From the Departments of Cell Biology and Orthopedics, Yale University School of Medicine, New Haven, Connecticut 06510
Received for publication, September 20, 2002, and in revised form, December 27, 2002
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ABSTRACT |
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Many G protein-coupled receptors undergo
endocytosis, but the mechanisms involved in endocytic sorting and
recycling remain to be fully elucidated. We found that the G
protein-coupled calcitonin receptor (CTR) undergoes tonic
internalization and accumulates within the cell. Using a fluorescence
loss in photobleaching assay, we classified these vesicles
functionally as recycling vesicles. In a two-hybrid screening, we found
that the actin-binding protein filamin interacted with the C-terminal
tail of the CTR. The degradation of the receptor was profoundly
increased in the absence of filamin or the CTR-filamin interaction. The
absence of filamin was also associated with a marked decrease in
recycling of the receptor from the endosomes to the cell surface.
In contrast, calcitonin-induced inhibition of spontaneous
filamin proteolysis was associated with increased recycling of the
receptor to the cell surface and decreased degradation of the CTR,
suggesting an important role for filamin in the endocytic sorting and
recycling of the internalized CTR.
The calcitonin receptor
(CTR)1 belongs to subclass B
of the G protein-coupled receptors (GPCRs). It binds calcitonin (CT), which acts on bone and kidney to maintain calcium homeostasis. It also
acts on the central nervous system, where it has anorexic and analgesic
effects (1). GPCRs comprise the largest known superfamily of cell
receptors. They mediate responses to the majority of known hormones,
neurotransmitters, and neuromodulators and also function as sensors for
extracellular ions, light, and pheromones. These facts make GPCRs in
general extremely interesting targets for therapeutic drugs. GPCRs
mediate signal transduction by serving as ligand-regulated guanine
nucleotide exchange factors for heterotrimeric GTP-binding proteins,
which in turn regulate several downstream effectors. Moreover, several
recent reports demonstrate the existence of G protein-independent
signaling pathways used by GPCRs (2).
Endocytosis is a common response to ligand stimulation that sequesters
the receptors by redistributing them from the cell membrane to
intracellular membranes (3). The internalized receptor can then either
recycle back to the cell surface or be targeted to lysosomes and
degraded, a process inducing down-regulation of the GPCR. There is a
body of evidence showing receptor- and cell-specific differences in the
mechanisms of GPCR endocytosis (3). A common mechanism involves ligand
binding and activation of the receptor followed by phosphorylation of
the GPCR by GPCR kinases, which leads to binding of In addition to the well characterized ligand-induced endocytosis, there
are reports of GPCRs undergoing tonic internalization (7-14).
Mechanisms underlying tonic endocytosis include agonist-independent Here we show that the CTR undergoes tonic internalization and recycling
to the cell surface. Efficient recycling was found to be dependent on
the interaction of the C-terminal tail of the CTR with the
actin-binding protein filamin. The absence of filamin or disruption of
the association between filamin and the C-terminal tail of the receptor
resulted in reduced CTR recycling back to the cell surface and the
rapid degradation of the CTR, whereas CT-induced inhibition of filamin
proteolysis increased recycling to the cell surface and decreased
degradation of the CTR.
Reagents and Antibodies--
Salmon calcitonin (sCT) was
purchased from Peninsula Laboratories, Inc. (Belmont, CA). MG-132 was
from Sigma. The monoclonal anti-HA antibody (F-7) was from Santa
Cruz Biotechnology, Inc. (Santa Cruz, CA), the monoclonal anti-GFP
antibody was from Clontech (Palo Alto, CA), the
monoclonal anti-FLAG antibody (M2) was from Sigma, the monoclonal
anti-filamin antibody was from Chemicon (Temecula, CA), and the
monoclonal anti-xpress© antibody was from Invitrogen. Enhanced
chemiluminescence solutions and nitrocellulose membranes were from
Amersham Biosciences and Schleicher & Schuell, respectively.
Cell Culture and Transient Transfections--
Minimal essential
medium, Dulbecco's modified Eagle's medium (DMEM), methionine-free
DMEM, fetal bovine serum (FBS), and newborn calf serum were purchased
from Invitrogen. All media were supplemented with 100 µg/ml
streptomycin and 100 units/ml penicillin. HEK 293 cells were cultured
as described before (15). A7 and M2 cells were a generous gift from Dr.
John Hartwig (Harvard University). Both cell lines were cultured in
minimal essential medium with 8% newborn calf serum and 2% FBS. The
medium of A7 cells contained 500 µg/ml G418 (Invitrogen). For
transient transfections, cells were grown to 60-70% confluence and
then transfected with FuGENE 6 (Roche Molecular Biochemicals) according
to the manufacturer's protocol.
DNA Constructs--
A cDNA encoding the rabbit CTR was
generated by PCR and cloned in the KpnI/HindIII
sites of the p3XFLAG-CMV-13 vector (Sigma) to obtain a CTR with a
C-terminal 3-fold FLAG tag. The same PCR product was cloned in the
KpnI/HindIII site of pEGFP-N1
(Clontech) to obtain a C-terminal GFP-tagged CTR.
