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Am J Physiol Cell Physiol 295: C1230 –C1237, 2008.
First published September 11, 2008; doi:10.1152/ajpcell.00240.2008.
Glucagon receptor recycling: role of carboxyl terminus, ␤-arrestins,
and cytoskeleton
Lada Krilov,1 Amy Nguyen,1 Teruo Miyazaki,1 Cecilia G. Unson,2 and Bernard Bouscarel1,3
1
Gastroenterology Research Laboratory, Digestive Diseases Center, Department of Biochemistry and Molecular Biology,
The George Washington University, Washington, District of Columbia; 2Laboratory of Molecular Biology and Biochemistry,
The Rockefeller University, New York, New York; and 3Department of Medicine, The George Washington University,
Washington, District of Columbia
Submitted 2 May 2008; accepted in final form 20 August 2008
of resensitization. Rab4 mediates rapid recycling of the transferrin and ␤2-adrenergic receptors (20, 21), whereas Rab11
mediates slower recycling through perinuclear recycling endosomes (21). ␤-Arrestins modulate postendocytic sorting of
some GPCRs, in particular recycling (16, 18, 29). The receptor’s carboxyl (COOH) terminus interacts with the cytoskeleton, which plays an important role in trafficking, signaling, and
allosteric regulation of many GPCRs (3). In particular, actin
filaments play a role in receptor recycling (6, 9, 30) and its
targeting to lysosomes (9, 13, 28) for degradation.
Previously, we and others have shown that glucagon
triggers desensitization and internalization of the GR (2, 5,
14). The current study was undertaken to elucidate the
pathways of postendocytic sorting of the GR, specifically,
receptor recycling and degradation. Using isolated Golden
Syrian hamster hepatocytes and/or GR stably transfected
human embryonic kidney (HEK)-293 cells, we have investigated 1) the timeline and routes of GR recycling; 2) the
roles of the cytoskeleton, ␤-arrestins, and the GR’s COOH
terminus in recycling; and 3) the subcellular site of GR
degradation.
MATERIALS AND METHODS
Materials
the basal level of glucagon is elevated and
the insulin-to-glucagon relationship is altered (19, 22, 25),
leading to hyperglycemia. Recently, there has been an increased interest in the glucagon receptor (GR) as a target in
diabetes management. Understanding the mechanisms of GR
desensitization and downregulation may facilitate identifying
novel therapeutic targets in diabetes management.
The GR is a prototypical Family B G protein-coupled
receptor (GPCR). After exposure to agonists, GPCRs are
desensitized and sequestered in the cytosol. Internalized receptors are either recycled to the plasma membrane or targeted for
degradation. Recycling of receptors is an important mechanism
Cell culture media, penicillin/streptomycin, L-glutamine, and
amino acids were from Cellgro (Kansas City, MO). Geneticin/G418,
cytochalasin D, cyclohexamide, tetracycline, sodium butyrate,
DABCO, and di(N-succinimidyl) 3,3⬘-dithiodipropionate were from
Sigma-Aldrich (St. Louis, MO). Glucagon was purchased from
Bachem (King of Prussia, PA). 125I-labeled glucagon was purchased
from Linco (St. Charles, MO). Phorbol myristate acetate, Mowiol,
MG-132, nocodazole, and poly-L-lysine were purchased from Calbiochem (San Diego, CA). Lipofectamine 2000 and OptiMEM were from
Invitrogen (Rockville, MD).
Plasmids. ␤-Arrestin1 DN (V53D) was kindly provided by Dr. Marc
Caron (Duke University, Durham, NC). ␤-Arrestin2 DN (arr2 319-418)
was a generous gift from Dr. Jeffrey L. Benovic (Thomas Jefferson
University, Philadelphia, PA). To obtain a fusion protein with a green
fluorescent protein (GFP) tag at the COOH terminus, the rat GR gene was
inserted into a TOPO-GFP plasmid (Stratagene, La Jolla, CA) (Krilov L,
Nguyen A, David M, Miyazaki T, Meng J, Unson CG, and Bouscarel B,
unpublished observations).
