| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
MRC Reproductive Biology Unit (M.V., L.A., A.H., A.M.M., P.L.T.,
K.A.E.) Centre for Reproductive Biology Edinburgh, EH3 9EW,
United Kingdom
Molecular Pharmacology Group (A.D.G.,
G.M.) Division of Biochemistry and Molecular Biology Institute
of Biomedical Life Sciences University of Glasgow Glasgow G12
QQ, United Kingdom
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Experiments measuring endocytotic events at the level of the receptor have been performed for the ß2-adrenergic receptor (6, 7, 8), TRH (9), and muscarinic (10) and angiotensin II type 1A (AT1A) receptors (11). After agonist activation these receptors internalize into intracellular endosomes via clathrin-coated pits and subsequently recycle back to the plasma membrane as functional receptors. The binding of ß-arrestins to GPCRs has been shown to be a convergent step of GPCR signaling with ß-arrestins acting as adaptor-like proteins regulating the rate and specificity of receptor internalization (12, 13). GPCR phosphorylation appears to be a prerequisite for arrestin-receptor interaction (14), and it is proposed that ß-arrestin binding to ligand-activated GPCRs specifically directs the phosphorylated receptors into clathrin-coated pits due to its ability to interact with both the receptor and clathrin. Mutagenesis studies have revealed that the N-terminal half of nonvisual arrestins has the ability to recognize the agonist-activated GPCRs while the clathrin-binding domain is located in the COOH terminus of the molecule (15, 16). The role of ß-arrestin in promoting GPCRs internalization has been further confirmed using ß-arrestin dominant negative mutants to impair receptor internalization (17). Recently, the jellyfish (Aqueora victoria) green fluorescent protein/ß-arrestin fusion protein has been introduced as a sensitive tool for real time visualization of the receptor-mediated translocation of ß-arrestin in living cells (18). Such a process has been demonstrated for a number of ligand-activated GPCRs (19).
Sites located within the intracellular receptor loops, and in particular the intracellular C-terminal region, have been shown to be important in GPCR internalization, as receptor mutants with truncated C-terminal tails or lacking putative G protein-coupled receptor kinase phosphorylation sites displayed impaired internalization (13, 20, 21). We have demonstrated that the addition of a functional intracellular C-terminal tail to the GnRH receptor significantly increased internalization rates (22).
The aim of this study was to examine endocytotic and recycling events of the GnRH receptor. Previous studies on internalization events connected with the GnRH receptor have employed radiolabeled ligand binding (23, 24, 25, 26, 27, 28), which is based on the assumption that ligand and receptor sort similarly through the endocytotic pathway. To test this assumption, we employed the receptor tagging methodology to determine the GnRH receptor endocytotic trafficking cycle at the level of the receptor. The role of ß-arrestin in promoting internalization in the GnRH receptor and in a GPCR possessing a C-terminal tail, the TRH receptor, was also determined.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Visualization and Cellular Localization of GnRH Receptors
GnRH receptor distribution was examined in HEK 293 cells
stably expressing HA-tagged GnRH receptors using indirect
immunofluorescent staining. Fluorescently labeled HA-tagged GnRH
receptors were predominantly distributed around the cell circumference
in unstimulated HEK 293-B5 cells. Permeabilized, nontreated cells (Fig. 1a
) showed similar fluorescent
distribution consistent with localization of the receptor to the cell
surface. GnRH agonist treatment (1 or 2 h at 37 C) elicited a
redistribution of cellular immunostaining indicative of GnRH receptor
internalization (Fig. 1b). The redistributed cytoplasmic signal showed
a vesicular pattern, and in some cells positive staining was observed
in the perinuclear region. TRH, another hypothalamic releasing hormone,
had no effect on GnRH receptor distribution (Fig. 1c). The same was
also observed for the GnRH receptor antagonist
[Ac-3,4-dehydro-Pro1,
D-p-F-Phe2,
D-Trp3,6]-GnRH(GnRH-antag) (Fig. 1d),
and pretreatment of cells with GnRH-antag prevented agonist-induced
internalization (Fig. 1e). No specific staining was observed in
untransfected HEK 293 cells (Fig. 1f).
|
) showed a similar distribution
pattern with transferrin receptor (Fig. 2b), and colocalization is
shown on Fig. 2c.
|
shows a comparison between
GnRH-induced internalization as quantified by radioligand binding and
ELISA. The two methods gave comparable results regarding
receptor/ligand internalization kinetics at 37 C in the presence or
absence of hypertonic sucrose medium. The agonist-induced
internalization process was almost completely suppressed at 4 C, and no
internalization was observed for GnRH-antag as determined by
radioligand assays (data not shown). ELISA measurements also
demonstrated that the level of surface-expressed receptor after
GnRH-antag treatment was comparable with the levels observed in
untreated cells (Fig. 4).
|
|
). Coincubation of cells with the
protein synthesis inhibitor, cycloheximide (10 µg/ml), inhibited
neither agonist-induced internalization nor receptor recycling (Table 1), showing that during the course of the experiment no substantial
de novo synthesis of the receptor occurred.