Both constructs contained a HA tag in the extracellular N terminus of
the receptor (after aa 29 of the original sequence). A construct
expressing the CTR with three tandem Myc epitope tags at the C
terminus was generated as described before (16). All CTR constructs
were compared with the wild-type CTR with respect to ligand binding,
phosphorylation after ligand stimulation, and cAMP generation and were
found to be indistinguishable. A fragment spanning the seventh
transmembrane domain and the C-terminal tail of the receptor (aa
373-474) was cloned between the HindIII and KpnI
sites of the p3XFLAG-CMV-13 vector after generation by PCR. The same
was done for the C-terminal tail without the seventh transmembrane
domain (aa 397-474). A filamin fragment spanning the
immunoglobulin-like repeats 20-23, obtained from a construct encoding
the human filamin cDNA (gift of Dr. John Hartwig, Harvard
University), was cloned in the XbaI/BamHI sites
of the pcDNA4HisMaxA vector (Invitrogen). All PCR-derived constructs were sequenced by the Yale Keck Sequencing Facility. The
HA-tagged Yeast Two-hybrid Screening--
A cDNA encoding the
C-terminal tail of the rabbit CTR (aa 397-474) was cloned in the bait
vector pBTM116 and used to screen a mouse osteoclast-like library in
pASV4, as described before (17). The DNA of the colonies that were
positive for both reporter assays was extracted and transformed in
Escherichia coli, strain DH5 Co-immunoprecipitation and Western Blotting--
Cells were
lysed in mRIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% IGEPAL, 1% sodium deoxycholate, 10 mM NaF, 1 µg/ml pepstatin, and 1 mM
phenylmethylsulfonyl fluoride) and incubated at 4 °C for 30 min.
Lysates were then centrifuged for 30 min at 4 °C, 16,000 × g, the protein concentrations were measured with the BCA
protein assay kit (Pierce) and equal amounts of protein were used for
immunoprecipitation. 30 µl of protein-G-agarose slurry and typically
5 µg of antibody were suspended in 500 µl of PBS and incubated for
1 h at 4 °C. The beads were washed three times in mRIPA buffer,
then 500 µg of lysate protein and bovine serum albumin (0.2%) (w/v)
were added, and the mix was incubated for 2 h at 4 °C. The
immune complexes on the beads were washed four times with washing
buffer containing 500 mM NaCl and 0.1% Triton X-100 and
once with PBS. Beads were boiled in 2× SDS-PAGE buffer, and samples
were electrophoresed on precast 10% SDS-PAGE gels (Invitrogen).
Proteins were transferred onto nitrocellulose. Nonspecific binding was
blocked by incubating the membranes in 5% non-fat milk in TBST buffer
(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1%
Tween 20) for 1 h. Membranes were incubated in the primary
antibody for 2 h, washed three times for 15 min in TBST, and
incubated for 1 h in 1:10,000 diluted horseradish
peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG antibody
(Promega). Blots were developed using the enhanced chemiluminescence
system from Amersham Biosciences.
Measurement of Receptor Cell Surface Expression and Endocytosis
Using FACS--
The surface expression of the CTR was measured by
fluorescence-activated cell sorter (FACS) analysis. Cells in 6-well
plates were trypsinized, and the trypsin activity was neutralized by addition of growth medium containing 10% dialyzed FBS. Cells were collected by centrifugation at 800 × g for 3 min. The
cell pellet was resuspended in 100 µl of ice-cold PBS. Usually about
3 × 105 cells were used for each experiment. Normal
goat IgG was added to a final concentration of 200 µg/ml. After 10 min of incubation, the anti-HA antibody was added to final
concentration of 10 µg/ml.
To measure cell surface expression, a 30-min incubation step was
followed by resuspending cells in 100 µl of PBS containing 50 µg/ml
phycoerythrin (PE)-conjugated goat anti-mouse IgG antibody (Molecular
Probes). After a final washing step, cells were resuspended in PBS
containing 2% formaldehyde to fix the sample. Bound antibody was
analyzed by fluorescence flow cytometry (FACScalibur; BD
Biosciences). Win MDI software, version 2.8, was used for data analysis.
To measure receptor endocytosis, a 30-min incubation step at 4 °C
was followed by two washes in 100 µl of ice-cold PBS. The washed
cells were resuspended in ice-cold DMEM, with or without 10 nM sCT. Cells were then quickly warmed to 37 °C in a
water bath to allow endocytosis of the receptor for different time
periods. Cells were then cooled to 4 °C, centrifuged, and
resuspended in ice-cold PBS containing 50 µg/ml PE-conjugated
goat anti-mouse IgG antibody (Molecular Probes). After a final washing
step, cells were resuspended in PBS containing 2% formaldehyde to fix
the sample, and bound antibody was analyzed by flow cytometry.
Analysis of Receptor Recycling Using FACS--
Cells were
prepared as described for the internalization assay, except that cells
were incubated for 30 min at 37 °C in the presence of the anti-HA
antibody to allow endocytosis of the CTR with the bound antibody. After
cooling to 4 °C, cells were incubated with an acid strip solution
(0.2 M acetic acid, 0.5 M NaCl, pH 3) for 3 min
to remove the surface-bound antibody. The acid strip reduced the amount
of PE-conjugated secondary antibody binding to levels similar to
controls without anti-HA antibody. Cells were then warmed to 37 °C
for different time periods to allow recycling of the CTR. PE staining
and FACS analysis were performed as described above.
Analysis of Receptor Endocytosis Using Cleavable
Biotin--
CTR-transfected cells in 10-cm dishes were washed twice
with ice-cold PBS and incubated with 0.5 mg/ml sulfo-NHS-S-S-biotin (Pierce) in PBS for 30 min at 4 °C to biotinylate cell surface proteins. Excess biotin was quenched by incubating in Tris-HCl, pH 7.4, (final concentration 50 mM) for 10 min at 4 °C. Cells were transferred to medium prewarmed to 37 °C with or without 10 nM sCT and incubated at 37 °C for various times to allow
endocytosis of the receptor and then chilled on ice to stop
endocytosis. Biotin was cleaved from proteins on the cell surface by
washing cells twice at 4 °C with 50 mM glutathione, 75 mM NaCl, 75 mM NaOH, 10% FBS. The cells were
then washed twice for 10 min at 4 °C in iodoacetamide buffer (50 mM iodoacetamide, 1% bovine serum albumin in PBS) to
quench residual glutathione. Cells were harvested in mRIPA supplemented
with 1 mg/ml iodoacetamide. The FLAG-tagged CTR was immunoprecipitated,
electrophoresed on 10% SDS-PAGE without reducing agent, and
transferred electrophoretically to nitrocellulose. The endocytosed,
biotinylated receptor was then detected using horseradish
peroxidase-conjugated avidin D (Vector Laboratories, Burlingame, CA).