Antibodies and dyes. The mouse anti-␤-actin antibody, mouse
anti-␤-tubulin antibody, and anti-mouse horseradish peroxidase
(HRP)-conjugated secondary antibody were from Sigma-Aldrich.
Address for reprint requests and other correspondence: B. Bouscarel, Dept.
of Biochemistry and Molecular Biology, George Washington Univ., 2300 Eye
St. NW, Washington, DC 20037 (e-mail: [email protected]).
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.
actin; G protein-coupled receptor; lysosome; Rab4; Rab11
IN TYPE 2 DIABETES,
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Krilov L, Nguyen A, Miyazaki T, Unson CG, Bouscarel B. Glucagon
receptor recycling: role of carboxyl terminus, ␤-arrestins, and cytoskeleton.
Am J Physiol Cell Physiol 295: C1230–C1237, 2008. First published September 11, 2008; doi:10.1152/ajpcell.00240.2008.—Glucagon receptor
(GR) activity and expression are altered in several diseases, including
Type 2 diabetes. Previously, we investigated the mechanism of GR
desensitization and internalization. The present study focused on the
fate of internalized GR. Using both hamster hepatocytes and human
embryonic kidney (HEK)-293 cells, we showed that internalized GR
recycled to the plasma membrane within 30 – 60 min following stimulation of the cells with 100 nM glucagon. In HEK-293 cells and
during recycling, GR colocalized with Rab4, Rab11, ␤-arrestin1,
␤-arrestin2, and actin filaments, in the cytosolic and/or perinuclear
domains. Glucagon treatment triggered redistribution of actin filaments from the plasma membrane to the cytosol. GR coimmunoprecipitated with ␤-actin in both hepatocytes and HEK-293 cells. Downregulation of ␤-arrestin1 and ␤-arrestin2 or disruption of the cytoskeleton inhibited recycling, but not internalization of GR. Deletion of the
GR carboxyl-terminal 70 amino acids abolished internalization of GR
in response to glucagon while deletion of the last 40 amino acids only
did not affect GR internalization and recycling. After exposure of the
cells to either high concentrations or prolonged duration of glucagon,
GR colocalized with lysosomes. GR degradation was inhibited by
lysosomal, but not proteosomal, inhibitors. In conclusion, GR recycles
through Rab4- and Rab11- positive vesicles. The actin cytoskeleton,
␤-arrestin1, ␤-arrestin2, and the receptor’s carboxyl terminus are
involved in recycling. Prolonged stimulation with glucagon targets
GR for degradation in lysosomes. Therefore, the present study provides a better understanding of the GR recycling mechanism, which
could become useful in the treatment of certain diseases, including
diabetes.
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The mouse anti-FLAG M2 antibody was purchased from Stratagene.
The rabbit anti-Rab4 was from Upstate (Lake Placid, New York), and
the rabbit anti-Rab11 was from Zymed (South San Francisco, CA).
The fluorescently labeled secondary antibodies, goat anti-rabbit Alexa
Fluor 568, and goat anti-mouse Alexa Fluor 488, as well as Lysotracker Red and Texas Red-phalloidin dyes, were from Molecular
Probes (Carlsbad, CA). The HRP-conjugated anti-rabbit antibody was
from Amersham Biosciences (Piscataway, NJ).
and 20 mM HEPES, pH 7.4). The unbound glucagon was washed out
with binding buffer, and the cells were lysed in 0.8 M NaOH.
Measurements were conducted in quadruplicate. In both methods,
high concentrations of unlabeled glucagon (⬎800 nM) were used to
estimate nonspecific binding. All values were corrected for nonspecific binding. Where indicated, the calculated values for internalization were normalized to the ones obtained for control samples (nontreated) and are expressed as percentage different from control, set
at 100%.