|
Effect of ß-Arrestin on Internalization Kinetics of the GnRH and
TRH Receptors
To address the role of ß-arrestin on the internalization
kinetics of GnRH and TRH receptors we 1) performed internalization
experiments in HEK 293 and COS-7 cells that express different levels of
endogenous ß-arrestins, 2) studied the effect of both WT
ß-arrestin and ß-arrestin dominant negative mutant [ß-arrestin
(319–418)] coexpression on internalization of GnRH and TRH receptors
in HEK 293 and COS-7 cells, respectively, and 3) followed the
agonist-induced receptor-mediated redistribution of ß-arrestin/green
fluorescent protein (GFP) conjugate to the plasma membrane. Studies
performed using HEK 293 cells stably expressing either the GnRH or TRH
receptors showed a striking difference between internalization kinetics
of the two receptor types. A rapid decrease of surface-bound ligand was
observed for TRH receptor with an estimated half-time
(t1/2) of 2.2 min after TRH treatment. Internalization
reached a steady state within the first 5–10 min after agonist
exposure, and after this period the proportion of the internalized
receptors was about 70% (data not shown). In contrast, the
disappearance of GnRH receptors from the cell surface was delayed, with
an estimated t1/2 of 20 min. Equilibrium between surface
and intracellular receptors was reached only after 1 h of
continuous agonist exposure with the estimated intracellular pool of
receptor being around 30% (Fig. 3
). The effect of different endogenous
levels of ß-arrestin on receptor internalization rate was evaluated
by performing experiments in COS-7 cells, which express about 70% less
total ß-arrestins/mg protein than HEK 293 cells (30). Under
conditions of low endogenous ß-arrestin expression, TRH receptor
internalization was significantly slower in COS-7 (t1/2 of
5.7 min) than in HEK 293 cells. However, the rate was significantly
increased by coexpression of ß-arrestin in COS-7 cells
(t1/2 of 3.1 min) (Fig. 5).
In contrast, coexpression of GnRH receptor with ß-arrestin in COS-7
cells had no effect on internalization kinetics (Fig. 5). Coexpression
with ß-arrestin in HEK 293 cells did not promote internalization in
either receptor (data not shown). We next assessed the ability of a
dominant negative ß-arrestin mutant (ß-arrestin (319–418)), which
has been previously characterized to retain clathrin binding but lack
the receptor binding activity (17), to impair receptor internalization.
COS-7 cells were transfected either with GnRH or TRH receptor alone or
together with different ß-arrestin constructs and the intracellular
receptor pool estimated after agonist treatment (15 min at 37 C). The
coexpression of ß-arrestin (319–418) substantially reduced the
effect of ß-arrestin on TRH receptor internalization in COS-7 cells
(Fig. 6). The coexpression of
ß-arrestin (319–418) only slightly decreased the basal
agonist-induced internalization in COS-7 cells, whereas a significant
decrease was observed in HEK 293 cells stably expressing the TRH
receptor (Fig. 7). No significant effect
of ß-arrestin construct coexpression on GnRH receptor internalization
was observed either in COS-7 or in HEK 293 cells (Figs. 6 and 7). In
addition, the ability of coexpressed ß-arrestin/GFP conjugate in
COS-7 cells to enhance the internalization of TRH, but not the GnRH
receptor, was also confirmed (Fig. 6).
|
|
|
. Comparable rate
constants derived from data using either quantitative radioligand
binding or ELISA measurements were obtained for HA-tagged GnRH receptor
in HEK 293-B5 cells [ke = 0.013 ± 0.001;
kr = 0.032 ± 0.001 min-1; ks
= 0; kx= 0.010 ± 0.001 min-1
(radioligand binding); and ke = 0.013 ± 0.001;
kr = 0.028 ± 0.002; ks = 0;
kx = 0.018 ± 0.001 min-1 (ELISA
measurements)], confirming the use of radioligand-binding assays for
assessing receptor intracellular trafficking. Similar rate constants
(ke = 0.012 ± 0.001; kr = 0.033 ±
0.003; ks = 0; kx = 0.008 ± 0.001
min-1) were also obtained for WT GnRH receptor stably
expressed in HEK 293-A2 cells. The GnRH receptor internalization rates
were similar in both cell types (HEK 293 and COS-7) and also unaffected
by different ß-arrestin expression levels (Table 2A). The
ke for TRH receptor in COS-7 cells was nearly 4-fold lower
than the corresponding value in HEK 293 cells. However, ß-arrestin
coexpression in COS-7 cells resulted in an almost 2-fold increase in
ke for TRH receptor (Table 2B). The obtained kx
values were also substantially higher for TRH- than GnRH receptor in
both cell types (Table 2, a and b). In addition, internalization rates
obtained with 
T3 cells (ke = 0.014 ± 0.002;
kr = 0.032 ± 0.005) and GH3
(ke = 0.181 ± 0.025; kr = 0.078 ±
0.006) cells, which endogenously express GnRH and TRH receptors,
respectively, were comparable to those observed for the respective
receptor types stably expressed in HEK 293 cells. In all calculations,
the fitted value of ks was not significantly different from
zero, confirming the absence of synthesis of either receptor during the
course of the experiment (data not shown).
|
). Confocal microscopy showed cytosolic distribution of
ß-arrestin/GFP in untreated HEK 293 cells expressing either TRH (Fig. 8a) or GnRH receptor (Fig. 8c). After
agonist stimulation, a time-dependent TRH-mediated redistribution of
ß-arrestin/GFP from the cytosol to the cell membrane was observed
(Fig. 8b), while no increase in membrane-associated fluorescence
indicative of ß-arrestin/GFP translocation was observed in GnRH
receptor-expressing cells even after prolonged exposure to GnRH (Fig. 8d). Similar results were obtained in COS-7 cells transiently
expressing either TRH or GnRH receptor (not shown).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The agonist-induced internalization time course for GnRH receptors was
further quantified by two different methods; radioligand binding and
ELISA, of which the latter is not influenced by the state of receptor/G
protein coupling (32). Relatively low internalization rates were
confirmed using both methods. Radioligand binding studies in both
primary (23) and immortalized T3–1 gonadotrope cells (24), as well
as in GH3 lactotropes stably transfected with the GnRH
receptor (25), also demonstrate a surface GnRH receptor loss over a
similar time frame.
