(Received for publication, September 12, 1994)
From the
Endocytosis of the gastrin releasing peptide receptor (GRP-R)
may regulate cellular responses to GRP. We observed endocytosis in
transfected epithelial cells by confocal microscopy using cyanine 3-GRP
(cyanine 3.18-labeled gastrin releasing peptide) and GRP-R antibodies.
At 4 °C, cy3-GRP and GRP-R were confined to the plasma membrane.
After 5 min at 37 °C, ligand and receptor were internalized into
early endosomes with fluorescein isothiocyanate-transferrin. After 10
min, cy3-GRP and GRP-R were in perinuclear vesicles, and at 60 min
cy3-GRP was in large, central vesicles, while GRP-R was at the cell
surface. We quantified surface GRP-R using an antibody to an
extracellular epitope and an I-labeled secondary
antibody. After exposure to GRP, there was a loss and subsequent
recovery of surface GRP-R. Recovery was unaffected by cycloheximide,
and thus independent of new protein synthesis, but was attenuated by
acidotropic agents, and therefore required endosomal acidification.
Internalization of
I-GRP, assessed using an acid wash,
was maximal after 10-20 min, and was clathrin-mediated since it
was inhibited by hyperosmolar sucrose and phenylarsine oxide. Thus, GRP
and its receptor are rapidly internalized into early endosomes and then
dissociate in an acidified compartment. GRP is probably degraded
whereas the GRP-R recycles.
Gastrin releasing peptide (GRP) ()is a widely
distributed neuropeptide with multiple functions which is structurally
related to the amphibian peptide bombesin(1) . GRP and bombesin
stimulate endocrine and exocrine secretions in several tissues, induce
contraction of smooth muscle, and are growth factors for normal cells
and cancer cell lines(1, 2, 3, 4) .
These actions are mediated by a high affinity interaction with the
seven transmembrane domain, G protein-coupled GRP receptor
(GRP-R)(5, 6) . Little is known about regulation of
the cellular location and signaling of the GRP-R. This regulation would
be expected to affect the responsiveness of cells to GRP.
The
response of many cells expressing the GRP-R desensitizes and
resensitizes after exposure to
agonist(7, 8, 9) . The mechanism of
desensitization is unknown, but may include phosphorylation of the
receptor with subsequent uncoupling from G proteins and physical
removal of the receptor from the plasma membrane by
endocytosis(8) . Another neuropeptide, substance P, induces
endocytosis and recycling of the neurokinin 1 receptor which coincides
with desensitization and resensitization of cellular responses to
substance P(10, 11) . Endocytosis of the thrombin
receptor also contributes to desensitization(12) . In contrast,
endocytosis of the -adrenergic receptor is not
involved in rapid desensitization, but is required for
resensitization(13, 14) . Thus, for several G-protein
coupled receptors, endocytosis may regulate cellular responses to
ligand.
Endocytosis of the GRP-R has usually been studied indirectly
by ligand binding experiments using I-labeled ligand and
an acid wash procedure to distinguish between cell surface and
internalized
peptide(15, 16, 17, 18) . However,
the intracellular compartment containing GRP and the GRP-R has not been
identified, and the mechanism of endocytosis and sorting has not been
examined. We directly observed endocytosis of both GRP and the GRP-R in
transfected cells using fluorescent GRP and receptor antibodies. The
results show that GRP and its receptor are rapidly internalized by a
clathrin-dependent mechanism into early endosomes. GRP is sorted from
its receptor in an acidified perinuclear compartment; GRP remains in
the cell whereas the GRP-R recycles to the plasma membrane.
To localize the internalized receptor, cells were incubated with 10-100 nM GRP-10 at 4 °C, washed, and incubated at 37 °C for 0-120 min. Cells were fixed with paraformaldehyde, and incubated in PBS containing 1% normal goat serum and 0.1% saponin for 3 periods of 5 min. All subsequent washes and antibody dilutions used this buffer. Cells were incubated with the GRP-R antiserum #9432 (1:50 for 4 h at 37 °C or 1:500 overnight at 4 °C), washed, incubated with a FITC- or rhodamine-labeled goat anti-rabbit IgG (1:50 for 2 h at room temperature), washed, and mounted.