Cell Culture and Transfection
Fluorescence Microscopy
Cells grown on poly-L-lysine-coated coverslips were fixed in 3.7%
paraformaldehyde for 10 min and permeabilized by a 15-min incubation with Triton X-100 at 37°C. The cells were then incubated in
blocking buffer (8% BSA-PBS) for 1 h. Primary and secondary
antibodies were diluted in 2% BSA-PBS buffer and incubated with the
cells for 1 h each, respectively. The coverslips were mounted onto
glass slides using Mowiol with DABCO. Images were taken with an
Olympus IX81 fluorescence microscope. For each frame, four to eight
planes were acquired and the projection images were deconvolved
using Slidebook 4.1 software (Intelligent Imaging Innovations,
Denver, CO).
Recycling Assay
Hepatocytes, HEK-GR, or HEK-Flag-GR cells were serum starved
for 1 h and then incubated with 100 nM glucagon for 30 min at 37°C.
Glucagon was removed by incubation with cold glycine buffer (pH 3),
followed by several washes in binding buffer. To allow for recycling
of receptors, the cells were returned to 37°C for the indicated period
of time.
Radioligand Binding Assay
Method A. Hepatocytes in suspension were incubated with 125Ilabeled glucagon (⬃10 nM) for 2 h at 4°C in binding buffer (DMEM
containing 20 mM HEPES, pH 7.4, supplemented with 1 mM PMSF
and 2% BSA). Cells were transferred to glass microfiber (GF/B) filters
(Whatman) presoaked in 4% BSA and were washed five times with
Tris-buffered saline (pH 7.4) to remove unbound 125I-labeled glucagon. Measurements were conducted in triplicate.
Method B. Internalization and recycling conditions for the HEK293 cells were similar to those used for the hepatocytes. For GR
binding determination, the cells were labeled with 125I-labeled glucagon for 2 h at 4°C in binding buffer (DMEM containing 0.1% BSA
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Role of Cytoskeleton in GR Trafficking
To assess the importance of cytoskeleton in GR internalization,
HEK-GR cells were serum starved for 30 min at 37°C, then incubated
for another 30 min at 37°C in the presence of either 10 ␮M nocodazole or 10 ␮M cytochalasin D to disrupt microtubules or actin
filaments, respectively, followed by 30-min treatment with 100 nM
glucagon at 37°C. The cells were then washed to remove unbound
glucagon, and the number of surface receptors was assessed by
binding as described above (method B). To explore the effect of
cytoskeletal disruption on GR recycling, HEK-GR cells were serum
starved for 60 min at 37°C, then incubated with 100 nM glucagon at
37°C for 30 min. The cells were then washed extensively, nocodazole
(10 ␮M) or cytochalasin D (10 ␮M) was added on ice, and the cells
were then returned to 37°C for 60 min to allow for recycling. Surface
GR was measured by binding (method B).
Immunoprecipitation and Western Blot Analysis
HEK-GR cells or hepatocytes were incubated with the cross-linker
1.25 mM di(N-succinimidyl) 3,3⬘-dithiodipropionate for 30 min at
room temperature, centrifuged, and the pellets resuspended in lysis
buffer containing 100 mM Tris 䡠 HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, and protease inhibitor cocktail
(Roche, Palo Alto, CA). For hepatocytes, membrane fractions were
separated from cytosolic fractions with ultracentrifugation at 100,000
g for 50 min at 4°C and were then resuspended in membrane isolation
buffer (50 mM Tris 䡠 HCl, pH 7.4, 2.5 mM MgCl2, 1 mM EDTA, 1
mM DTT, 5 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail).