Endocytosis by a clathrin-mediated pathway is temperature dependent and can also be inhibited by hypertonic sucrose (33), which induces abnormal clathrin polymerization and subsequently reduces the number of clathrin-coated pits. Another well established marker for endocytosis via clathrin-coated pits is the transferrin receptor (34). We observed that GnRH-induced internalization was inhibited both at 4 C and in the presence of sucrose. The location of redistributed GnRH receptors also overlapped substantially with that of the transferrin receptors. Therefore, our results suggest that agonist-triggered endocytosis of the GnRH receptor probably occurs via a clathrin-dependent mechanism.
After internalization, receptors can either be sorted into endosomes for recycling back to the cell surface or, alternatively, may undergo degradation within lysozomes (31). After agonist removal, GnRH receptors reappeared at the cell surface, a process unaltered by cycloheximide, suggesting that GnRH receptor recycling from endosomes, rather than de novo receptor synthesis, was involved. However, if recycling had occurred only after agonist removal, we would have anticipated a linear increase in the intracellular pool of receptor. Our observations show that this is not the case, and the recycling data confirmed that in the continuous presence of agonist, a dynamic equilibrium between endocytosed and recycling receptors was reached. GPCR internalization is now thought to be associated primarily with receptor resensitization, which is achieved through endosomal sorting and recycling of functional receptor back to the plasma membrane (4, 35). Recycling of the GnRH receptor was inhibited by NH4Cl, indicating the involvement of acidified endosomal compartments in receptor trafficking, as has been previously shown for ß2-adrenergic (4) and angiotensin receptors (11).
The efficiency of GPCRs endocytosis is not only receptor- but also cell type-dependent and should reflect differences in endogenous levels of certain intracellular proteins. HEK 293 and COS-7 cells express high and low levels of ß-arrestins, respectively (30), and thus provide us with an opportunity for examining the effect of different endogenous ß-arrestin expression levels on GPCR internalization dynamics in a natural cellular environment. Therefore, we determined internalization kinetics for two functionally distinct members of the GPCR family; the GnRH receptor and also the TRH receptor, which possesses a functional carboxy-terminal tail. Additional reasons to choose TRH receptor were that its endocytosis displays characteristics of the clathrin-mediated pathway (9), and the importance of the carboxy-terminal tail in agonist-induced internalization was previously confirmed using this receptor (20).
The ke and kr values were similar for the WT and epitope-tagged GnRH receptor stably expressed in HEK 293 cells and for WT GnRH receptor transiently expressed in COS-7 cells, and this ke is also comparable with a reported value for WT GnRH receptor in COS-7 cells (28). Considering the reported differences in endogenous ß-arrestin levels between the two cell types, a higher GnRH internalization rate in HEK 293 than in COS-7 cells was expected, which was not the case. The rate constants for the GnRH receptor in both cell types were much lower than those obtained for TRH receptor and also those reported for some other GPCRs (8, 36); however, they are still within the range of internalization rates reported for clathrin-mediated endocytosis in nonneuronal cells (37). TRH receptor internalization rates were cell type dependent: lower in COS-7 than in HEK 293 cells; however, overexpression of ß-arrestin in COS-7 cells enhanced TRH receptor kinetics to levels approaching those in HEK 293 cells. The receptor degradation can increase in the presence of agonist (36); therefore, it is necessary to take degradation into account in any attempt to model intracellular trafficking. The rate of TRH receptor degradation (kx) was higher than kx for GnRH receptor in both cell types, whereas the synthesis rate constant (ks) was negligible for either receptor type. A loss of approximately 10% of surface GnRH receptor was observed during the recycling experiments in the absence or presence of the protein synthesis inhibitor cycloheximide. This result is in close agreement with the kx and ks values for GnRH receptor obtained from the four-compartment model, which predicts that 30–40% of receptor in the endosomal compartment is destined for degradation, and no new synthesis of receptor occurs during the course of the experiment.
The GnRH receptor internalization kinetics in the two cell types were similar, and no obvious role of ß-arrestin in this process was observed. This observation was further supported by results obtained with ß-arrestin dominant negative mutant [ß-arrestin (319–418)], which efficiently impaired agonist-induced internalization of the TRH but not of the GnRH receptor. The applicability of this construct for studying agonist-induced GPCRs internalization in different cell lines has been previously determined (17). In addition, we constructed the ß-arrestin/GFP fusion protein, which enabled us to assess translocation of ß-arrestin/GFP complex to the plasma membrane in response to GPCR activation. The construct was fully functional with respect to its ability to promote TRH receptor internalization, and the time- and agonist-dependent redistribution of ß-arrestin/GFP to the plasma membrane was easily observed by confocal microscopy. However, no evident redistribution of ß-arrestin/GFP was observed in cells expressing the GnRH receptor. The results obtained with ß-arrestin constructs could suggest either that GnRH receptor internalization is not ß-arrestin-dependent or that ß-arrestin, which preferentially binds phosphorylated receptor (14), has a low affinity for GnRH receptor due to the lack of a tail and therefore of potential phosphorylation sites. Truncation of the carboxy-terminal tail of the ß2-adrenergic receptor has been shown to reduce ligand-induced internalization by 50%, an effect that is reversed when the receptor is coexpressed with ß-arrestin, and coexpression of the WT receptor with inactive mutants of ß-arrestin reduced internalization by 70% (13). Similarly, the removal of the phosphorylation sites in the carboxyl tail of the ß2-adrenergic receptor reduces receptor internalization to levels comparable to those observed for the GnRH receptor (30). This observation indicates that GPCR kinase-mediated phosphorylation is not absolutely required as a signal initiating internalization, but only as a factor determining its rate.