Metabolic Labeling, Pulse-Chase
Experiments--
CTR-FLAG-transfected cells were washed twice with PBS
and preincubated in methionine-free labeling DMEM medium, containing 10% dialyzed FBS (Invitrogen) for 30 min.
[35S]-Methionine (Amersham Biosciences) was added to a
final activity of 150 µCi/ml. Cells were incubated for 90 min, then
washed twice with chase medium (DMEM with 10% FBS, 2 mM
methionine), and incubated in chase medium for the indicated time
periods. After the chase, cells were washed twice with ice-cold PBS and
lysed in mRIPA buffer. The CTR was immunoprecipitated and
electrophoresed on 10% SDS-PAGE gel. The gel was dried in a gel dryer
at 80 °C for 2 h and exposed to Eastman Kodak Co. MR x-ray film
for at least 24 h. The x-ray film was scanned, and the intensity
of the corresponding bands was quantified using the NIH Scion Image
1.62c software, checking that the bands of interest were in the linear
range of the x-ray film.
Fluorescence Loss in Photobleaching (FLIP)
Experiments--
Cells were seeded in 35-mm glass-bottom dishes
(MatTek Corp., Ashland, MA) and 24 h later were transfected
with the GFP-tagged CTR constructs as described above. To acquire the
images, the dish was placed in a heated stage (DH-35; Warner
Instruments, Hamden, CT) in a Zeiss LSM 510 confocal microscope
(Zeiss, Jena, Germany) to maintain a temperature of 37 °C. The
culture medium was replaced by 37 °C Na+-HEPES buffer
(135 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 10 mM glucose, and 10 mM HEPES, pH 7.3, 290 mosmol/liter). All photobleaching experiments were carried out in the
presence of 25 µM cycloheximide added 30 min before the
start of the recordings to rule out contribution from new protein
synthesis. A 63 × 1.25 numerical aperture water immersion
objective was used. GFP was excited at 488 nm by an argon laser.
Emission was measured in the green channel from 505-530 nm.
Photobleaching was obtained by scanning the region of interest (a
hand-drawn box, the contours of which surrounded the area to bleach) at
75% power and 100% transmission. Imaging was done by scanning the
whole cell with attenuated transmission (1%). The fluorescence
intensity in membrane areas of the bleached cell distant from the
bleached spot, and as a control, the fluorescence intensity of the
membrane of a neighboring, unbleached cell were measured every 6 s
for 10 min after the bleaching. This procedure was repeated four times.
Data were transferred to Excel, and for each data point the
fluorescence intensity was expressed as percentage of the initial mean
fluorescence intensity before bleaching.
Statistical Analysis--
For analysis of the statistical
significance of the observed differences, data from all independent
experiments were pooled, mean values and S.E. were calculated, and a
Student's t was test performed. A significant difference
was assumed when p was <0.05.
The Calcitonin Receptor Undergoes Tonic Endocytosis and Recycling
Back to the Cell Surface--
To study the ligand-independent and
ligand-induced endocytosis of the CTR, we transfected HEK 293 cells
with the CTR-FLAG construct and surface-labeled the cells with a
cleavable biotin compound. Internalization of the receptor was allowed
to proceed for different time periods, and the biotin remaining on the
cell surface was stripped. The cells were lysed, and the FLAG-tagged CTR were immunoprecipitated and blotted with avidin (Fig.
1A) to detect the endocytosed
CTR. We found that a substantial fraction of the CTR was internalized
in the absence of ligand, whereas the presence of CT caused only a
slightly higher amount of CTR internalization. To confirm this result
with a more quantitative method, we performed a flow cytometry-based
internalization assay. HEK 293 cells were transfected with C-terminally
GFP-tagged, N-terminally HA-tagged CTR. After serum starving for at
least 12 h, cells were incubated at 4 °C in the presence of an
anti-HA antibody to label the extracellular HA tag. After this binding
step, cells were quickly warmed to 37 °C and incubated in the
presence or absence of 10 nM sCT for different time periods
to allow endocytosis. The remaining anti-HA antibody bound to the CTR
at the surface was then labeled with the PE-conjugated secondary
antibody, and the cell-associated PE was quantified by FACS. This
method also showed that a substantial fraction of the CTR on the
surface was internalized in the absence of ligand, although more
receptor was internalized in the presence of the ligand, with the
difference statistically significant at 30 and 60 min (Fig.
1B). Co-expression of the dominant-negative dynamin I-K44A
mutant, which blocks endocytosis via clathrin-coated pits (18), failed
to reduce tonic- or ligand-induced endocytosis of the CTR (data not
shown).
To determine whether the internalized CTR is recycled back to the cell
surface, we allowed endocytosis of the CTR to proceed in the presence
of the anti-HA antibody and then stripped surface-bound HA-antibody
with an acid wash. The cells were then re-warmed for different time
periods. Although the amount of antibody reappearing on the surface was
clearly less than what had been endocytosed, consistent with the
assumption by other investigators that non-covalently bound antibodies
would be lost as the internalized receptor passes through acidified
internal compartments (14), we observed a clear increase in the amount
of antibody on the cell surface after re-warming, indicating that at
least some of the internalized CTR-antibody complex survived the
passage through the endocytic vesicles and was recycled to the cell
surface. The amount of antibody-decorated CTR on the cell surface
reached a steady state by about 20 min. When CT was present during both
internalization and recycling periods, significantly more of the CTR
was recycled at 40 min (Fig. 1C). These data, together with
the result shown in Fig. 1B, suggest that ligand binding
induces both more internalization and more recycling of the CTR.