To quantify
internalized peptide, cells were incubated with 400,000 cpm of I-GRP and 2 nM unlabeled GRP-27 for 60 min at 4
°C, and washed 3 times at 4 °C. Warm medium was added, and the
cells were incubated at 37 °C for 0-60 min, and washed 3
times with ice-cold buffer. To separate cell-surface (acid-sensitive)
from internalized (acid-resistant)
I-GRP, cells were
incubated in 1 ml of ice-cold 0.2 M acetic acid, 500 mM NaCl (pH 2.5) for 5 min, lysed, and the acid-sensitive and
acid-resistant pools were counted(11, 19) .
The effects of cycloheximide, brefeldin A, and various acidotropic
agents on recovery of cell-surface GRP-R were examined. Brefeldin A
causes disassembly of the Golgi apparatus and mixing with the
endoplasmic reticulum, and induces alterations in the morphological
appearance of endosomes and lysosomes(25) . Acidotropic agents
used included bafilomycin A, an inhibitor of vacuolar-type
H
-ATPase(26) , and monensin, which prevents
intracellular degradation of
I-substance P(11) .
Cells were preincubated with 70 µM cycloheximide, 1
µM bafilomycin A
, 50 µM monensin,
or 10 µg/ml brefeldin A for 30-60 min at 37 °C before
addition of GRP, and the drugs were included in all solutions during
the experiment. Control cells were incubated with appropriate carrier
solutions.
Figure 1:
Purification
and characterization of cy3-GRP. A, Lys GRP-27 was
labeled with cyanine 3.18, and purified by HPLC. Peak 2 was used in all
experiments. B, peak 2 (1.75 nM) injected at the arrow induced a prompt increase in
[Ca
]
, in KNRK Flag
GRP-R cells loaded with fura-2/AM. C, flow cytometry of cells
after equilibrium binding of cy3-GRP indicated that the mean intensity
of cyanine 3 in KNRK Flag GRP-R cells was 1 log greater than that of
KNRK CMV cells.
The specificity of cy3-GRP binding to KNKR Flag GRP-R cells and the homogeneity of the cell population were examined by flow cytometry and fluorescence microscopy. KNRK Flag GRP-R cells incubated with cy3-GRP yielded at least 1 log fold greater intensity in the cyanine 3 channel than KNRK CMV control cells (Fig. 1C). At 4 °C, a strong, crisp signal was localized to the cell surface (Fig. 2A). No fluorescent signal was detected when KNRK Flag GRP-R cells were preincubated with 1 µM GRP-10 for 30 min at 4 °C, and then incubated with both cy3-GRP and 1 µM GRP-10 (Fig. 2B), or when KNRK-CMV cells were incubated with cy3-GRP. Since preincubation with excess unlabeled GRP blocked binding, cy3-GRP binds the GRP-R specifically in KNRK-Flag GRP-R cells.
Figure 2: Specific binding of cy3-GRP to KNRK Flag GRP-R cells. A, cells were incubated with 100 nM cy3-GRP for 60 min at 4 °C, washed and fixed. B, cells were preincubated with 1 µM unlabeled GRP-10 for 30 min at 4 °C, and then incubated with 100 nM cy3-GRP plus 1 µM unlabeled GRP-10 as in A. Scale bar = 10 µm.
Biotinylated GRP-27 is also biologically active, and specifically interacts with the GRP-R on Swiss 3T3 cells(28) . However, use of this ligand requires a secondary detection system, which can cause artifacts in fluorescence microscopy and flow analysis, and necessitates extensive controls. Since cy3-GRP is directly visualized, it can be used for flow analysis and sorting of receptor bearing cells based directly on receptor affinity or number, and is also ideally suited for direct observation of peptide internalization in living cells.
Figure 3: Internalization of the cy3-GRP by live KNRK Flag GRP-R cells. Cells were incubated with 100 nM cy3-GRP for 60 min at 4 °C, washed, and incubated on an inverted microscope at 37 °C (A-D) or 18 °C (E-H). Sequential images of the same cells (A-D or E-H) were captured after 0 (A and E), 2 (B and F), 5 (C and G), or 20 (D and H) min of warming. Scale bar = 5 µm.