The obvious question is can clathrin-dependent endocytosis be ß-arrestin independent, since ß-arrestins specifically target activated receptor into clathrin-coated vesicles? In HEK 293 cells expressing AT1A receptor, agonist-promoted sequestration of this receptor was shown to be ß-arrestin independent, although it can be recruited to this pathway by overexpression of ß-arrestin (38). Furthermore, it has been suggested that AT1A receptor internalization is clathrin-mediated, since colocalization with transferrin receptor in the same cell line was observed (11). Similarly, we showed that the GnRH receptor undergoes agonist-induced receptor internalization (albeit at a slower rate than other GPCRs) probably via a clathrin-dependent mechanism and provided evidence for the dynamic nature of this process. However, it seems likely that the GnRH receptor endocytotic pathway is ß-arrestin and cell type independent as overexpression of ß-arrestin in different cell types does not affect internalization kinetics.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cell Culture
Cell lines were routinely grown in complete DMEM containing 10%
(vol/vol) heat inactivated FCS, glutamine (0.3 mg/ml), penicillin (100
IU/ml), and streptomycin (100 µg/ml) and incubated at 37 C in a
humidified atmosphere of 5% (vol/vol) CO2 in air.
Derivation of Epitope-Tagged GnRH Receptor-Expressing Cell
Lines
A double-stranded oligonucleotide fragment corresponding to the
sequence of the HA tag (YPYDVPDYA) was synthesized and ligated in
frame, into the amino terminus of the rat GnRH receptor in the vector
pcDNA3. Linearized HA-tagged GnRH receptor plasmid DNA was then stably
transfected into human embryonal 293 cells (HEK 293) cells using
Transfectam. Receptor-containing clones were then selected after the
treatment of cell cultures with geneticin (1 mg/ml), expanded, and
maintained in complete DMEM containing geneticin (500 µg/ml).
HA-tagged GnRH receptor-containing clones were subsequently identified
using previously described GnRH receptor ligand-binding assays (39) and
total inositol phosphate assays (40). The clone HEK 293-B5, expressing
the HA-tagged GnRH receptor, was chosen for further study.
Detection and Visualization of HA-Tagged GnRH Receptors
HA-tagged GnRH receptors were detected using indirect
immunocytochemistry on cell monolayers. Trypsinized cells were plated
into eight-well chamber slides at a density of 2.5 x
104 cells per well in complete DMEM. After 2 days, cells
were washed twice with 0.01 M PBS, pH 7.4, and treated as
required in HEPES-modified DMEM with 0.1% BSA, pH 7.4, before fixing
with freshly prepared 4% paraformaldehyde for 30 min at room
temperature. To reduce the nonspecific binding, cells were incubated in
blocking solution (PBS containing 1% BSA and 10% normal serum, pH
7.4) for 30 min. If permeabilized cells were required, to enable the
visualization of internalized HA-tagged GnRH receptors, nonionic
detergent (Nonidet P-40) was added to the blocking solution to a final
concentration of 0.2%. Subsequently, cells were washed with PBS (three
times) before incubating with primary antibody in blocking solution
overnight at 4 C. After washing (four times in PBS) cells were
incubated with goat antimouse FITC-conjugated secondary antibody (20
µg/ml) for 60 min at room temperature. Slides were then washed (four
times in PBS), mounted in Citifluor, and sealed with coverslips. Cells
were examined under an oil immersion objective (x60) using a Zeiss LMS
510 confocal laser microscope and a filter selective for FITC
fluorescence. Optical sections (0.45 µm) were taken, and
representative sections corresponding to the middle of the cells were
presented. After indirect immunofluorescent staining, no specific
fluorescence was observed in untransfected HEK 293 cells, or in HEK
293-B5 cells treated with secondary FITC-linked goat antimouse IgG
antibody only.
Colocalization of HA-Tagged GnRH Receptors with Transferrin
Receptor
For colocalization of epitope-tagged receptor with transferrin
receptor, the cells were first treated (1 µM GnRH for
1 h at 37 C), fixed, and incubated with both rabbit polyclonal
anti-HA antibody and mouse antihuman transferrin receptor antibody
overnight at 4 C. The epitope-tagged receptors and the transferrin
receptors were detected using antirabbit FITC and antimouse Texas
Red-conjugated secondary antibody (as described previously). Cells were
then viewed as previously described using filters selective for either
rhodamine or fluorescein fluorescence.
Total IP Assays
Assays were performed in 24-well plates containing 1.5 x
105 cells per well, and total IPs were extracted and
separated as described previously (40).
Iodination of GnRH Agonist and Antagonist
Iodinated radiolabeled GnRH analogs were prepared using the
glucose oxidase/lactoperoxidase method and purified by chromatography
on a Sephadex G-25 column in 0.01 M acetic acid/0.1% BSA.
The specific activities of the
125I-des-Gly10,[D-Trp6]-GnRH
(GnRH agonist) and 125I-[Ac-3,4-dehydro-Pro1,
D-p-F-Phe2,
D-Trp3,6]-GnRH (GnRH-antag) tracers were 53
µCi/µg and 28.2 µCi/µg, respectively, and were calculated from
self-displacement assays using either rat pituitary homogenates or
COS-7 cells transiently transfected with the WT GnRH receptor cDNA.