We then determined the intracellular localization of the tonically
internalized CTR, using a CTR fused at the C-terminal to GFP. Confocal
microscopy showed substantial accumulation of the CTR-GFP within the
cell (Fig. 2A, left
panel). The transferrin receptor, which is constitutively
internalized and recycled back to the cell surface, colocalized with
the CTR at the cell surface and in a vesicle-rich area in the center of
the cell (data not shown). To determine whether the CTR was recycling
to the cell surface from this region, we used the FLIP method in cells
expressing the CTR-GFP by repeated photobleaching of the intracellular
CTR-GFP accumulations. Fig. 2 shows a typical experiment. The
intracellular accumulation of CTR-GFP was photobleached in one cell
whereas an adjacent unbleached cell served as a control. The
fluorescence intensity at the surface of the bleached cell began to
decrease about 10 min after the initial bleaching and eventually
decreased to about 50% of the initial level. The fluorescence of the
neighboring unbleached cell was unchanged. This result strongly
suggests that the constitutively endocytosed CTR is recycled back to
the cell surface via the vesicle-rich area in the center of the
cell.
The C-terminal Tail of the Calcitonin Receptor Constitutively
Interacts with the Actin-binding Protein, Filamin--
We suspected
that an association of the C-terminal tail of the CTR with a cellular
protein is necessary for targeting the CTR to the recycling
compartment. To identify proteins that associate with the C-terminal
tail of the CTR and might thereby promote the recycling of the
receptor, we screened an osteoclast-like cell yeast two-hybrid library
(17) with the CTR C-terminal tail (aa 398-474). Screening of
106 independent clones yielded five positive clones, one of
which encoded the C-terminal region of filamin A, analogous to aa
2129-2647 of human filamin A, which contains the 19th to 24th
immunoglobulin-like repeats and the second hinge region (19) (Fig.
3A).
Truncation mutants were used to map the region that binds to the
cytoplasmic tail of the CTR. The fragments spanning aa 2129-2458 or aa
2129-2330 were active in the yeast two-hybrid assay, suggesting that
the primary interacting region is between aa 2129 and 2330, corresponding to the immunoglobulin-like repeats 20 and 21. However, the activation was weaker than that of the original filamin clone, suggesting either that the smaller fragment folds differently or that
sequences elsewhere in the original clone stabilized the binding to the
CTR.
The location of the filamin binding site on the CTR C-terminal domain
was further refined using the yeast two-hybrid system (Fig.
3B). The strong activation in the reporter assays achieved by the complete C-terminal tail (aa 397-474) was seen only with a
fragment spanning aa 422-474. The smaller fragment spanning aa
447-474 was also positive in both reporter assays, although less so
than the full-length tail. This region contains no sequences that
correspond to known protein binding domains such as PDZ, Src homology
2, or Src homology 3 domains. The CTR fragments more proximal to the
seventh transmembrane domain (aa 397-447 and aa 397-422) also showed
a consistent but much smaller activation in the reporter assays. The
results suggest that aa 447-474 span the primary binding site for
filamin but that other more proximal regions could be also contributing
to stabilizing the filamin-CTR association.
To show that the interaction of the CTR and filamin occurs in mammalian
cells, we immunoprecipitated C-terminally FLAG-tagged CTR from HEK 293 cells and blotted for filamin (Fig. 3C) or
immunoprecipitated endogenous filamin and blotted for FLAG (data not
shown). Both proteins were present in the immune complexes, regardless
of the antibody used in the isolation, confirming an interaction of
filamin and the CTR in mammalian cells. Treating the cells with sCT had no detectable effect on the amount of filamin that
co-immunoprecipitated with the CTR (Fig. 3C), indicating
that the association between the CTR and filamin is not regulated by
CTR-induced signaling effectors.
To further confirm the CTR-filamin interaction, we examined the effect
of overexpressing xpress©-tagged filamin repeats 20 to 23, the
FLAG-tagged CTR cytoplasmic tail, or the FLAG-tagged CTR cytoplasmic
tail plus 7th transmembrane domain on the interaction between the
full-length CTR and filamin. Each of the fragments strongly reduced the
co-immunoprecipitation of endogenous filamin with the Myc-tagged CTR
(Fig. 3D).
The Interaction of the Calcitonin Receptor with Filamin Reduces the
Degradation of the Receptor--
To investigate the functional
relevance of the association between filamin and the CTR we used M2
cells, which express no filamin A, and A7 cells, which are M2 cells
stably transfected with filamin A (20). We transfected these cell lines
with the CTR and analyzed CT-induced signal transduction by the CTR in the absence of filamin. We found no significant differences in the
ligand-induced generation of cAMP, increase in intracellular calcium
concentrations, or ERK phosphorylation in M2 and A7 cells (data not
shown), suggesting that the association with filamin is not required
for these signaling pathways of the CTR.
However, Western blots indicated that there was less CTR in
the M2 cells than in the A7 cells (Fig.
4A), and both confocal microscopy (Fig. 4B) and FACS analysis (Fig. 4C)
indicated that there was less surface expression of the CTR in M2 cells
than in the filamin-containing A7 cells. The lower expression of the CTR in the absence of filamin could result from lower de
novo synthesis or from faster degradation of the CTR. To
distinguish between these possibilities, we performed a pulse-chase
analysis of CTR degradation in M2 and A7 cells (Fig. 4D).