In parallel experiments, after
equilibrium binding of cy3-GRP (Fig. 3E), the
temperature was raised to 18 °C, which is permissive for
endocytosis but does not allow delivery to lysosomes (29) .
Within 2 min at 18 °C, cy3-GRP was internalized into small vesicles
beneath the cell surface (Fig. 3F). Cy3-GRP remained in
small, peripherally located vesicles when the cells were maintained at
18 °C for up to 40 min (Fig. 3H, 20 min). When
these same cells were warmed to 37 °C, cy3-GRP entered larger
vesicles in a perinuclear region, suggesting that the larger more
central vesicles are lysosomes. This agrees with previous studies
showing that internalized I-GRP is degraded in pancreatic
acinar cells by a mechanism partially sensitive to the acidotropic
agent chloroquine(17) .
Fluorescently-labeled transferrin, epidermal growth factor, and low density lipoprotein have been used to study endocytosis(22) . In contrast, small fluorescent peptides have not been widely used since they usually can be labeled on only 1 residue and therefore fluoresce weakly. Cy3-GRP not only generated a bright signal, but enabled sequential visualization of the same cell, which is often difficult with a rapidly photobleached fluorophore such as FITC. Based on our observations with cy3-GRP, we selected specific time points to examine endocytosis of GRP and its receptor in more detail.
We incubated KNRK Flag GRP-R cells with cy3-GRP and fixed the cells at various times after warming to 37 °C to (a) examine the uniformity of endocytosis in multiple cells; (b) investigate in detail the intracellular distribution of the peptide; and (c) make direct comparisons between these results and those showing the distribution of internalized GRP-R. In all cells examined, cy3-GRP was confined to the plasma membrane at 4 °C (Fig. 4A), and internalized into superficial vesicles after 5 min at 37 °C (Fig. 4B). After 10 min, cy3-GRP was predominantly localized in larger vesicles although also present in small superficial vesicles (Fig. 4C). By 30 min, cy3-GRP was found in large vesicles near the nucleus. At 60 and 120 min, cy3-GRP was observed in large, central vesicles and there was also diffuse central staining (Fig. 4D). Labeling of the plasma membrane was not observed at the later time points, suggesting that GRP did not recycle to the cell surface.
Figure 4: Internalization of the cy3-GRP by KNRK Flag GRP-R cells. Cells were incubated with 100 nM cy3-GRP for 60 min at 4 °C, washed, and incubated at 37 °C for 0 (A), 5 (B), 10 (C), or 60 (D) min. Cells were fixed and observed. Scale bar = 10 µm.
We compared the distribution of the GRP-R with that of cy3-GRP at the same times. KNRK Flag GRP-R cells were incubated with unlabeled GRP-10 at 4 °C, and warmed to 37 °C. At 4 °C, GRP-R immunoreactivity, like cy3-GRP, was confined to the plasma membrane (Fig. 5A). After 5 min at 37 °C, GRP-R was detected in numerous small vesicles located beneath the cell surface, similar to those containing cy3-GRP (Fig. 5B). After 10 min, the GRP-R was present in vesicles in a perinuclear region (Fig. 5C). After 30 and 60 min the intracellular staining had declined, and the plasma membrane was again strongly stained (Fig. 5D). This recovery of surface GRP-R immunoreactivity is in contrast to the distribution of cy3-GRP, which remained inside the cell.
Figure 5: Internalization of the GRP-R in KNRK Flag GRP-R cells. Cells were incubated with 10 nM GRP-10 for 60 min at 4 °C, washed, and incubated at 37 °C for 0 (A), 5 (B), 10 (C), or 30 (D) min. Cells were fixed and incubated with the GRP-R antibody. Scale bar = 10 µm.
