreacted fluorophore was quenched with
diethylaminopropylamine. The newly synthesized fluorescent probe,
rhodamine-Gly-[(Nle28,31)CCK-26-32]-phenethyl
ester (Rho-OPE), was purified by reverse-phase HPLC (27) and was
characterized and quantified by amino acid analysis.
Cell and tissue preparations.
CHO-CCKR cells expressing the rat type A CCK receptor have previously
been established and fully characterized (11). CHO-CCKR cells were
grown in Ham's F-12 medium in a 37°C humidified incubator containing 5% CO2. Two days
before experimental manipulation, cells were plated on glass coverslips
and grown to ~80% confluence for receptor distribution studies and
to 50% confluence for photobleaching recovery experiments.
For native cell morphological studies, dispersed rat pancreatic acini
were prepared from male Sprague-Dawley rats (125-150 g) by
sequential enzymatic and mechanical dissociation of pancreatic tissue
(17). For competition binding studies, enriched rat pancreatic plasma
membranes were prepared as we reported (25). All procedures involving
animals were approved by the Mayo Clinic Animal Care and Use Committee.
CCK receptor binding.
The CCK analog
D-Tyr-Gly-[(Nle28,31)CCK-26-33]
was radioiodinated oxidatively and purified by reverse-phase HPLC to a
specific radioactivity of 2,000 Ci/mmol, as previously described (27). Samples (total volume 500 µl) of membrane diluted in
Krebs-Ringer-HEPES medium (KRH) containing (in mM) 25 HEPES, pH 7.4, 104 NaCl, 5 KCl, 1.2 MgSO4, 2 CaCl2, 1 KH2PO4,
and 1 phenylmethylsulfonyl fluoride, as well as 0.2% BSA and 0.01%
soybean trypsin inhibitor, were incubated with 18-22 pM
125I-labeled
D-Tyr-Gly-[(Nle28,31)CCK-26-33]
plus appropriate concentrations of OPE or Rho-OPE for 1 h at room
temperature. A Skatron cell harvester (Sterling, VA) with
receptor-binding filter mats was used to separate bound from free
radioligand, and bound radioligand was quantified using a gamma
spectrometer. Nonsaturable binding of
125I-D-Tyr-Gly-[(Nle28,31)CCK-26-33]
represented the amount of radioactivity associated with membranes when
the medium contained 1 µM nonradioactive competing peptide. Values
for the Michaelis-Menten inhibition constant
(Ki) were
determined by the LIGAND program (22), and data were graphed using the
Prism graphics program (GraphPad, San Diego, CA).
Morphological localization of CCK receptors on CHO-CCKR cells.
CHO-CCKR cells grown on coverslips were washed three times at 37°C
with PBS (in mM: 1.5 NaH2PO4,
8 Na2HPO4,
0.145 NaCl, 0.1 MgCl2, and 0.08 CaCl2, pH 7.4). Cells were washed
and equilibrated for 10 min with iced PBS at 4°C to inhibit
endocytosis and were then incubated with 10, 50, or 100 nM Rho-OPE for
1 h at 4°C. For receptor distribution studies, the cells were
washed quickly with iced PBS and immediately placed in freshly prepared
fixative (2% paraformaldehyde in PBS, pH 7.4) for 30 min at room
temperature. After fixation, the coverslips were washed three times
with PBS and then mounted on glass slides. Cells were examined using an inverted Zeiss microscope equipped for epifluorescence (Oberkochen, Germany). A 50-W mercury lamp was used for illumination. Specimens were
photographed using a 35-mm camera with Tmax 3200 film (Eastman Kodak,
Rochester, NY). For control studies, labeling was attempted with
untransfected CHO cells or with the CHO-CCKR cells in the presence of
nonfluorescent competing ligand.
For studies to examine the effect of receptor affinity state on
internalization, CHO-CCKR cells grown on coverslips were incubated with
50 nM Rho-OPE, 50 nM Rho-OPE plus 5 µM L-364718, or 50 nM Rho-OPE
plus 10 µM CCK-8 for 1 h at 4°C, as described above. They were
then washed once with 37°C PBS and placed in a 37°C shaking water bath for times ranging from 5 to 30 min. After the appropriate time had elapsed, cells were fixed and prepared for microscopic observation.
To disrupt the first step of receptor-mediated endocytosis in which
receptors cluster in clathrin-coated pits, cells were incubated in
hypertonic (0.4 M) sucrose in PBS, as previously described (4, 12, 31).
CHO-CCKR cells grown on coverslips were washed three times at 37°C
with hypertonic medium (0.4 M sucrose in PBS), equilibrated with
hypertonic medium in a shaking water bath for 10 min at 37°C, and
then labeled with 50 nM Rho-OPE in the shaking water bath. At the
appropriate time point, samples were washed with 37°C hypertonic
medium and fixed and prepared for microscopic observation.
Morphological localization of CCK receptors on pancreatic acinar
cells.