The synthesis of the CTR was similar in both cell lines (data not
shown), but the extent of degradation of the CTR in the M2 cells was
about twice that in A7 cells (Fig. 4D, upper
panel). Half of the labeled receptor in the M2 cells was degraded
by 3 h, whereas more than 60% of the labeled receptor was still
present in the A7 cells at 6 h (Fig. 4D, upper
panel). To rule out the possibility that the accelerated
degradation was not specific for the CTR, we expressed another GPCR,
the
We next asked whether disrupting the filamin/CTR association also
increased the degradation of the CTR. Filamin repeats 20-23 were
co-expressed with the CTR in A7 and M2 cells, and the CTR degradation
was measured in a pulse-chase assay (Fig. 4E). The co-expression of the filamin fragment significantly increased the
amount of degradation of the CTR in A7 cells. In contrast, and as
expected, the expression of this fragment had little effect on the
degradation of the CTR in M2 cells.
We investigated the pathways involved in the degradation of
the CTR by treating pulse-labeled M2 and A7 cells with chloroquine or
NH4Cl, both of which inhibit lysosomal degradation, or with MG132, a proteasome inhibitor. MG132 significantly inhibited the degradation of the receptor in M2 cells. Chloroquine and
NH4Cl also slightly inhibited the degradation of the CTR in
both cell lines (Fig. 4E), although the results failed to
reach statistical significance, suggesting that the lysosomal pathway
might also be involved in the degradation of the CTR in these cells.
Filamin Is Required for Efficient Recycling of the
Calcitonin Receptor--
The increased degradation of the CTR in the
absence of its association with filamin could result from an increased
rate of tonic endocytosis of the CTR. Flow cytometric analysis (Fig.
5A) clearly showed similar
ligand-independent internalization in both M2 and A7 cells. We next
examined the CTR recycling in the M2 and A7 cells and found
significantly less of the CTR recycled to the surface in M2 cells than
in A7 cells (Fig. 5B).
We next did a FLIP analysis of CTR-GFP trafficking in M2 and A7 cells
to examine the recycling of the CTR from the perinuclear vesicle
compartment in the absence of filamin. In A7 cells, the fluorescence
intensity of the cell membrane decreased after photobleaching of the
intracellular CTR-GFP-containing endosomes (Fig.
6, upper panel), suggesting
that the CTR recycles back to the cell surface from the photobleached
endosomes in A7 cells as it does in the HEK 293 cells. In contrast,
photobleaching the intensely fluorescent vesicles in M2 cells caused a
much smaller decrease in the fluorescence intensity of the cell
membrane (Fig. 6, lower panel), indicating that fewer of the
CTR in the photobleached endosomes recycle to the surface in the
absence of filamin. The more rapid degradation of the CTR in the
absence of filamin may therefore be related because of a reduced
efficiency of recycling from the perinuclear vesicles.
Calcitonin Affects the Degradation of Its Receptor by Inhibiting
Filamin Proteolysis--
Our initial results (Fig. 1C)
suggested that CT promoted the recycling of the internalized CTR to the
cell surface. We considered the possibility that CTR signaling
specifically promotes the filamin-dependent recycling of
the internalized CTR, thereby protecting it from degradation. We
therefore determined how CT treatment affected the degradation of
pulse-labeled CTRs in A7 and M2 cells. Exposing the cells to 10 nM CT resulted in about 30% decrease in the amount of
degradation in the filamin-containing A7 cells but had no significant effect on the degradation in the filamin-free M2 cells (Fig.
7A).
Cleavage of filamin between the actin binding site and the CTR binding
site by calpain would result in uncoupling of the CTR from the actin
cytoskeleton (21). Given that Marzia et al. (22) have
demonstrated that CT inhibited calpain activity and induced a transient
decrease in the constitutive production of a 190-kDa fragment in
osteoclasts, we determined whether CT affected filamin fragmentation in
HEK 293 cells in a manner similar to what has been observed in
osteoclasts. In untreated HEK 293 cells, as in osteoclasts, the 190-kDa
proteolytic fragment was clearly detected. Brief exposure to CT (5 min)
caused the amount of the fragment to decrease (about 55% of baseline
level), suggesting that CT is inhibiting calpain activity. The amount
of the fragment slowly increased, approaching basal levels between 45 and 60 min (Fig. 7B).
The calpain inhibitor calpeptin also reduced the rate of degradation of
the pulse-labeled CTR in the A7 cells but not in M2 cells (Fig.
7A). Together these results suggest that constitutive calpain-catalyzed cleavage of filamin occurs at a low rate and that
inhibiting that cleavage reduces CTR degradation in a
filamin-dependent manner.
Although numerous studies have provided detailed information on
the molecular mechanisms of endocytosis of GPCRs (23), much less is
known about the endosomal sorting and recycling of these receptors. The
mechanisms involved in tonic receptor internalization and recycling,
which is observed for a growing number of GPCRs (7-14), are of
particular interest, because efficient sorting and recycling of the
internalized receptor is required to avoid extensive degradation of the
receptor and to maintain appropriate numbers of receptors at the cell
surface. The sorting of tonically endocytosed receptors also provides a
mechanism by which cells regulate the level of surface expression of
these receptors.
In this paper we show that the CTR is constitutively internalized and
recycles to the cell surface. Our results offer insight into several
aspects of CTR internalization and recycling. CTR recycling is
decreased, and its degradation is increased when the CTR cannot bind
filamin. In filamin-containing cells, the CTR appears to be recycled
through a population of perinuclear vesicles that also stain for the
transferrin receptor.
Several different experiments suggest that the transit time for
internalized receptors to return to the surface is about 10 min. Both
the loss of antibody-decorated CTR from the cell surface and the
reappearance of antibody-decorated CTR after acid wash approach a
steady state at this time, and the decrease in surface membrane
fluorescence in the FLIP experiment, which reflects the insertion of
bleached receptors at the surface, begins 8 to 10 min after the initial photobleaching.