Protein concentration in the lysates was determined by the BCA assay
(Pierce, Rockford, IL). Lysates containing 400 ␮g protein were
precleared with 50 ␮l anti-mouse agarose beads (E-Biosciences, San
Diego, CA) for 30 min, centrifuged, and the supernatants incubated
with the anti-FLAG (5 ␮g) or anti-GR (ST-18) antibody overnight at
4°C. The precipitates were incubated with 50 ␮l agarose beads for
1.5 h. The beads were washed and the pellets were resuspended in 20
␮l lysis buffer containing glycoprotein denaturation buffer (New
England Biolabs, Ipswich, MA) and were incubated for 30 min at
37°C to inactivate the cross-linking reagent. The proteins were separated on an 8 –16% Tris-glycine gradient gel (Invitrogen) and transferred to nitrocellulose Hybond membranes (Amersham Biosciences).
The membranes were blocked in 5% milk dissolved in wash buffer
containing 25 mM Tris, 150 mM NaCl, and 0.1% Tween 20 for 1 h.
The primary and HRP-conjugated secondary antibodies were incubated with the membrane for 1 h, and the bands visualized using
Western Lightning ECL detection reagent (PerkinElmer, Boston,
MA). The blots were scanned, and densitometric analysis was conducted using ImageQuant software (CGRB Core Laboratories, Amersham Biosciences).
GR Degradation
HEK-GR cells were serum starved for 1 h and were incubated with
25 ␮M cycloheximide and 100 nM glucagon in the presence or
absence of lysosomal inhibitor chloroquine (200 ␮M, Sigma-Aldrich)
and proteosomal inhibitor MG-132 (20 ␮M) for 2– 8 h. At the end of
the incubation period, total cell lysates were prepared and immunoblotting was conducted as described above. For live-cell microscopy,
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Hepatocytes were isolated from male Golden Syrian hamsters
(100 –130 g body wt) using the previously described collagenase
perfusion technique (4). All procedures involving animals, including
liver cell isolation, have been approved by the Institutional Animal
Care and Use Committee at George Washington University. The cells
were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal calf serum, 200 ␮M L-glutamine, MEM
nonessential and essential amino acids, penicillin (5,000 IU/ml) and
streptomycin (5,000 ␮g/ml). HEK-293 cells stably expressing the rat
GR [HEK-GR (12)] were maintained in DMEM supplemented with
10% serum, 200 ␮M L-glutamine, MEM nonessential and essential
amino acids, penicillin (5,000 IU/ml), and streptomycin (5,000 ␮g/
ml). HEK-293 cells stably expressing a tetracycline-inducible FLAGtagged GR [HEK-Flag-GR (26)] were maintained in DMEM/F-12
(50:50) supplemented with 200 ␮g/ml geneticin/G418, 10% fetal
calf serum, and penicillin-streptomycin. The GR expression was
induced in DMEM/F-12 supplemented with 5 mM sodium butyrate, 2 ␮g/ml tetracycline, 10% serum, penicillin (5,000 IU/ml),
and streptomycin (5,000 ␮g/ml) for 24 h. The cells were transfected with 4 – 8 ␮g plasmid DNA using Lipofectamine 2000,
following the manufacturer’s instructions.
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HEK-293 cells transfected with GR-GFP were seeded onto glassbottom dishes (MatTek, Ashland, MA). The cells were incubated with
50 nM Lysotracker Red in serum-free medium for 30 min to label
lysosomes, were washed, and were treated with 100 or 1,000 nM
glucagon for 1– 4 h. Images were taken at 30-min intervals for up
to 4 h.
Statistical Analysis
Except as otherwise indicated, the results are expressed as means ⫾
SE. The statistical significance was determined by ANOVA and
Bonferroni posttest using GraphPad Prism 4 software (GraphPad
Software, San Diego, CA). P ⬍ 0.05 was considered significant.
RESULTS
Radioligand binding assays were performed to investigate the
fate of the internalized GR. In both hamster hepatocytes (Fig. 1A)
and GR stably transfected HEK-293 (HEK-GR) cells (Fig. 1B),
30 – 40% of receptors internalized after 30-min treatment with 100
nM glucagon. In transfected HEK-293 cells, ⬃70% of internal-
Fig. 1. Glucagon receptor (GR) recycles to the plasma membrane in a ␤-arrestin-dependent manner: A: hamster hepatocytes were incubated without (control,
ctrl) and with 100 nM glucagon (gluc) for 30 min (30⬘) at 37°C, washed, and then incubated or not at 37° for 120 min recycling (rec). GR expression on the
cell surface was determined by radioligand binding (method A). The results shown are means ⫾ SE of 4 independent experiments. *Significantly different: P ⬍
0.05. B: human embryonic kidney (HEK)-293 cells transfected with GR (HEK-GR) were incubated without (ctrl) and with 100 nM glucagon for 30 min at 37°C.