Freshly prepared dispersed rat pancreatic acini were collected by
centrifugation at 300 rpm for 3 min and then washed and resuspended in
iced KRH enriched with 2.5 mM
D-glucose, essential and
nonessential amino acids, and 2 mM glutamine. Acini were incubated with
concentrations ranging from 30 to 100 nM Rho-OPE at 4°C for 20 min
or at 37°C for 5-30 min. In selected experiments, the pH was
lowered to attempt to dissociate the ligand from the receptor by
washing Rho-OPE-treated acini with iced glycine buffer (50 mM glycine
and 150 mM NaCl, pH 3.0) for 30 s and then resuspending the acini in
iced KRH medium. Cells were observed and photographed immediately
following sample preparation using a Zeiss Axiophot microscope equipped
for epifluorescence. Samples were illuminated with a 75-W xenon lamp
with a 10-nm bandpass excitation filter centered at 546 nm and a 590-nm
emission filter. Photographs were taken with a 35-mm camera using
Hypertech film (Microfluor, Stony Brook, NY).
Fluorescence photobleaching recovery measurements.
Fluorescence photobleaching recovery measurements were carried out
following the procedure previously described (30). This was performed
both in CHO-CCKR cells and dispersed rat pancreatic acinar cells. The
CHO-CCKR cells were plated on no. 1 22-mm square glass coverslips
(Baxter, McGaw Park, IL) in individual 35-mm polystyrene dishes (Becton
Dickinson, Lincoln Park, NJ), where they were fluorescently labeled.
Coverslips were then placed cell side down over a 20-µl drop of
4°C PBS on a chilled glass slide, blotted, and sealed. The acinar
cells were labeled in suspension and then placed on a chilled glass
slide and covered with a no. 0 22-mm square glass coverslip (Baxter).
Photobleaching recovery measurements were made immediately following
sample preparation. Fluorescence was excited by the 514-nm line of an
Innova 90 argon laser (Coherent, Palo Alto, CA). Laser illumination
intensity was controlled using the first-order diffraction from an
acoustooptic modulator (IntraAction, Bellwood, IL). The beam then
passed through a spatial filter and then into a Zeiss Axioplan
microscope equipped for epifluorescence. A ×100 (1.3 numerical
aperture) oil objective was used to focus the beam onto the sample. The
1/e2 radius of
the Gaussian beam profile was ~0.5 µm on the sample. Fluorescence
emitted from the sample was detected continuously using an avalanche
diode single-photon counting module (EG&G Opto-electronics, Vaudreuil,
PQ, Canada) interfaced to a 33-MHz 80486 microcomputer. Optimization of
the plane of focus was conducted as described elsewhere (30).
Fluorescence intensities were quantified by accumulating photon counts
for 25 ms/data point. During fluorescence recovery experiments, samples
were maintained at 10 ± 1°C using a temperature-controlled
stage. To minimize the effects of receptor clustering and
redistribution following ligand occupation, all readings were taken
immediately following sample preparation, while fluorescence appeared
to be homogeneously distributed over the plasma membrane. Control
studies (30) showed that ligand binding and dissociation kinetics did
not contribute appreciably to the recovery observed.
For analysis, fluorescence photobleaching recovery data were expressed
as the fractional fluorescence recovery,
f(t), such that
|
(1)
|
where
t is time,
F(t) is the time-dependent
fluorescence intensity, F(+0) is the fluorescence immediately after
photobleaching, and F(
) is the prebleach fluorescence.
The series solution for
F(t)/F(
) when
t
+0, derived for a Gaussian laser
beam profile illuminating fluorophores diffusing in two dimensions, was
used to fit the fractional recovery curves (1). In this
case
|
(2)
|
where
0 is the immobilized
fraction of fluorophores, K is the
bleaching depth given by F(+0)/F(
) = (1
e
K)/K,
and
1 is the fraction of
fluorophores undergoing lateral diffusion with characteristic
relaxation time
D (39).
Equation 2 models a system with mobile and
immobile components of fluorophores, with all fluorophores having
identical photobleaching rates. A least squares protocol with equality
and inequality constraints gave the best choices for the linear
parameters,
0 and
1, for each choice of the
nonlinear parameter,
D. The
best choice for
D was located
by grid search.
The mobile fraction (R) of
fluorophores in the cell membrane is then given by
|
(3)
|
and
the lateral diffusion coefficient
(D) is given by
|
(4)
|
where
is the laser beam half width at
e
2 height
such that
0.25 µm.
Statistical analysis.
The number of replicate experiments is noted for each presentation of
data. Values are expressed as means ± SE of experimental replicates. Significant differences were determined by the Mann-Whitney nonparametric test of unpaired values, with
P < 0.05 considered to be significant.
 |
RESULTS |
Binding affinities of the fluorescent partial agonist.
The fluorescent partial agonist Rho-OPE bound to CCK receptors on rat
pancreatic membranes specifically and with high affinity (Fig.
1). Addition of the
fluorophore did not markedly change the binding affinity of Rho-OPE
(Ki = 7.3 ± 2.4 nM) compared with OPE
(Ki = 4.4 ± 1.0 nM). Analysis of the data indicated a best fit for a
single-site affinity model, which is similar to binding of OPE to rat
pancreatic membranes (6).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1.