We used confocal microscopy to confirm that the punctate pattern of staining was due to internalization of the receptor into vesicles rather than its aggregation in the plasma membrane, and to more precisely examine the distribution of the receptor. At 4 °C, GRP-R immunoreactivity was confined to the plasma membrane in optical sections just below the plasma membrane and through the center of the cell (Fig. 6A). No large pools of preformed intracellular GRP-R were detectable. After 5 min at 37 °C, the GRP-R was localized to numerous vesicles located immediately beneath the plasma membrane, and to small vesicles in the perinuclear region (Fig. 6B). After 10 min, the GRP-R was predominantly found in larger perinuclear vesicles (Fig. 6C). In contrast to cy3-GRP, which was completely depleted from the cell surface after 10 min at 37 °C (Fig. 4C), residual GRP-R remained at the plasma membrane. After 60 min, the GRP-R was localized to the plasma membrane and to small vesicles between the nucleus and plasma membrane (Fig. 6D).
Figure 6:
Confocal photomicrographs showing
internalization of the GRP-R in KNRK GRPR cells. Cells were incubated
with 10 nM GRP-10 for 60 min at 4 °C, washed, and
incubated at 37 °C for 0 (A), 5 (B), 10 (C), or 60 (D) min. Cells were fixed and incubated
with the GRP-R antibody. The figure shows optical sections at
3.5-µm intervals through the base (left panels) and
center (right panels) of the cells. Scale bar = 10 µm.
Thus, the
primary steady-state location of the GRP-R in KNRK cells is the plasma
membrane. Incubation with GRP and warming to 37 °C alters the
steady state distribution by causing internalization. Initially,
cy3-GRP and the GRP-R are internalized into superficial vesicles which
rapidly progress to a perinuclear region where cy3-GRP and the GRP-R
are sorted into distinct compartments. After 60 min the GRP-R resumes
the steady state distribution, whereas cy3-GRP remains inside the cell.
We have previously shown that substance P and the neurokinin 1 receptor
are internalized into the same endosomes and then sorted in a
perinuclear region, leading to degradation of substance P and recycling
of the neurokinin 1 receptor(10, 11) . ()Therefore, it is probable that GRP is also degraded in a
perinuclear region and its receptor returns to the cell surface.
Figure 7: Confocal photomicrographs showing internalization of cy3-GRP and FITC-transferrin by KNRK Flag GRP-R cells. Cells were incubated with 100 nM cy3-GRP and 17 µM FITC-transferrin for 60 min at 4 °C, washed, and then incubated for 10 min at 37 °C. Cells were fixed and observed using fluorescein filters to detect FITC-transferrin (A) and rhodamine filters to detect cy3-GRP (B). The image in C is the superimposition of images from A and B. Colocalization of FITC-transferrin and cy3-GRP is indicated by arrows pointing to the yellow vesicles. Scale bar = 10 µm.
Figure 8:
Internalization of I-GRP by
KNRK Flag GRP-R cells. A, cells were incubated with
I-GRP for 60 min at 4 °C, washed, and incubated at 37
°C. An acid wash was used to separate acid-sensitive (cell surface)
and acid-resistant (internalized) fractions. B, the effects of
0.45 M sucrose and 80 µM phenylarsine oxide on
internalization of
I-GRP in KNRK Flag GRP-R cells.
Binding is expressed as a percentage of the counts added to the cells,
normalized to a cell count of 100,000. Results are of duplicate
observations from n = three
experiments.
To quantify cell surface GRP-R,
we incubated non-permeabilized KNRK Flag GRP-R cells with an antibody
to the extracellular Flag epitope followed by a I-labeled
secondary antibody. Nonspecific binding was measured by incubating KNRK
CMV cells with the Flag antibody, and was 6.8 ± 1.0% of that
measured in KNRK Flag GRP-R cells (triplicate observations, n = 9 experiments). Specific binding of the Flag antibody to
KNRK Flag GRP-R cells that were incubated with or without GRP-10 for 60
min at 4 °C was the same. Therefore, GRP binding did not affect the
ability of the Flag antibody to bind the receptor. When KNRK Flag GRP-R
cells were incubated with 100 nM GRP-10 at 4 °C, and
warmed to 37 °C for various times, there was a marked reduction in
cell surface Flag immunoreactivity which then returned to baseline
levels (Fig. 9A). After 10 min at 37 °C, the
surface immunoreactivity had declined to 77.3 ± 2.7% (triplicate
observations, n = 13 experiments) of control levels (no
GRP-10), and after 60 min at 37 °C had returned to 92.08 ±
1.86% (n = 10 experiments) of the control. The loss of
surface Flag immunoreactivity at 10 min was dependent on the
concentration of ligand and was maximal at 1 µM GRP-10.