For recycling, the cells were washed and then returned to 37°C for 30 min. GR expression on the cell surface was determined by radioligand binding (method
B). The results shown are means ⫾ SE of 4 independent experiments. ***Significantly different: P ⬍ 0.001. C: HEK-GR cells were transfected with ␤-arrestin1
(␤-arr1) and ␤-arrestin2 (␤-arr2) dominant-negative (DN) mutants or with an empty vector (EV). Recycling was assessed as described in B. Data shown represent
means ⫾ SE of 3 independent experiments. **Significantly different from control: P ⬍ 0.01; ***significantly different from control: P ⬍ 0.001; ##significantly
different: P ⬍ 0.01. D: HEK-GR cells were transiently cotransfected with both ␤-arrestin1 and ␤-arrestin2 DN mutants or empty vector. After 30-min treatment
with 100 nM glucagon at 37°C, the cells were washed and incubated up to 120 min at 37°C. The cell surface GR expression level was determined by radioligand
binding (method B). Data represent means ⫾ SE of 4 independent experiments. **Significantly different from control: P ⬍ 0.01; ***significantly different from
control: P ⬍ 0.001; #significantly different from 30-min glucagon; ##significantly different: P ⬍ 0.05. The data were analyzed by one-way ANOVA and
Bonferroni posttest using Prism software (GraphPad Software, San Diego, CA).
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GR Recycles to the Plasma Membrane
in a ␤-Arrestin-Dependent Manner
ized receptors were recovered on the cell surface 30 min after the
removal of glucagon (Fig. 1B) and nearly 100% after 60 min (data
not shown), while 120 min were necessary to observe full recovery of GRs on the cell surface of hepatocytes (Fig. 1A). Previously, we have shown that treatment of HEK-293-GR with
glucagon induced translocation of ␤-arrestins to the perinuclear
region in living cells and that GRs colocalized with both ␤-arrestin1 and ␤-arrestin2. Overexpression or downregulation of either
␤-arrestin1 or ␤-arrestin2 did not significantly affect GR internalization (Krilov L, Nguyen A, David M, Miyazaki T, Meng J,
Unson CG, and Bouscarel B, unpublished observations). To
investigate the role of ␤-arrestins in recycling, HEK-GR cells
were transfected with ␤-arrestin1 and ␤-arrestin2 dominant-negative (DN) mutants. Downregulation of at least ␤-arrestin1 significantly reduced GR recycling, and a trend is observed with
␤-arrestin2 DN (Fig. 1C). To verify whether this was a complete
block or merely a delay of recycling, a time-course experiment
was conducted in HEK-GR cells cotransfected with DN ␤-arrestin1 and DN ␤-arrestin2 mutants. While in control cells (HEK-GR
transfected with an empty vector), the GR continued to recycle to
the plasma membrane over time, reaching a plateau at 60 min, in
the presence of DN ␤-arrestins, GR recycling was inhibited even
after 120 min (Fig. 1D).
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Fig. 2. Role of the COOH terminus in GR
internalization and recycling: HEK-293T cells
transfected with T7a (A) or T7b (B) containing
plasmids were incubated in the absence (control)
and presence of 100 nM glucagon for 30 min at
37°C. For recycling, the cells were washed and
placed at 37°C for an additional 30 min. Surface
GR was determined by radioligand binding assay (method B). The results shown are means ⫾
SE of 3– 4 independent experiments. The data
were analyzed by one-way ANOVA and Bonferroni posttest. *,**,***Significantly different
from respective control: P ⬍ 0.05, P ⬍ 0.01,
and P ⬍ 0.001, respectively. ###Significantly
different from 30-min glucagon: P ⬍ 0.001.