Competition for binding of
125I-labeled
D-Tyr-Gly[(Nle28,31)-
CCK-26-33] to pancreatic membranes by OPE and Rho-OPE.
Membranes were incubated with 18-22 pM radioligand and increasing
concentrations of Rho-OPE and OPE; 100% binding represents specific
binding in absence of competing peptide. Nonspecific binding,
determined in presence of 1 µM nonradioactive competing peptide,
represented <15% of total binding. Inhibition constant was 7.3 ± 2.4 nM for Rho-OPE and 4.4 ± 1.0 nM for OPE. Values are means ± SE for 3 separate experiments performed in duplicate.
|
|
Morphological characterization of fluorescent partial agonist.
The specificity of binding Rho-OPE to CCK receptors was further
characterized by morphological assessment. CHO-CCKR cells incubated
with 50 nM Rho-OPE at 4°C displayed fluorescent labeling diffusely
distributed over the surface of the membrane (Fig.
2A), and
this labeling was completely eliminated in cells incubated with Rho-OPE
in the presence of 100-fold molar excess unlabeled OPE (Fig.
2B). Further evidence that the
fluorescent labeling of the plasmalemma observed was specific to CCK
receptors and not general membrane staining was provided by the absence
of fluorescence signal on non-receptor-bearing CHO cells incubated
under identical conditions (Fig.
2C).

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 2.
Specific labeling of CHO-CCKR cells with Rho-OPE at 4°C. Shown are
fluorescence images of CHO-CCKR cells incubated for 1 h at 4°C with
50 nM Rho-OPE (A), CHO-CCKR cells
incubated for 1 h at 4°C with 50 nM Rho-OPE + 5 µM nonfluorescent
OPE (B), and non-receptor-bearing
CHO cells incubated for 1 h at 4°C with 50 nM Rho-OPE
(C). Images are representative of 3 separate experiments.
|
|
Internalization of CCK receptors on CHO-CCKR cells following
occupation by partial agonist.
CCK receptors occupied by Rho-OPE were rapidly moved into intracellular
destinations following incubations at 37°C. Initially (at 4°C),
the fluorescence signal appeared to be distributed uniformly over the
surface of the plasma membrane (Fig.
3A).
After 5 min at 37°C, the fluorescence was observed in a punctate
pattern resembling endosomal structures, both near the plasma membrane
and deep within the cell. By 30 min, most of the fluorescence had moved
to structures near the nucleus, with a small proportion of fluorescence
remaining near the plasma membrane. This internalization pathway is
morphologically similar to that observed for CCK receptors occupied by
the full agonist Rho-CCK (Fig. 3,
D-F).
Because our previous studies with Rho-CCK showed predominantly
agonist-induced endocytosis initiated by the clustering of receptors in
clathrin-coated pits (31), we employed the established technique of
using hypertonic medium to prevent the formation of clathrin-coated
pits in an attempt to disrupt the internalization observed (4, 12).
Cells treated with hypertonic sucrose during incubations with 50 nM
Rho-OPE at 37°C did not internalize CCK receptors. The fluorescent
staining continued to be diffuse at the level of the plasma membrane
after 5 and 10 min (Fig. 4,
A and
B). After 30 min at 37°C, the
signal remained associated with the plasma membrane, although a more punctate distribution of the fluorescence was observed (Fig.
4C). These observations suggest that
the internalization of receptors occupied by the partial agonist
requires functional clathrin-coated pits.

View larger version (107K):
[in this window]
[in a new window]
|
Fig. 3.
Time course of internalization of Rho-OPE-occupied cholecystokinin
(CCK) receptors in CHO-CCKR cells. Cells were incubated with 50 nM
fluorescent ligand for 1 h at 4°C. Coverslips for
time
0 point were washed with 4°C PBS
and fixed immediately after incubations at 4°C with fluorescent
ligand. Coverslips for later time points were washed with 37°C PBS
and maintained at 37°C in a shaking water bath for indicated times.
Shown are images of cells incubated with 50 nM Rho-OPE for 0 (A), 5 (B), and 30 (C) min and images of cells
incubated with 50 nM Rho-CCK for 0 (D), 5 (E), and 30 (F) min. Data are representative of
3 separate experiments.
|
|

View larger version (65K):
[in this window]
[in a new window]
|
Fig. 4.
Disruption of clathrin-dependent endocytosis by hypertonic sucrose
treatment. CHO-CCKR cells were pretreated with 0.4 M sucrose in PBS
before and during incubations with 50 nM Rho-OPE at 37°C. Shown are
fluorescence images of cells after incubations for 5 (A), 10 (B), and 30 (C) min. Images are representative
of 3 separate experiments.
|
|
Much work has been performed to characterize interactions between
phenethyl ester analogs of CCK and the high- and low-affinity states of
the CCK receptor (16, 18, 34, 36, 40). We therefore examined the effect
of competing nonfluorescent ligands known to independently affect the
occupation of each of these affinity states of the CCK receptor by
Rho-OPE. Because CCK-8 has a greater affinity than OPE for receptors in
the high-affinity state, internalization studies were performed on
cells exposed to Rho-OPE for 30 min at 37°C in the presence of
100-fold molar excess of competing nonfluorescent CCK-8. The prominent
perinuclear fluorescence representing intracellular ligand was not
eliminated in these cells. When the competing concentration of CCK-8
was increased to a 200-fold molar excess (10 µM), the perinuclear staining persisted (Fig.