In addition, the steady state levels of antibody-decorated CTR in the
internalization experiments (40-60% of initial levels; see Fig.
1B) suggest that on average, about half the receptors that
are actively recycling are present on the surface at any one time. The
steady state level of antibody-decorated CTR that reappears on the cell
surface following the acid strip is necessarily lower, because only a
fraction of the initially labeled receptors are internalized and thus
protected from the stripping, and they will become progressively
diluted with undecorated receptors as recycling and internalization
progresses. Moreover, some of the antibodies may dissociate from
the receptors during the internalization and recycling.
The role of filamin in CTR recycling appears to involve the regulation
of the transition from the intracellular compartment to the surface.
The rates of internalization in the M2 and A7 cells, reflected in the
initial slope of the internalization curves (Fig. 5A), are
indistinguishable, whereas the return of the CTR to the surface in the
filamin-deficient M2 cells is less than half the normal level in both
the antibody-tagging and the FLIP experiments. The amount of CTR
degradation also changes by about 2-fold in the absence of filamin,
approximately doubling in the M2 cells. The increased degradation could
be a consequence of the presumed accumulation of the receptor in an
intracellular compartment as a consequence of the impaired recycling.
However, we cannot exclude that the accelerated degradation and
decrease in recycling via the perinuclear compartment is because of an increased active sorting of the CTR to degradative compartments when it
is not bound to filamin. Our data do not allow us to distinguish between these possibilities.
Other proteins that regulate GPCR recycling and degradation have been
described. Cong et al. (24) found that the
N-ethylmaleimide sensitive factor binds to the C-terminal
tail of the Although we did not find a CT-dependent change in the
CTR-filamin interaction, we did observe a CT-induced decrease in CTR degradation. The CT-induced increase in the amount of antibody-tagged CTR that recycled to the surface following an acid strip might be a
consequence of less degradation. On the other hand, the steady-state level of antibody-tagged CTR was somewhat lower in CT-treated cells,
possibly reflecting an overall net retention of CTR in intracellular
compartments under this condition. Further experiments will be
necessary to answer these questions.
We also observed a transient CT-induced decrease in the cleavage of
filamin. Cleavage of filamin by calpain separates the CTR binding site
from the N-terminal actin binding domain of filamin, thereby disrupting
the connection between the internalized CTR and actin. Inhibition of
calpain-induced filamin cleavage could thus increase CTR recycling and
consequently inhibit the degradation of the receptor, as illustrated in
Fig. 8. The inhibition of CTR degradation
by calpeptin provides further support for this hypothesis. The
inhibition of filamin proteolysis by CT might involve an inhibition of
calpain because of phosphorylation by cAMP-dependent
protein kinase (27) or phosphorylation of filamin itself (28).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestins to
the receptor and thereby to the uncoupling from the heterotrimeric G
protein (4-6).
-arrestin binding or tyrosine-based endocytic motifs (12, 13). Although the reasons for tonic internalization of GPCRs are not understood, it is clear that tonic receptor internalization must be
compensated by constant recycling of the receptor back to the cell
surface if surface expression of the receptor is to be maintained. However, much less is known about endosomal sorting and recycling processes of GPCRs than about the molecular events leading to GPCR endocytosis.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor was a gift of Dr. Brian
Kobilka (Stanford University).
. The library plasmid
was isolated, amplified, and retransformed in L40 cells already
containing the bait plasmid to confirm the positive result.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The calcitonin receptor undergoes tonic
endocytosis. A, CTR-FLAG-transfected HEK 293 cells were
labeled with the membrane-impermeable sulfo-NHS-S-S-biotin. Cells were
then transferred to medium prewarmed to 37 °C with or without 10 nM sCT and incubated for 5 to 60 min to allow endocytosis
of the receptor. The biotin attached to proteins still remaining on the
cell surface was cleaved by washing cells twice at 4 °C with a
glutathione strip solution. The FLAG-tagged CTR receptor was
immunoprecipitated (IP), and biotinylated receptors were
detected using horseradish peroxidase-conjugated avidin D
(top). The membrane was then stripped and reprobed with
anti-FLAG antibody (bottom). Identical results were obtained
from three independent experiments. IB, immunoblot.
B, serum-starved HEK 293 cells transfected with the CTR-GFP
construct were incubated with a monoclonal anti-HA antibody at 4 °C
to label the extracellular HA-tag in the receptor construct. After
washing, cells were incubated with ( ) or without (
) 10 nM sCT. Cells were then quickly warmed to 37 °C allow
endocytosis of the receptor for different time periods. Measurement of
the HA-antibody bound to the cell surface was performed as described
under "Experimental Procedures." The data represent the means of
four independent experiments, and the error bars represent
the S.E. of the different experiments. *, p < 0.05. C, serum-starved HEK 293 cells transfected with the CTR-GFP
construct were incubated with anti-HA antibody to internalize
antibody-decorated CTR as described under "Experimental
Procedures." After the endocytosis of the antibody-labeled CTR, the
cell were cooled to 4 °C, the antibody bound to the CTR at the cell
surface was removed by an acid strip, and the cells were then warmed to
37 °C for different time periods to allow recycling of the CTR
incubated with (
) or without (
) 10 nM sCT and then
processed for FACS analysis as described under "Experimental
Procedures." The fluorescence intensity at each time point was
normalized to the fluorescence prior to the acid strip. The data
represent the means of five independent experiments, and the
error bars represent the S.E. of the different experiments.
*, p < 0.05.
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Fig. 2.