Cytoskeleton Is Involved in GR Trafficking
Human GR truncated at the COOH terminus has a significantly reduced internalization rate compared with its wild-type
counterpart (5). The role of the rat GR COOH terminus in
receptor internalization and trafficking has not been studied
thus far. HEK-293 cells were transfected with GR truncation
mutants T7a and T7b lacking the last 70 and 42 COOHterminal amino acids, respectively (27). Control cells were
transfected with wild-type GR. Treatment with glucagon for 30
min caused a small (20%) but significant decrease in the
number of T7a receptors at the cell surface (Fig. 2A), Under the
same conditions, ⬃40% of T7b receptors were internalized
(Fig. 2B). Furthermore, at 30 min after removal of glucagon,
only T7b receptors significantly recycled to the plasma membrane (Fig. 2B) and to the same extent as the wild-type
counterpart (Fig. 1B).
Recent studies suggest that actin acts as a spatial organizer
of endocytic hot spots and as a carrier of endocytic vesicles (8).
To investigate the possible interaction of GR with actin filaments, HEK-Flag-GR cells were stained with the F-actinspecific label phalloidin (Fig. 3A). In the absence of glucagon
(control), GR and F-actin were colocalized at the plasma
membrane (Fig. 3A, merged). As early as 5 min after glucagon
(100 nM) treatment, the distribution of F-actin at the plasma
membrane became discontinuous and punctuate. GR translocated from the plasma membrane to the cytosol and at 15 and
30 min after treatment, colocalized with F-actin (Fig. 3A,
merged). In HEK-Flag-GR total cell lysate, GR coprecipitated
with ␤-actin, a major constituent of actin filaments, in control
cells and in cells treated with 100 nM glucagon for 10 min. At
20 min of glucagon treatment, the association of ␤-actin and
GR was not detectable (Fig. 3B). Likewise, a time-dependent
Fig. 3. Actin cytoskeleton is involved in GR trafficking. A: HEK-Flag-GR cells were treated with 100 nM glucagon for the indicated period of time. GR was
labeled with anti-FLAG antibody (green), and F-actin was labeled with Texas Red-phalloidin (red). The images shown are representative of 3 independent
experiments. B and C: HEK-Flag-GR cells and hamster hepatocytes, respectively, were treated with 100 nM glucagon for the indicated time. Total Flag-GR (B)
and plasma membrane GR (PM; C) were immunoprecipitated (IP) using anti-FLAG antibody and anti-GR antibody (ST-18), respectively, followed by
immunoblot analysis of GR and ␤-actin as described in MATERIALS AND METHODS. The images shown are representative of 3 independent experiments.
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Carboxyl Terminus Is Required for GR Internalization
and Recycling
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association of GR and ␤-actin was noted in hepatocytes when
GR was immunoprecipitated from the plasma membrane (Fig.
3C). To further investigate the involvement of the cytoskeleton
in GR trafficking, actin filaments and microtubules were pharmacologically disrupted. As shown in Fig. 4A, 30-min treatment with 10 ␮M nocodazole and 10 ␮M cytochalasin D
disrupted microtubule organizing centers (arrows) and actin
filaments (arrows), respectively. While disruption of actin
filaments or microtubules did not affect GR internalization
(Fig. 4B), it significantly reduced GR recycling by up to 20%
(Fig. 4C).
GR Colocalizes With Rab4 and Rab11
To elucidate the recycling route used by the GR, HEKFlag-GR cells were immunostained with anti-Rab4 and antiRab11 antibodies (Fig. 5, A and B). In control cells, GR was
localized predominantly at the plasma membrane, whereas
Fig. 5. GR colocalizes with Rab4 (A) and
Rab11 (B). HEK-Flag-GR cells were treated
with 100 nM glucagon for 30 min, washed,
further incubated for up to 60 min at 37°C,
fixed, and permeabilized. GR was immunostained with anti-FLAG antibody (green), antiRab4 (red), or anti-Rab11 (red). Images shown
are representative of 3 independent experiments. Recyc, recycling.