5B),
although the signal was considerably less intense than that observed in
the absence of competing CCK-8 (Fig.
5A). Because essentially all
receptors in the high-affinity state were occupied by nonfluorescent
CCK-8, the signal observed was likely due to internalization of
receptors in their native low-affinity state. Experiments were also
performed using the antagonist L-364718, which binds strongly to CCK
receptors in both high- and low-affinity states. All observed
fluorescence was eliminated in cells incubated for 30 min at 37°C
with 50 nM Rho-OPE plus 5 µM L-364718 (100-fold excess; Fig.
5C).

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of receptor affinity state on internalization of CCK receptors.
CHO-CCKR cells were incubated for 1 h at 4°C with 50 nM Rho-CCK in
presence or absence of indicated nonfluorescent competing ligand and
then warmed to 37°C with PBS and maintained at 37°C for 30 min
in a shaking water bath. Shown are images of cells incubated with 50 nM
Rho-OPE (A), 50 nM Rho-OPE + 10 µM
CCK octapeptide (CCK-8; B), and 50 nM Rho-OPE + 5 µM L-364718 (C).
Images are representative of 3 separate experiments.
|
|
Cellular handling of partial agonist-occupied receptor in the native
milieu.
Examination of the cellular handling of the partial agonist was also
performed in native pancreatic acinar cells. Acinar cells exposed to 30 nM Rho-OPE at 4°C displayed a diffuse pattern of fluorescence over
the basolateral membrane (Fig.
6A),
similar to that observed in acini incubated with the full agonist
Rho-CCK. After the temperature was raised to 37°C, cells incubated
with 30 nM Rho-OPE continued to display a similar pattern of
fluorescence; no fluorescence moving to the cell interior was observed
(Fig. 6B, 5 min; Fig.
6C, 30 min). Notably, the membrane
fluorescence persisted even after the acini were washed with acidic
glycine medium in an attempt to dissociate the partial agonist ligand from the receptor. This treatment was, however, effective in
eliminating all fluorescence signal from cells that had been incubated
with Rho-OPE at 4°C (Fig.
7A). The
resistance to acid washing after incubation at 37°C (Fig.
7B) was similar to that previously
observed on acini incubated with the full agonist Rho-CCK (30).

View larger version (54K):
[in this window]
[in a new window]
|
Fig. 6.
Cellular handling of Rho-OPE-occupied receptors on native acinar cells.
Dispersed rat pancreatic acini were labeled with 30 nM Rho-OPE for 20 min at 4°C (A), 5 min at
37°C (B), and 30 min at 37°C
(C). Images are representative of 3 separate experiments.
|
|

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 7.
Insulation of Rho-OPE-occupied CCK receptors in acid-resistant
compartments on acinar cell surface. Dispersed pancreatic acini were
incubated with 30 nM Rho-OPE at 4 or 37°C and then washed with
acidic glycine. Fluorescent labeling was essentially eliminated after
acidic glycine washes of acini incubated with Rho-OPE for 20 min at
4°C (A), whereas intense
fluorescence signal remained after acid washes of acini incubated with
Rho-OPE at 37°C (B). Images are
representative of 2 separate experiments.
|
|
Lateral mobility characteristics of Rho-OPE-occupied CCK receptors.
Fluorescence recovery after photobleaching was used to measure the rate
at which CCK receptors occupied by Rho-OPE moved laterally in the plane
of the plasma membrane. CCK receptors on CHO-CCKR cells incubated with
Rho-OPE were laterally mobile, with a diffusion coefficient on the
order of 1 × 10
10
cm2/s, as shown in Table
1. The value of
D determined at 50 nM Rho-OPE (1.08 ± 0.24 × 10
10
cm2/s) was equivalent to the value
previously determined for CCK receptors on CHO-CCKR cells occupied by
50 nM Rho-CCK (1.4 ± 0.2 × 10
10
cm2/s) (30). Also, the fraction of
mobile receptors on these cells occupied by the Rho-OPE
(R = 0.88 ± 0.03) was equivalent
to that for the same concentration of Rho-CCK
(R = 0.88 ± 0.02) in our previous work (30). Values for D and
R did not change significantly as the
Rho-OPE concentration was varied between 10 and 100 nM.
The D value for 100 nM
Rho-OPE-occupied receptors on pancreatic acinar cells (0.72 ± 0.19 × 10
10
cm2/s) was not significantly
different from the value of D
determined for this concentration of this fluorescent ligand used with
CHO-CCKR cells (P > 0.05). The
R determined on acini (0.58 ± 0.07) was marginally lower than that determined on CHO-CCKR cells
(P = 0.06). Unlike the comparison of
data for effects of partial and full agonists on the CHO-CCKR cells,
which were quite similar, the comparison of acinar cell data for these
two ligands reflected differences. Occupation of the acinar cell
receptors with 50 nM Rho-CCK at 4°C was reported to have a higher
D (1.7 ± 0.3 × 10
10
cm2/s) and a lower
R (0.17 ± 0.05) (30). Some of this
apparent difference could be methodological, since the previous data
were analyzed manually using a graphical determination of recovery half
times. In contrast, the current work uses a mathematical model and
fitting routine to analyze the data and determine values for
D and
R. The model calculation and fitting
routine are likely more accurate at quantifying the slower recoveries
reported here.