Assays were incubated for 2 h at 4 C before filtration through a
cell harvester using Whatman GFB filter paper.
Receptor Internalization and Recycling Assays
Receptor internalization and recycling assays were based on
protocols described by Lauffenburger and Linderman (41). Briefly, HEK
B5–293 cells were plated at a density of 1.5 x 105
cells per well in 24-well plates. After 2 days, cells were washed once
with assay medium (HEPES-modified DMEM with 0.1% BSA, pH 7.4) before
being incubated with 125I-labeled GnRH agonist or
antagonist (100,000 cpm/well) in 0.5 ml assay medium for time intervals
ranging from 5 min to 2 h at either 4 C or 37 C. To test the
effect of hypertonic medium, assays were also performed in the presence
of sucrose; in this case, the cells were pretreated with assay medium
containing 0.4 M sucrose for 20 min at 37 C, the sucrose
concentration being maintained during the ligand treatment. At
appropriate times, cells were transferred onto ice and washed twice
with ice-cold PBS. Subsequently, the extracellular receptor-associated
ligand was removed by washing once with 1 ml of acid solution (50
mM acetic acid and 150 mM NaCl, pH 2.8) for 12
min. The acid wash was collected to determine the surface-bound
radioactivity, and the internalized radioactivity was determined after
solubilizing the cells in 0.2 M NaOH and 1% SDS (NaOH/SDS)
solution. Nonspecific binding for each time point was determined under
the same conditions in the presence of 10 µM unlabeled
agonist or antagonist. After subtraction of nonspecific binding, the
internalized radioactivity was expressed as a percentage of the total
binding at that time interval. All time points were performed in
triplicate for at least three separate experiments. To compare the
agonist-induced internalization kinetics of the GnRH receptor with
another member of the GPCR superfamily, HEK E2–293 cells, which stably
express WT TRH receptor (42), were also assayed under identical
conditions using 3H-labeled TRH agonist. To evaluate the
effect of ß-arrestin on internalization, COS-7 cells and HEK 293
cells stably expressing either GnRH or TRH receptor (2 x
106/100 mm dish) were transiently transfected with various
constructs using Transfectam. Cells were then split into 24-well
plates, and internalization assays were performed with the appropriate
labeled agonist as described above.
For the recycling experiment, HEK 293-B5 cells were preincubated with 125I-labeled GnRH agonist (1 h at 37 C) to get a substantial intracellular pool, followed by an acid wash to remove surface-bound ligand; cells were then incubated in medium containing saturating concentrations of unlabeled agonist for varying periods of time (5–60 min at 37 C) to monitor the decrease in the intracellular pool due to recycling of the ligand. To test whether the recycling proceeded through acidified endosomal compartments, 25 mM NH4Cl was added to the incubation medium during the recycling period, and the decrease in the intracellular receptor pool was monitored as described above. From the data obtained, the endocytosis (ke), recycling (kr), synthesis (ks), and degradation (kx) rate constants were calculated using the four-compartment model described by Koenig and Edwardson (31).
ELISA
ELISA assays for the measurement of surface-expressed HA-tagged
GnRH receptors and quantification of receptor internalization were
based on the method described previously (43). Cells were plated out at
a density of 5 x 104 cells per well in 48-well
plates. After 2 days, cells were treated as required in either
HEPES-modified DMEM with 0.1% BSA, pH 7.4, or hypertonic sucrose
medium for varying periods of time at 37 C before fixing with freshly
prepared 4% paraformaldehyde for 10 min at room temperature. Cells
were then washed three times in PBS, blocked (PBS containing 10%
normal serum, pH 7.4), and incubated with a 1:200 dilution of primary
anti-HA monoclonal antibody in blocking buffer overnight at 4 C.
Subsequently, cells were washed with PBS (three times) and incubated
for 1 h at 37 C in a 1:2000 dilution of a horseradish
peroxidase-conjugated sheep antimouse IgG. After final washes in PBS
(six times) and 0.9% NaCl (once), the reaction was developed using the
3,3',5,5'-tetramethylbenzidine liquid substrate system. The enzymatic
reaction was stopped after 30 min at room temperature with 0.5 N
H2SO4, and a 100 µl sample was taken for
colorimetric measurement at 450 nm using a Labsystem multiscan MCC/340
reader. For the recycling experiment, a preliminary treatment (1
µM GnRH for 2 h at 37 C) was followed by an acid
wash (50 mM acetic acid and 150 mM NaCl, pH
2.8) for 5 min to remove surface-bound ligand, and the cells were
incubated in serum-free HEPES-modified DMEM/BSA medium at 37 C for
varying periods of time (5 min to 1 h) to monitor the recovery of
surface-expressed receptors. Cycloheximide (10 µg/ml), when used, was
present throughout the whole experiment. Untransfected HEK 293 cells
were assayed concurrently to determine background. All experiments were
done in triplicate.