The recovery rate of surface Flag immunoreactivity was not altered by
ligand concentration.
Figure 9:
Quantification of surface Flag
immunoreactivity in KNRK Flag GRP-R cells. A, cells were
incubated with 100 nM GRP-10 for 60 min at 4 °C, washed,
warmed to 37 °C for 0-60 min, and incubated at 4 °C with
the Flag antibody and I-sheep anti-mouse IG. At each time
point, specific Flag binding is expressed as a percentage of binding to
control cells not treated with GRP. Results are of triplicate
observations from n = 3-13 experiments. B, to study the mechanism of endocytosis, cells were treated with
0.45 M sucrose or 80 µM phenylarsine oxide, and
after equilibrium binding of 100 nM GRP-10 at 4 °C, were
warmed to 37 °C for 10 min. To study the return in surface GRP-R,
cells were treated 70 µM cycloheximide, 50 µM monensin, 1 µM bafilomycin A
, or 10
µg/ml brefeldin A, and after GRP binding, warmed to 37 °C for
60 min. Surface GRP-R were detected as described in A. Each
experimental group had its own control not exposed to GRP, to correct
for any effects of these agents on the assay. Results are from
triplicate observations from n = three to nine
experiments. *, <0.05 compared to the 10-min carrier. **, <0.05
compared to the 60-min carrier.
Together, these results show that GRP and the GRP-R are maximally internalized within 10-20 min at 37 °C. However, whereas most of the specifically bound ligand is internalized, a considerable amount of receptor remains at the cell surface even after exposure to high concentrations of GRP (1 µM). Residual surface receptors may be in a low affinity state and unable to bind ligand.
Similarly, treating cells with hyperosmolar sucrose or phenylarsine oxide inhibited the decline in cell surface Flag immunoreactivity. When carrier-treated KNRK Flag GRP-R cells were incubated with 100 nM GRP-10 for at 4 °C, washed, and warmed to 37 °C for 10 min, surface Flag immunoreactivity had declined to 84.7 ± 1.0% (triplicate observations, n = four experiments) of control levels (no GRP-10). In cells treated with 0.45 M sucrose or 80 µM phenylarsine oxide, surface Flag immunoreactivity was 96.1 ± 2.0 and 100.5 ± 2.7% of control levels, respectively. In parallel experiments, KNRK Flag GRP-R cells treated with sucrose or phenylarsine oxide were processed to localize the GRP-R by immunocytochemistry and examined by confocal microscopy. Internalization of GRP-R immunoreactivity into early endosomes was abolished by both agents (Fig. 10).
Figure 10: Confocal photomicrographs showing the effects of hyperosmolar sucrose and phenylarsine oxide on internalization of the GRP-R in KNRK GRPR cells. Cells were incubated with 10 nM GRP-10 for 60 min at 4 °C, washed, and incubated at 37 °C for 10 min. Cells were immediately fixed and incubated with the GRP-R antibody. A, control cells; B, cells treated with 0.45 M sucrose; C, cells treated with 80 µM phenylarsine oxide. Scale bar = 5 µm.
Therefore, GRP and its receptor are internalized by clathrin-coated
pits. This is a common theme for seven transmembrane domain receptors,
since ligand-induced endocytosis of the neurokinin 1,
-adrenergic, and thrombin receptors is mediated by
clathrin(11, 12, 13) . A clathrin-independent
mode of internalization exists for
glycosylphosphatidylinositol-anchored proteins, such as the folate
receptor(33) . The folate receptor clusters in caveolae with
underlying caveolin enabling bound folate to enter the cytoplasm.
Ligands internalized through both clathrin-dependent and
clathrin-independent mechanisms move into endosomes containing the
transferrin receptor and follow a common
pathway(34, 35) .
We conclude that GRP induces clathrin-dependent internalization of the GRP-R into early endosomes. GRP and its receptor are sorted in an acidified, perinuclear compartment. GRP remains inside the cell, where it is probably degraded, whereas the receptor recycles to the plasma membrane. Internalization and recycling of the GRP-R may modulate the cellular response to GRP.