Notably, incubation of the acini at 37°C resulted in a significant
reduction in the value of D
(decreasing from 0.72 ± 0.19 × 10
10
cm2/s after incubations at 4°C
to 0.15 ± 0.06 ×10
10
cm2/s after incubations at
37°C; P = 0.008), while the
fraction of partial agonist-occupied receptors that were mobile
remained constant. This, also, is different from the results previously
reported for the same receptor occupied with the full agonist Rho-CCK
(30). Under identical conditions, the agonist-occupied receptor was felt to become immobilized on the cell surface.
 |
DISCUSSION |
The present work illustrates that partial agonist occupation of the CCK
receptor results in the same types of receptor trafficking as are
stimulated by occupation of these receptors with the natural full
agonist, CCK. This occurs despite the differences in signaling initiated by these distinct peptides. In CHO-CCKR cells, CCK-occupied receptors rapidly undergo receptor-mediated endocytosis, leading to
redistribution to an area near the cell nucleus (31). At the level of
fluorescence microscopy, after occupation of the CCK receptor on these
cells by the partial agonist Rho-OPE, receptor lateral mobility and
cellular distribution were very similar to these parameters observed
after receptor occupation with the full agonist Rho-CCK (31). In prior
ultrastructural studies with these cells, we showed that the
predominant pathway for endocytosis of receptors occupied by full
agonists was the clathrin-dependent pathway (31). The effect of
hypertonic sucrose treatment to block internalization of the CCK
receptors occupied with OPE suggested that this same pathway was
predominant for partial agonist-stimulated receptor internalization as well.
Similarities between receptor trafficking in response to agonist and
partial agonist were also demonstrated in the native pancreatic acinar
cell. This is a unique and special environment for the CCK receptor, in
which agonist occupation elicits desensitization by a process termed
insulation rather than internalization (30). In this process, the
agonist-occupied receptor becomes immobilized in a unique environment
depleted of G proteins. In the present study, most receptors occupied
by Rho-OPE also did not internalize but remained diffusely distributed
on the acinar cell membrane. There was, however, a change in their acid
lability. The Rho-OPE ligand, which was dissociated from the receptor
by acidic medium after binding at 4°C, became resistant to this
procedure after incubation at 37°C. This was associated with a
prominent decrease in the rate of lateral mobility of the partial
agonist-occupied receptors in the plane of the plasma membrane. This is
analogous to previous observations with the native full agonist (30)
when occupied receptors became resistant to acid dissociation and
lateral movement was slowed beyond the limit of the technique to
quantify movement. Thus both agonist and partial agonist initiate
conformational changes that "insulate" the receptor (30).
A key reason to explore receptor trafficking after occupation with
partial agonist was to correlate the regulation of this behavior with a
subset of the cellular events initiated by receptor occupation with
full agonist. We already know that stimulation with OPE results in
substantially less CCK receptor phosphorylation than stimulation with
CCK (10). The latter results in a stoichiometry of >5 mol
phosphate/mol receptor (23), with evidence for action of both PKC and
staurosporine-insensitive kinases (10, 23). In contrast, CCK receptor
phosphorylation in response to OPE results in a stoichiometry of <1
mol phosphate/mol receptor. Recent data for a CCK receptor mutant that
is not phosphorylated in response to CCK and is internalized normally
suggest that CCK receptor internalization can occur independently of
its state of phosphorylation (28).
It has been unclear how important the components of signaling might be
for receptor trafficking. We recently demonstrated that CCK receptors
on CHO-CCKR cells do not internalize constitutively but depend on
ligand occupation to stimulate that process (5). Of particular
interest, occupation with an antagonist was shown to be sufficient to
stimulate internalization of CCK receptors on those cells (29). In that
work, we constructed a fluorescent analog of JMV-179, a CCK analog
previously demonstrated to be an antagonist, that incorporates a
D-Trp in the position of
L-Trp-30 and a carboxy-terminal
phenethyl ester (26, 26a, 41). However, only a limited number (37%) of
the antagonist-occupied receptors were internalized (29). Those data
were interpreted as supporting receptor trafficking that is independent
of classical signaling events.
It is noteworthy that the degree of CCK receptor internalization
observed in the current report after receptor occupation with partial
agonist was much greater than that observed after occupation with
either the peptidyl or nonpeptidyl antagonists previously studied (29).
Internalization of the CHO-CCKR CCK receptors was essentially complete
after occupation by both a full agonist (31) and a partial agonist.
This supports the interpretation that efficient receptor
internalization can occur that is independent of the PLC cascade of
signaling events. It continues to be unclear whether any signaling
events are critical for this process. It might be that the
conformational change in the receptor that correlates with partial
agonist activity also supports the key molecular interaction with a
heretofore unidentified protein that leads to internalization and
insulation, which is less well supported by antagonist occupation of
the receptor.