Construction of the ß-Arrestin-GFP Expression Construct
Production and subcloning of the ß-arrestin/GFP fusion protein
were performed in two separate steps. In the first step, the coding
sequence of a modified form of GFP (44) was altered by PCR
amplification. Using the amino-terminal primer
5'-CCGCTCGAGAGTAAAGGAGAAGAACTTTTCAC-3' a XhoI
restriction site (underlined) was introduced adjacent to the
sequence of codon 2 of GFP. The ATG initiator codon was removed. Using
the carboxyl-terminal primer
5'-TGCTCTAGATTATTTGTATAGTTCATCCATGCC-3' an XbaI
restriction site (underlined) was introduced after the stop
codon. The amplified fragment of GFP digested with XhoI and
XbaI was ligated into the pcDNA3 expression vector
(Invitrogen, San Diego, CA) digested with XhoI and
XbaI. To obtain the ß-arrestin/GFP fusion protein, the
coding sequence of ß-arrestin (45) was amplified by PCR. Using the
amino-terminal primer
5'-AAAAAAGCTTTCTACCATGGGCGACAAAGGGACAC-3', a
HindIII restriction site (underlined) and a
partial Kozak site were introduced in front of the initiator Met of
ß-arrestin. Using the carboxyl-terminal primer
5'-AACTCGAGTCTGTTGTTGAGGTGTGGAGAGC-3' a XhoI
restriction site (underlined) was introduced just in front
of the stop codon of ß-arrestin. Finally, the GFP construct in pcDNA3
was digested with XhoI and HindIII and was
ligated together with the PCR product of the ß-arrestin amplification
that was digested with HindIII and XhoI. The open
reading frame so produced represents the coding sequence of
ß-arrestin/GFP. This construct was fully sequenced before its
expression and analysis. HEK 293 cells (1.5 x 106/60
mm dish) stably expressing either GnRH (B5 cells) or TRH (E2 cells)
receptor were transfected with 2.5 µg of ß-arrestin/GFP cDNA using
Transfectam. After 24 h, cells were plated into eight-well chamber
slides, and treatments were carried out 48–72 h after transfection.
The cells were then fixed with 4% paraformaldehyde, mounted, and
sealed with coverslips. Confocal microscopy was performed as described
previously.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
M. Vrecl is financially supported by the Ministry of Science and Technology of the Republic of Slovenia.
Received for publication July 10, 1998. Accepted for publication August 18, 1998.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
T3–1 cells. Endocrinology 135:1165–1170[Abstract]
T3–1 and HEK-293 cells expressing the GnRH receptor.
Endocrinology 136:5228–5231[Abstract]
2-adrenergic receptors. Mol Pharmacol 51:711–720T3–1 pituitary
gonadotroph cell. J Endocrinol 136:51–58
This article has been cited by other articles:
 |
|
 |
|
  B. W. Jones and P. M. Hinkle Subcellular Trafficking of the TRH Receptor: Effect of Phosphorylation Mol. Endocrinol., September 1, 2009; 23(9): 1466 - 1478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Finch, C. J. Caunt, S. P. Armstrong, and C. A. McArdle Agonist-induced internalization and downregulation of gonadotropin-releasing hormone receptors Am J Physiol Cell Physiol, January 1, 2009; 297(3): C591 - C600. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Pawson, E. Faccenda, S. Maudsley, Z.-L. Lu, Z. Naor, and R. P. Millar Mammalian Type I Gonadotropin-Releasing Hormone Receptors Undergo Slow, Constitutive, Agonist-Independent Internalization Endocrinology, March 1, 2008; 149(3): 1415 - 1422. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Svendsen, M. Vrecl, T. M. Ellis, A. Heding, J. B. Kristensen, J. D. Wade, R. A. D. Bathgate, P. De Meyts, and J. Nohr Cooperative Binding of Insulin-Like Peptide 3 to a Dimeric Relaxin Family Peptide Receptor 2 Endocrinology, March 1, 2008; 149(3): 1113 - 1120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. C. Kong, A. Hasbi, M. Mattocks, T. Fan, B. F. O'Dowd, and S. R. George Regulation of D1 Dopamine Receptor Trafficking and Signaling by Caveolin-1 Mol. Pharmacol., November 1, 2007; 72(5): 1157 - 1170. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Jorgensen, V. Kubale, M. Vrecl, T. W. Schwartz, and C. E. Elling Oxyntomodulin Differentially Affects Glucagon-Like Peptide-1 Receptor beta-Arrestin Recruitment and Signaling through G{alpha} J. Pharmacol. Exp. Ther., July 1, 2007; 322(1): 148 - 154. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. W. Jones, G. J. Song, E. K. Greuber, and P. M. Hinkle Phosphorylation of the Endogenous Thyrotropin-releasing Hormone Receptor in Pituitary GH3 Cells and Pituitary Tissue Revealed by Phosphosite-specific Antibodies J. Biol. Chem., April 27, 2007; 282(17): 12893 - 12906. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Breit, K. Wolff, H. Kalwa, H. Jarry, T. Buch, and T. Gudermann The Natural Inverse Agonist Agouti-related Protein Induces Arrestin-mediated Endocytosis of Melanocortin-3 and -4 Receptors J. Biol. Chem., December 8, 2006; 281(49): 37447 - 37456. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. R Sedgley, A. R Finch, C. J Caunt, and C. A McArdle Intracellular gonadotropin-releasing hormone receptors in breast cancer and gonadotrope lineage cells J. Endocrinol., December 1, 2006; 191(3): 625 - 636. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. J. Caunt, A. R. Finch, K. R. Sedgley, L. Oakley, L. M. Luttrell, and C. A. McArdle Arrestin-mediated ERK Activation by Gonadotropin-releasing Hormone Receptors: RECEPTOR-SPECIFIC ACTIVATION MECHANISMS AND COMPARTMENTALIZATION J. Biol. Chem., February 3, 2006; 281(5): 2701 - 2710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. W. Jones and P. M. Hinkle {beta}-Arrestin Mediates Desensitization and Internalization but Does Not Affect Dephosphorylation of the Thyrotropin-releasing Hormone Receptor J. Biol. Chem., November 18, 2005; 280(46): 38346 - 38354. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. N Hislop, C. J Caunt, K. R Sedgley, E. Kelly, S. Mundell, L. D Green, and C. A McArdle Internalization of gonadotropin-releasing hormone receptors (GnRHRs): does arrestin binding to the C-terminal tail target GnRHRs for dynamin-dependent internalization? J. Mol. Endocrinol., August 1, 2005; 35(1): 177 - 189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. K. Cheng and P. C. K. Leung Molecular Biology of Gonadotropin-Releasing Hormone (GnRH)-I, GnRH-II, and Their Receptors in Humans Endocr. Rev., April 1, 2005; 26(2): 283 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Ronacher, N. Matsiliza, N. Nkwanyana, A. J. Pawson, T. Adam, C. A. Flanagan, R. P. Millar, and A. A. Katz Serine Residues 338 and 339 in the Carboxyl-Terminal Tail of the Type II Gonadotropin-Releasing Hormone Receptor Are Critical for {beta}-Arrestin-Independent Internalization Endocrinology, October 1, 2004; 145(10): 4480 - 4488. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Popova and M. M. Rasenick Clathrin-mediated Endocytosis of m3 Muscarinic Receptors: ROLES FOR G{beta}{gamma} AND TUBULIN J. Biol. Chem., July 16, 2004; 279(29): 30410 - 30418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Vrecl, R. Jorgensen, A. Pogacnik, and A. Heding Development of a BRET2 Screening Assay Using {beta}-Arrestin 2 Mutants J Biomol Screen, June 1, 2004; 9(4): 322 - 333. [Abstract] [PDF]
|
|
|
|
|
|
R. P. Millar, Z.-L. Lu, A. J. Pawson, C. A. Flanagan, K. Morgan, and S. R. Maudsley Gonadotropin-Releasing Hormone Receptors Endocr. Rev., April 1, 2004; 25(2): 235 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ulloa-Aguirre, J. A. Janovick, A. Leanos-Miranda, and P. M. Conn Misrouted cell surface GnRH receptors as a disease aetiology for congenital isolated hypogonadotrophic hypogonadism Hum. Reprod. Update, March 1, 2004; 10(2): 177 - 192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. E. C. Miles, A. C. Hanyaloglu, J. R. Dromey, K. D. G. Pfleger, and K. A. Eidne Gonadotropin-Releasing Hormone Receptor-Mediated Growth Suppression of Immortalized L{beta}T2 Gonadotrope and Stable HEK293 Cell Lines Endocrinology, January 1, 2004; 145(1): 194 - 204. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. P. Brothers, J. A. Janovick, and P. M. Conn Unexpected Effects of Epitope and Chimeric Tags on Gonadotropin-Releasing Hormone Receptors: Implications for Understanding the Molecular Etiology of Hypogonadotropic Hypogonadism J. Clin. Endocrinol. Metab., December 1, 2003; 88(12): 6107 - 6112. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Pawson, S. R. Maudsley, J. Lopes, A. A. Katz, Y.-M. Sun, J. S. Davidson, and R. P. Millar Multiple Determinants for Rapid Agonist-Induced Internalization of a Nonmammalian Gonadotropin-Releasing Hormone Receptor: A Putative Palmitoylation Site and Threonine Doublet within the Carboxyl-Terminal Tail Are Critical Endocrinology, September 1, 2003; 144(9): 3860 - 3871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. Navratil, S. P. Bliss, K. A. Berghorn, J. M. Haughian, T. A. Farmerie, J. K. Graham, C. M. Clay, and M. S. Roberson Constitutive Localization of the Gonadotropin-releasing Hormone (GnRH) Receptor to Low Density Membrane Microdomains Is Necessary for GnRH Signaling to ERK J. Biol. Chem., August 22, 2003; 278(34): 31593 - 31602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. Haisenleder, H. A. Ferris, and M. A. Shupnik The Calcium Component of Gonadotropin-Releasing Hormone-Stimulated Luteinizing Hormone Subunit Gene Transcription Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase Type II Endocrinology, June 1, 2003; 144(6): 2409 - 2416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. H. Shah, M. P. Farshori, A. Jambusaria, and K. J. Catt Roles of Src and Epidermal Growth Factor Receptor Transactivation in Transient and Sustained ERK1/2 Responses to Gonadotropin-releasing Hormone Receptor Activation J. Biol. Chem., May 23, 2003; 278(21): 19118 - 19126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Hanyaloglu, R. M. Seeber, T. A. Kohout, R. J. Lefkowitz, and K. A. Eidne Homo- and Hetero-oligomerization of Thyrotropin-releasing Hormone (TRH) Receptor Subtypes. DIFFERENTIAL REGULATION OF beta -ARRESTINS 1 AND 2 J. Biol. Chem., December 20, 2002; 277(52): 50422 - 50430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Conn, A. Leanos-Miranda, and J. A. Janovick Protein Origami: Therapeutic Rescue of Misfolded Gene Products Mol. Interv., September 1, 2002; 2(5): 308 - 316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. J. Dolph Book Review: Arrestin: Roles in the Life and Death of Retinal Neurons Neuroscientist, August 1, 2002; 8(4): 347 - 355. [Abstract] [PDF]