The constitutively endocytosed calcitonin
receptor accumulates intracellularly and recycles back to the cell
surface. A, HEK 293 cells transfected with the
GFP-tagged CTR construct were placed in a heated stage in a confocal
microscope to maintain the temperature at 37 °C. The accumulation of
CTR-containing vesicles was photobleached as described under
"Experimental Procedures." The fluorescence intensity of the
bleached area, the surface membrane of the bleached cell distant from
the bleached area, and, as a control, the fluorescence intensity of the
membrane of a neighboring unbleached cell were measured every 6 s
for 8 min after the bleaching. This procedure was repeated four times.
The micrographs show the same cells at the beginning
(left panel) and end (right panel) of the
experiment. B, the time course of the change in fluorescence
intensity in the different regions. The results are typical of five
independent experiments.
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Fig. 3.
Filamin interacts with the C-terminal tail of
the CTR. A, schematic diagram of the fragments of
filamin encoded by the cDNA initially cloned by the yeast
two-hybrid screening and truncations of this fragment used to map the
filamin binding site for the C-terminal tail of the CTR.
ABD, actin binding domain; H1 and H2,
hinge regions 1 and 2. The numbers within boxes represent
the number of the immunoglobulin-like repeats of human filamin (19),
the numbers at the beginning and end
of the lines refer to aa positions in the human full-length
filamin sequence (33). Both CTR and filamin constructs were
co-transformed in yeast strain L40 and assayed for growth on medium
lacking histidine and for -galactosidase activity. ++ indicates a
strong interaction based on the assay parameters. + represents also
growing on selective medium and activation in the
-galactosidase
assay but less activity compared with ++. + represents a very weak
activation of the
-galactosidase activity.
indicates failure of
growth and the absence of a
-galactosidase activity. B,
schematic diagram of the fragments of the CTR that interacted with
filamin 2129-2642 in the two-hybrid screening. The numbers
refer to residues in the rabbit CTR (34). C, HEK 293 cells
were transfected with the FLAG-tagged CTR construct or with empty
vector. Cells were stimulated with 10 nM sCT for the
indicated time periods and lysed, and the CTR was immunoprecipitated
(IP). The immune complexes were immunoblotted
(IB) with the anti-filamin antibody (upper
panel). The membrane was stripped and re-blotted with the M2
anti-FLAG antibody (middle panel). Total cell lysates
(TCL) were immunoblotted with the anti-filamin antibody
(lower panel). Identical results were obtained from four
independent experiments. D, HEK 293 cells were
co-transfected with a C-terminally Myc-tagged CTR construct in
combination with a vector encoding the immunoglobulin-like repeats
20-23 of filamin with a xpress© tag (lane 3), a vector
encoding the 7th transmembrane domain and the C-terminal tail of the
CTR with a FLAG tag (lane 4), or a vector encoding the
C-terminal tail of the CTR with a FLAG tag (lane 5). The
cells were lysed, and the CTR was immunoprecipitated with the anti-Myc
antibody. The immune complexes were immunoblotted with the anti-filamin
antibody (upper panel), and the blot was stripped and
re-blotted for Myc to check the immunoprecipitation (second
panel). The lysates used for the immunoprecipitations were blotted
for filamin (third panel) and with the anti-xpress©
antibody (lane 3) and M2 anti-FLAG antibody (fourth panel).
Identical results were obtained from three independent
experiments.
2-adrenergic receptor, in M2 and A7 cells and examined its degradation by pulse-chase analysis. In contrast to the
results obtained with the CTR, we found essentially no difference in
the degradation of the
2-adrenergic receptor in the two
cell lines (Fig. 4D, lower panel).
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Fig. 4.
Expression and degradation of the calcitonin
receptor is dependent on the interaction with filamin.
A, M2 and A7 cells were transfected with the CTR-FLAG
construct, cells were lysed, and proteins were separated by SDS-PAGE
and transferred to nitrocellulose. The membrane was immunoblotted
(IB) with the anti-FLAG antibody (upper panel)
and then stripped and reblotted for actin (lower panel).
Identical results were obtained from three independent experiments.
B, A7 (left panel) and M2 (right
panel) cells were seeded on glass coverslips and transfected with
the GFP-tagged CTR construct. 36-48 h later, cells were analyzed by
confocal microscopy. The bars represent 10 µm.
C, for quantification of the CTR cell surface expression, M2
cells and A7 cells were transfected with the GFP-tagged CTR construct,
and the surface expression was measured by FACS using the antibody
against the extracellular HA tag at the N-terminal of the CTR construct
as described under "Experimental Procedures." The mean fluorescence
of PE, reflecting the amount of the HA antibody binding, was used as a
measure of the receptor cell surface expression. The mean values of
four independent samples in M2 and A7 cells are shown *,
p < 0.05. D, A7 ( ) and M2 (
) cells
were labeled with [35S]methionine. The cells were lysed,
and the CTR was immunoprecipitated and subjected to SDS-PAGE. The dried
gel was autoradiographed, and the labeled CTR was quantified by
densitometry (upper panel). The slopes of the linear
regressions in M2 and A7 cells were significantly different
(p < 0.05). The degradation of the
2-adrenergic receptor was analyzed in a similar manner
(lower panel). The data represent the means of three
independent experiments, and the error bars represent the
S.E. of the different experiments. E, pulse-chase analysis
was performed as described with proteolysis inhibitors (40 µM MG132, 150 µM chloroquine, 10 mM NH4Cl) present during a 3-h chase period. In
addition, the filamin 20-23 fragment, which inhibits the binding of
the CTR to filamin, was co-expressed, and its effect on CTR degradation
were examined in the same way. The data represent the means of five
independent experiments, and the error bars represent the
S.E. of the different experiments. All data in the different
experiments were normalized to the amount of degraded CTR in the
untreated control in the same cell line. In untreated A7 cells, 32 ± 9% was degraded compared with 55 ± 12% in untreated M2
cells. *, p < 0.05.
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Fig. 5.