There is another interesting observation in the current work that
relates to receptor affinity states. For some G protein-coupled receptors, internalization has been correlated with G protein association and the high-affinity state of the receptor (2, 37). Here,
we can rule that out for the CCK receptor. Competition for the
high-affinity binding by coincubation with nonfluorescent CCK did not
prevent the prominent internalization of Rho-OPE. In contrast,
nonfluorescent peptidyl and nonpeptidyl antagonists that are known to
effectively occupy both high- and low-affinity states of the CCK
receptor completely eliminated the internalization of the Rho-OPE.
Previous, less direct studies have, however, suggested that only the
high-affinity CCK receptor is internalized (20). Like the CCK receptor,
the angiotensin receptor has been shown to be internalized
independently of G protein coupling and signal transduction (13).
Desensitization of agonist-stimulated receptors provides a mechanism to
protect the cell from potential overstimulation. In the absence of
receptor phosphorylation, it was possible that a partial agonist could
have bypassed this safety mechanism. That was somewhat supported by the
previous observation that, unlike CCK, JMV-180 treatment did not lead
to desensitization of the pancreatic acinar cell (19). In that work,
however, the response being assayed was the ability of carbamylcholine
to stimulate amylase secretion. That measurement would of course be
unaffected by CCK receptor internalization or insulation, which could
be protective of overstimulation by agonists acting at the CCK
receptor. There is no way for a hydrophilic natural ligand of the CCK
receptor to access its binding site after the receptor is moved into
the cell. This suggests that partial agonist stimulation is capable of
desensitizing the CCK receptor by a mechanism that is independent of
receptor phosphorylation.
 |
ACKNOWLEDGEMENTS |
We acknowledge the contribution to select experiments by Dr. R. Rentsch, the excellent technical assistance of E. Hadac and E. Holicky,
and the excellent secretarial assistance of S. Erickson.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants
DK-32878 (to L. J. Miller) and AR-39288 (to T. P. Burghardt) and
Training Grant DK-09078 (to B. F. Roettger) and by the Fiterman Foundation.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: L. J. Miller, Center for Basic Research
in Digestive Diseases, Guggenheim 17, Mayo Clinic, Rochester, MN 55905.
Received 28 September 1998; accepted in final form 7 December
1998.
 |
REFERENCES |
1.
Axelrod, D.,
D. E. Koppel,
J. Schlessinger,
E. L. Elson,
and
W. W. Webb.
Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.
Biophys. J.
16:
1055-1069,
1976[Abstract].
2.
Benya, R. V.,
M. Akeson,
J. Mrozinski,
R. T. Jensen,
and
J. F. Battey.
Internalization of the gastrin-releasing peptide receptor is mediated by both phospholipase C-dependent and -independent processes.
Mol. Pharmacol.
46:
495-501,
1994[Abstract].
3.
Black, J.
A personal view of pharmacology.
Annu. Rev. Pharmacol. Toxicol.
36:
1-33,
1996[Medline].
4.
Daukas, G.,
and
S. H. Zigmond.
Inhibition of receptor-mediated but not fluid-phase endocytosis in polymorphonuclear leukocytes.
J. Cell Biol.
101:
1673-1679,
1985[Abstract].
5.
De Toledo, C. F.,
B. F. Roettger,
C. Morys-Wortmann,
W. E. Schmidt,
and
L. J. Miller.
Cellular handling of unoccupied and agonist-stimulated cholecystokinin receptor determined by immunolocalization.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G488-G497,
1997[Abstract/Free Full Text].
6.
Gaisano, H. Y.,
U. G. Klueppelberg,
D. I. Pinon,
M. A. Pfenning,
S. P. Powers,
and
L. J. Miller.
Novel tool for the study of cholecystokinin-stimulated pancreatic enzyme secretion.
J. Clin. Invest.
83:
321-325,
1989[Medline].
7.
Gaisano, H. Y.,
and
L. J. Miller.
Complex role of protein kinase C in mediating the supramaximal inhibition of pancreatic secretion observed with cholecystokinin.
Biochem. Biophys. Res. Commun.
187:
498-506,
1992[Medline].
8.
Gaisano, H. Y.,
and
L. J. Miller.
Low concentrations of protein kinase C-activating agonists suppress cholecystokinin-OPE-evoked Ca2+ mobilization in rat pancreatic acini.
Pancreas
9:
450-453,
1994[Medline].
9.
Galas, M. C.,
M. F. Lignon,
M. Rodriguez,
C. Mendre,
P. Fulcrand,
J. Laur,
and
J. Martinez.
Structure-activity relationship studies on cholecystokinin: analogues with partial agonist activity.
Am. J. Physiol.
254 (Gastrointest. Liver Physiol. 17):
G176-G182,
1988[Abstract/Free Full Text].
10.
Gates, L. K.,
C. D. Ulrich,
and
L. J. Miller.
Multiple kinases phosphorylate the pancreatic cholecystokinin receptor in an agonist-dependent manner.
Am. J. Physiol.