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Fig. 4. Role of cytoskeleton in GR internalization and recycling. A: HEK-GR cells were treated
with either 10 ␮M nocodazole or 10 ␮M cytochalasin D for 30 min at 37°C. The cells were permeabilized and immunostained with either the
mouse anti-␤-tubulin antibody or the mouse anti␤-actin antibody. The arrows indicate starlike
structures of microtubules (top) and actin filaments (bottom). The images shown are representative of 3 independent experiments: B: HEK-GR
cells were treated with either 10 ␮M nocodazole
(Noc) or 10 ␮M cytochalasin D (Cyt D) for 30
min at 37°C, followed by 30-min incubation in the
absence (control) and presence of 100 nM glucagon at 37°C. Surface binding was determined by
radioligand binding assay (method B). The data
shown are means ⫾ SE of 3 independent experiments. C: after glucagon treatment, the cells were
washed and incubated without (control) and with
either 10 ␮M nocodazole or 10 ␮M cytochalasin
D and returned to 37°C for 30 min. Surface
binding was determined by radioligand binding
assay (method B). Data represent means ⫾ SE of
3 independent experiments. Data were analyzed
by one-way ANOVA and Bonferroni posttest.
*Significantly different from control: P ⬍ 0.05.
***Significantly different from control: P ⬍
0.001.
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Rab4 and Rab11 were distributed uniformly throughout the
cytosol (Fig. 5, A and B, control overlay). After 30 min of
treatment with 100 nM glucagon, GR accumulated inside the
cell and colocalized with Rab11 (arrows, Fig. 5B) but not with
Rab4 (Fig. 5A). At 10 min of recycling, GR colocalized with
both Rab4 and Rab11 in the cytosol. As recycling progressed,
Rab4 and Rab11 translocated toward the plasma membrane. At
30 and 60 min of recycling, colocalization of GR with Rab4
and Rab11 was predominantly observed in the perimembrane
region, at cell-cell junctions, and at the plasma membrane (Fig.
5, A and B).
GR Is Degraded in Lysosomes
Prolonged treatment of cells with specific agonist frequently
results in degradation of the respective receptors. In a preliminary binding study (data not shown), it was determined that in
HEK-GR cells, glucagon triggered GR degradation as early as
3 h after treatment, and the level of GR remained stable for up
to 8 h. Lysosomes were labeled with Lysotracker Red in live
HEK-293 cells transfected with GR-GFP (Fig. 6A). Upon
treatment with 1 ␮M glucagon for 2 h or with 0.1 ␮M glucagon
for 4 h, we observed colocalization of GR and lysosomes (Fig.
Fig. 7. Schematic representation of GR trafficking: Upon
binding glucagon (G), the GR is internalized. After short-term
stimulation with glucagon, the GR recycles to the plasma
membrane through Rab4- and Rab11-positive vesicles in a
␤-arrestin (AR)-dependent manner. Prolonged stimulation directs GR to degradation in lysosomes (LY) and possibly
proteosomes (PS). RV, recycling vesicle; RE, recycling endosome; Chlor, chloroquine; LE, late endosome.
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Fig. 6. GR is degraded in lysosomes. A: HEK-293 cells were transfected with GR-green fluorescent protein. Lysosomes were labeled with Lysotracker Red as
described in MATERIALS AND METHODS and treated with the indicated concentration of glucagon (glu) for up to 4 h. Images were taken every 30 min.
Representative images from 5–7 independent experiments are shown. B: HEK-GR cells were serum starved for 30 min and then treated with 25 ␮M
cyclohexamide, without and with either 20 ␮M MG-132 (MG) or 200 ␮M chloroquine (Chl) for 30 min at 37°C, followed by 100 nM glucagon for 5 h at 37°C.