Tonic internalization of the calcitonin
receptor is unchanged in the presence or absence of filamin, but
recycling in the absence of filamin is altered. A,
internalization of GFP-tagged CTR in unstimulated A7 ( ) or M2 (
)
cells was measured by FACS as described under "Experimental
Procedures." The data represent the means of five independent
experiments, and the error bars represent the S.E. of the
different experiments. B, A7 and M2 cells were incubated for
30 min at 37 °C in the presence of the anti-HA antibody to allow
endocytosis of the CTR with the bound antibody. After cooling to
4 °C, surface-bound antibody was removed with an acid wash as
described under "Experimental Procedures." Cells were then warmed
to 37 °C for different time periods to allow recycling of the
internalized CTR to the cell surface. PE staining and FACS analysis was
done as described under "Experimental Procedures." The fluorescence
intensity at each time point was normalized to the starting
fluorescence prior to the acid wash. The data represent the means of
five independent experiments, and the error bars represent
the S.E. of the different experiments. *, p < 0.05.
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Fig. 6.
Recycling of the calcitonin receptor to the
cell surface is markedly reduced in the absence of filamin. A7
cells (upper panel) or M2 cells (lower panel)
were transfected with the GFP-tagged CTR construct. The FLIP experiment
was done as described under "Experimental Procedures." The
panels show the time course of the fluorescence intensity in
the regions of the surface membrane and the intracellular bleached
area. The fluorescence intensities are normalized to the fluorescence
intensity at the beginning of the experiment. The figure shows the
result of a typical experiment of five separate experiments. Identical
results were obtained from five independent experiments.
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Fig. 7.
CT inhibits CTR degradation and filamin
proteolysis. A, to test the effect of CT on CTR
degradation, A7 and M2 cells transfected with the CTR-FLAG were
pulse-labeled with [35S]methionine as described under
"Experimental Procedures," and the amount of labeled CTR was
determined 90 min after the pulse labeling as described for Fig. 4. The
data represent the means of three independent experiments, and the
error bars represent the S.E. of the different experiments.
A Student's t test was used to compare the undegraded
fraction in the treated cells with control cells. *, p < 0.05. B, HEK 293 cells transfected with the CTR-FLAG
construct were treated with 10 nM sCT for 5 and 45 min.
Cell lysates were prepared for immunoblotting (IB) as
described under "Experimental Procedures." The membrane was
immunoblotted with the anti-filamin antibody (lower panel).
The 190-kDa filamin fragment was quantified by gel densitometry. The
numbers below the panel represent the percentage
of the 190-kDa filamin fragment compared with the untreated control.
Identical results were obtained from three independent experiments. The
mean values for the densitometry for all three experiments were 59%
after 5 min and 83% after 45 min.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor and showed that
binding of this protein, known to be involved in membrane fusion
processes, is required for recycling of the
2-adrenergic
receptor. Whistler et al. (25) recently described a protein
(GPCR-associated sorting protein, GASP) that targets several GPCR,
including the
2-adrenergic receptor and
-opiod
receptor, to lysosomal degradation. The same group (26) has identified
another protein, EBP-50, that links the
2-adrenergic receptor to F-actin. This linkage, which is required for recycling of
the
2-adrenergic receptor to the cell surface, may be
regulated by ligand-induced phosphorylation of the
2-adrenergic receptor.
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Fig. 8.
Model for the role of filamin in regulating
the cellular trafficking of the calcitonin receptor. The CTR
undergoes constitutive endocytosis and is targeted to recycling
endosomes. The association of the C-terminal tail of the receptor to
filamin is necessary for efficient recycling of the CTR to the cell
surface.
The dopamine D2 and D3 receptors (29, 30) and the calcium sensing receptor (31, 32) have also been reported to interact with filamin. Interestingly, the functional consequences of the association seem to be different for the individual GPCRs. For the Gi-coupled D2 receptor, there was less inhibition of adenylyl cyclase reported in the absence of filamin (29), possibly because the receptor was inefficiently expressed on the cell surface of filamin-free cells (30). In the case of the calcium sensing receptor, the absence of filamin or the disruption of the interaction with the receptor abolishes ERK phosphorylation. We failed to find a difference in the CTR-induced ERK phosphorylation in M2 and A7 cells. Differences in the mitogen-activated protein kinase activation pathways for the two receptors might underlie these different responses.
In conclusion, the CTR is tonically endocytosed and recycled to the
cell surface via a mechanism that depends in part on an interaction
between the CTR and filamin. Because receptor trafficking is shown to
be an important regulator of GPCR function (23), it is likely that the
mechanism shown in this study, with regulation of CTR degradation and
recycling via the association with intact filamin, contributes
significantly to the specific biological response induced by the CTR
and possibly also other GPCRs.
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. John Hartwig for
providing the A7 and M2 cell line. We are also indebted to Dr. Brian
Kobilka for providing the HA-tagged 2-adrenergic
receptor construct.
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FOOTNOTES |
---|
* This work was supported in part by Grant SE 999/1-1 from the Deutsche Forschungsgemeinschaft (to T. S.) and Grant DE-0724 from the National Institutes of Health (to R. B.).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. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed:
Berufsgenossenschaftliche Klinik Bergmannsheil, Burkle-de-la-Camp-Platz
1, 44789 Bochum, Germany. Tel.: 0049-234-3020; E-mail:
thomas_seck@hotmail.com.
Published, JBC Papers in Press, January 16, 2003, DOI 10.1074/jbc.M209655200
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ABBREVIATIONS |
---|
The abbreviations used are: CTR, calcitonin receptor; aa, amino acid; CT, calcitonin; sCT, salmon CT; ERK, extracellular signal-regulated kinase; FLIP, fluorescence loss in photobleaching; GFP, green fluorescent protein; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; mRIPA, modified radioimmune precipitation assay; PE, phycoerythrin; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter.
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