264 (Gastrointest. Liver Physiol. 27):
G840-G847,
1993[Abstract/Free Full Text].
11.
Hadac, E. M.,
D. V. Ghanekar,
E. L. Holicky,
D. I. Pinon,
R. W. Dougherty,
and
L. J. Miller.
Relationship between native and recombinant cholecystokinin receptors: role of differential glycosylation.
Pancreas
13:
130-139,
1996[Medline].
12.
Heuser, J. E.,
and
R. G. W. Anderson.
Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation.
J. Cell Biol.
108:
389-400,
1989[Abstract].
13.
Hunyady, L.,
A. J. Baukal,
T. Balla,
and
K. J. Catt.
Independence of type I angiotensin II receptor endocytosis from G protein coupling and signal transduction.
J. Biol. Chem.
269:
24798-24804,
1994[Abstract/Free Full Text].
14.
Klueppelberg, U. G.,
L. K. Gates,
F. S. Gorelick,
and
L. J. Miller.
Agonist-regulated phosphorylation of the pancreatic cholecystokinin receptor.
J. Biol. Chem.
266:
2403-2408,
1991[Abstract/Free Full Text].
15.
Lignon, M.,
M. Galas,
M. Rodriquez,
G. Gaillon,
and
J. Martinez.
A CCK-analogue that exhibits part of the CCK response on rat pancreatic acini: relationships among amylase release, receptor affinity and phosphoinositide breakdown.
In: Gastrin and Cholecystokinin. Chemistry, Physiology, and Pharmacology, edited by J. P. Bali,
and J. Martinez. New York: Elsevier Science, 1987, p. 57-60.
16.
Lignon, M. F.,
M. C. Galas,
M. Rodriguez,
and
J. Martinez.
Correlation between phospholipid breakdown, intracellular calcium mobilization and enzyme secretion in rat pancreatic acini treated with Boc-[Nle28, Nle31]-CCK-7 and JMV180, two cholecystokinin analogues.
Cell. Signal.
2:
339-346,
1990[Medline].
17.
Lutz, M. P.,
S. L. Sutor,
R. T. Abraham,
and
L. J. Miller.
A role for cholecystokinin-stimulated protein tyrosine phosphorylation in regulated secretion by the pancreatic acinar cell.
J. Biol. Chem.
268:
11119-11124,
1993[Abstract/Free Full Text].
18.
Matozaki, T.,
B. Goke,
Y. Tsunoda,
M. Rodriguez,
J. Martinez,
and
J. A. Williams.
Two functionally distinct cholecystokinin receptors show different modes of action on Ca2+ mobilization and phospholipid hydrolysis in isolated rat pancreatic acini. Studies using a new cholecystokinin analog, JMV-180.
J. Biol. Chem.
265:
6247-6254,
1990[Abstract/Free Full Text].
19.
Menozzi, D.,
H. A. Stark,
J. Martinez,
R. T. Jensen,
and
J. D. Gardner.
Cholecystokinin (CCK)-induced desensitization of pancreatic enzyme secretion is mediated by low affinity CCK receptors.
Peptides
10:
337-341,
1989[Medline].
20.
Menozzi, D.,
R. Vinayek,
R. T. Jensen,
and
J. D. Gardner.
Down-regulation and recycling of high affinity cholecystokinin receptors on pancreatic acinar cells.
J. Biol. Chem.
266:
10385-10391,
1991[Abstract/Free Full Text].
21.
Molero, X.,
and
L. J. Miller.
The gall bladder cholecystokinin receptor exists in two guanine nucleotide-binding protein-regulated affinity states.
Mol. Pharmacol.
39:
150-156,
1991[Abstract].
22.
Munson, P. J.,
and
D. Rodbard.
LIGAND: a versatile computerized approach for characterization of ligand-binding systems.
Anal. Biochem.
107:
220-239,
1980[Medline].
23.
Ozcelebi, F.,
and
L. J. Miller.
Phosphopeptide mapping of cholecystokinin receptors on agonist-stimulated native pancreatic acinar cells.
J. Biol. Chem.
270:
3435-3441,
1995[Abstract/Free Full Text].
24.
Ozcelebi, F.,
R. V. Rao,
E. Holicky,
B. J. Madden,
D. J. McCormick,
and
L. J. Miller.
Phosphorylation of cholecystokinin receptors expressed on Chinese hamster ovary cells: similarities and differences relative to native pancreatic acinar cells.
J. Biol. Chem.
271:
3750-3755,
1996[Abstract/Free Full Text].
25.
Pearson, R. K.,
and
L. J. Miller.
Affinity labeling of a novel cholecystokinin-binding protein in rat pancreatic plasmalemma using new short probes for the receptor.
J. Biol. Chem.
262:
869-876,
1987[Abstract/Free Full Text].
26.
Poirot, S. S.,
C. Escrieut,
M. Dufresne,
J. Martinez,
M. Bouisson,
N. Vaysse,
and
D. Fourmy.
Photoaffinity labeling of rat pancreatic cholecystokinin type A receptor antagonist binding sites demonstrates the presence of a truncated cholecystokinin type A receptor.
Mol. Pharmacol.