Total lysates were prepared, and Western blot analysis was conducted as described in MATERIALS AND METHODS. The image shown is representative of 3
independent experiments. C: protein levels for the anti-GR and anti-␤-actin bands were determined by densitometric analysis using STORM phosphoimager and
ImageQuant software. The amount of GR protein was normalized to the amount of ␤-actin in each lane, and the obtained values are expressed as percentage
of control (nontreated cells). Data shown are means ⫾ SE of 3 independent experiments. The data were analyzed by one-way ANOVA and Bonferroni posttest.
*Significantly different from control: P ⬍ 0.05. **Significantly different from control: P ⬍ 0.01.
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6A, 2 h and 4 h). No colocalization was noted in the absence of
glucagon (control). Western blot analysis of the total cell lysate
(HEK-GR cells) was conducted to quantify the amount of GR
in cells. To prevent de novo protein synthesis, the cells were
pretreated with 25 ␮M cyclohexamide for 1 h before incubation of the cells with 100 nM glucagon for 5 h in serum-free
media (Fig. 6, B and C). Densitometric analysis indicated that
5-h glucagon treatment reduced the amount of GR by ⬃40%,
in the presence and absence of 20 ␮M proteosomal inhibitor
MG-132. In contrast, in the presence of 200 ␮M lysosomal
inhibitor chloroquine, the GR expression level was similar to
that of control (Fig. 6, B and C). These results indicate that
prolonged treatment with glucagon targets GRs to degradation
at least partially in lysosomes.
Results of the present study show that internalized GR
recycles to the plasma membrane in both hepatocytes and
transfected HEK-293 cells. We noted that expression of
␤-arrestin1 and ␤-arrestin2 DN mutants diminishes recycling
of the GR. This is in agreement with the study of Vines et al.
(29), who demonstrated that ␤-arrestins are required for recycling of the N-formyl peptide receptor. In contrast, other
studies have suggested that stable interactions with ␤-arrestins
abrogate receptor GPCR recycling (17).
The COOH terminus plays a critical role in desensitization
of many GPCRs and mediates postendocytic sorting (24). The
expression and function of the rat GR truncation mutants T7a
and T7b are similar to their wild-type counterpart in terms of
affinity for glucagon and ability to stimulate cAMP production
(27). Here we show that the T7a mutant, which lacks the entire
COOH terminus (last 70 amino acids), leads to a significant
although reduced GR internalization but prevents GR recycling. Under the same conditions, the GR T7b mutant, which
lacks approximately half of the COOH terminus (last 40 amino
acids), internalized and recycled to the same level as the
wild-type counterpart. It is of interest to mention that the
human GR lacking the last 56 or 62 amino acids of the COOH
terminus had reduced receptor internalization compared with
its wild-type counterpart, while deletion of only the last 24
amino acids of the COOH terminus did not have a great impact
on receptor internalization (5). Mutation of seven serine residues to alanine in the COOH terminus of the human GR
significantly reduced GR internalization (5), suggesting that
phosphorylation plays a critical role in ligand-induced GR
internalization. On the basis of the amino acid sequence of the
rat GR carboxyl terminus (27), results of the present study are
suggesting a maximum of three serines and one threonine to be
important in the rat GR internalization. Therefore, at least part
of the GR COOH terminus plays a critical role in receptor
internalization. Clearly, further studies are required to identify
the serine(s) and/or threonine(s) that are phosphorylated and
therefore responsible for rat GR internalization induced by
glucagon, as well as the functional domains of the COOH
terminus involved in recycling and interaction with the cytoskeleton. However, dephosphorylation of GR is not required
for recycling (data not shown), as was also observed with the
related parathyroid hormone receptor (7).
Actin filaments and microtubules are established regulators
of protein endocytosis and trafficking (1), and their importance
AJP-Cell Physiol • VOL
GRANTS
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant DK-56108.
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DISCUSSION
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