45:
599-607,
1994[Abstract].
26a.
Poirot, S. S.,
C. Hadjiivanova,
C. Escrieut,
M. Dufresne,
J. Martinez,
N. Vaysse,
and
D. Fourmy.
Study of the states and populations of the rat pancreatic cholecystokinin receptor using the full peptide antagonist JMV 179.
Eur. J. Biochem.
212:
529-538,
1993[Abstract].
27.
Powers, S. P.,
D. I. Pinon,
and
L. J. Miller.
Use of N,O-bis-Fmoc-D-Tyr-ONSu for introduction of an oxidative iodination site into cholecystokinin family peptides.
Int. J. Pept. Protein Res.
31:
429-434,
1988[Medline].
28.
Rao, R. V.,
B. F. Roettger,
E. M. Hadac,
and
L. J. Miller.
Roles of cholecystokinin receptor phosphorylation in agonist-stimulated desensitization of pancreatic acinar cells and receptor-bearing Chinese hamster ovary cholecystokinin receptor cells.
Mol. Pharmacol.
51:
185-192,
1997[Abstract/Free Full Text].
29.
Roettger, B. F.,
D. Ghanekar,
R. Rao,
C. Toledo,
J. Yingling,
D. Pinon,
and
L. J. Miller.
Antagonist-stimulated internalization of the G protein-coupled cholecystokinin receptor.
Mol. Pharmacol.
51:
357-362,
1997[Abstract/Free Full Text].
30.
Roettger, B. F.,
R. U. Rentsch,
E. M. Hadac,
E. H. Hellen,
T. P. Burghardt,
and
L. J. Miller.
Insulation of a G protein-coupled receptor on the plasmalemmal surface of the pancreatic acinar cell.
J. Cell Biol.
130:
579-590,
1995[Abstract].
31.
Roettger, B. F.,
R. U. Rentsch,
D. Pinon,
E. Holicky,
E. Hadac,
J. M. Larkin,
and
L. J. Miller.
Dual pathways of internalization of the cholecystokinin receptor.
J. Cell Biol.
128:
1029-1042,
1995[Abstract].
32.
Rowley, W. H.,
S. Sato,
S. C. Huang,
D. M. Collado Escobar,
M. A. Beaven,
L. H. Wang,
J. Martinez,
J. D. Gardner,
and
R. T. Jensen.
Cholecystokinin-induced formation of inositol phosphates in pancreatic acini.
Am. J. Physiol.
259 (Gastrointest. Liver Physiol. 22):
G655-G665,
1990[Abstract/Free Full Text].
33.
Saluja, A. K.,
M. Saluja,
H. Printz,
A. Zavertnik,
A. Sengupta,
and
M. L. Steer.
Experimental pancreatitis is mediated by low-affinity cholecystokinin receptors that inhibit digestive enzyme secretion.
Proc. Natl. Acad. Sci. USA
86:
8968-8971,
1989[Abstract].
34.
Sato, S.,
H. A. Stark,
J. Martinez,
M. A. Beaven,
R. T. Jensen,
and
J. D. Gardner.
Receptor occupation, calcium mobilization, and amylase release in pancreatic acini: effect of CCK-JMV-180.
Am. J. Physiol.
257 (Gastrointest. Liver Physiol. 20):
G202-G209,
1989[Abstract/Free Full Text].
36.
Stark, H. A.,
C. M. Sharp,
V. E. Sutliff,
J. Martinez,
R. T. Jensen,
and
J. D. Gardner.
CCK-JMV-180: a peptide that distinguishes high-affinity cholecystokinin receptors from low-affinity cholecystokinin receptors.
Biochim. Biophys. Acta
1010:
145-150,
1989[Medline].
37.
Tseng, M.-J.,
S. Coon,
E. Stuenkel,
V. Struk,
and
C. D. Logsdon.
Influence of second and third cytoplasmic loops on binding, internalization, and coupling of chimeric bombesin/m3 muscarinic receptors.
J. Biol. Chem.
270:
17884-17891,
1995[Abstract/Free Full Text].
38.
Tsunoda, Y.,
H. Yoshida,
and
C. Owyang.
Structural requirements of CCK analogues to differentiate second messengers and pancreatic secretion.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G8-G19,
1996[Abstract/Free Full Text].
39.
Van Zoelen, E. J. J.,
L. G. J. Tertoolen,
and
S. W. DeLaat.
Simple computer method for evaluation of lateral diffusioin coefficients from fluorescence photobleaching recovery kinetics.
Biophys. J.
42:
103-108,
1983[Abstract].
40.
Yule, D. I.,
and
J. A. Williams.
U73122 inhibits Ca2+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells.
J. Biol. Chem.
267:
13830-13835,
1992[Abstract/Free Full Text].
41.
Yule, D. I.,
and
J. A. Williams.
CCK antagonists reveal that CCK-8 and JMV-180 interact with different sites on the rat pancreatic acinar cell CCKA receptor.
Peptides
15:
1045-1051,
1994[Medline].
Am J Physiol Cell Physiol 276(3):C539-C547
0002-9513/99 $5.00
Copyright © 1999 the American Physiological Society