|
|
|
|
|
|
J. A. Janovick, G. Maya-Nunez, and P. M. Conn Rescue of Hypogonadotropic Hypogonadism-Causing and Manufactured GnRH Receptor Mutants by a Specific Protein-Folding Template: Misrouted Proteins as a Novel Disease Etiology and Therapeutic Target J. Clin. Endocrinol. Metab., July 1, 2002; 87(7): 3255 - 3262. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Sandoval, H. Curtis, and L.F. Congote Enhanced proliferative effects of a baculovirus-produced fusion protein of insulin-like growth factor and {alpha}1-proteinase inhibitor and improved anti-elastase activity of the inhibitor with glutamate at position 351 Protein Eng. Des. Sel., May 1, 2002; 15(5): 413 - 418. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hilairet, C. Belanger, J. Bertrand, A. Laperriere, S. M. Foord, and M. Bouvier Agonist-promoted Internalization of a Ternary Complex between Calcitonin Receptor-like Receptor, Receptor Activity-modifying Protein 1 (RAMP1), and beta -Arrestin J. Biol. Chem., November 2, 2001; 276(45): 42182 - 42190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. N. Hislop, H. M. Everest, A. Flynn, T. Harding, J. B. Uney, B. E. Troskie, R. P. Millar, and C. A. McArdle Differential Internalization of Mammalian and Non-mammalian Gonadotropin-releasing Hormone Receptors. UNCOUPLING OF DYNAMIN-DEPENDENT INTERNALIZATION FROM MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING J. Biol. Chem., October 19, 2001; 276(43): 39685 - 39694. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Hashizume, W.-H. Yang, C. M. Clay, and T. M. Nett Internalization Rates of Murine and Ovine Gonadotropin-Releasing Hormone Receptors Biol Reprod, March 1, 2001; 64(3): 898 - 903. [Abstract] [Full Text]
|
|
|
|
|
|
D. A. Groarke, T. Drmota, D. S. Bahia, N. A. Evans, S. Wilson, and G. Milligan Analysis of the C-Terminal Tail of the Rat Thyrotropin-Releasing Hormone Receptor-1 in Interactions and Cointernalization with {beta}-Arrestin 1-Green Fluorescent Protein Mol. Pharmacol., February 1, 2001; 59(2): 375 - 385. [Abstract] [Full Text]
|
|
|
|
 |
|
 Z. Lenkei, A. Beaudet, N. Chartrel, N. De Mota, T. Irinopoulou, B. Braun, H. Vaudry, and C. Llorens-Cortes A Highly Sensitive Quantitative Cytosensor Technique for the Identification of Receptor Ligands in Tissue Extracts J. Histochem. Cytochem., November 1, 2000; 48(11): 1553 - 1564. [Abstract] [Full Text]
|
|
|
|
|
|
R. Grosse, S. Roelle, A. Herrlich, J. Hohn, and T. Gudermann Epidermal Growth Factor Receptor Tyrosine Kinase Mediates Ras Activation by Gonadotropin-releasing Hormone J. Biol. Chem., April 14, 2000; 275(16): 12251 - 12260. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C.-C. Tseng and X.-Y. Zhang Role of G Protein-Coupled Receptor Kinases in Glucose-Dependent Insulinotropic Polypeptide Receptor Signaling Endocrinology, March 1, 2000; 141(3): 947 - 952. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Heding, M. Vrecl, A. C. Hanyaloglu, R. Sellar, P. L. Taylor, and K. A. Eidne The Rat Gonadotropin-Releasing Hormone Receptor Internalizes via a {beta}-Arrestin-Independent, but Dynamin-Dependent, Pathway: Addition of a Carboxyl-Terminal Tail Confers {beta}-Arrestin Dependency Endocrinology, January 1, 2000; 141(1): 299 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. B. Willars, A. Heding, M. Vrecl, R. Sellar, M. Blomenrohr, S. R. Nahorski, and K. A. Eidne Lack of a C-terminal Tail in the Mammalian Gonadotropin-releasing Hormone Receptor Confers Resistance to Agonist-dependent Phosphorylation and Rapid Desensitization J. Biol. Chem., October 15, 1999; 274(42): 30146 - 30153. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Cornea, J. A. Janovick, X. Lin, and P. M. Conn Simultaneous and Independent Visualization of the Gonadotropin-Releasing Hormone Receptor and Its Ligand: Evidence for Independent Processing and Recycling in Living Cells Endocrinology, September 1, 1999; 140(9): 4272 - 4280. [Abstract] [Full Text]
|
|
|
|
|
|
S. A. Laporte, R. H. Oakley, J. A. Holt, L. S. Barak, and M. G. Caron The Interaction of beta -Arrestin with the AP-2 Adaptor Is Required for the Clustering of beta 2-Adrenergic Receptor into Clathrin-coated Pits J. Biol. Chem., July 21, 2000; 275(30): 23120 - 23126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Cornea, J. A. Janovick, G. Maya-Nunez, and P. M. Conn Gonadotropin-releasing Hormone Receptor Microaggregation. RATE MONITORED BY FLUORESCENCE RESONANCE ENERGY TRANSFER J. Biol. Chem., January 12, 2001; 276(3): 2153 - 2158. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. C. Hanyaloglu, M. Vrecl, K. M. Kroeger, L. E. C. Miles, H. Qian, W. G. Thomas, and K. A. Eidne Casein Kinase II Sites in the Intracellular C-terminal Domain of the Thyrotropin-releasing Hormone Receptor and Chimeric Gonadotropin-releasing Hormone Receptors Contribute to beta -Arrestin-dependent Internalization J. Biol. Chem., May 18, 2001; 276(21): 18066 - 18074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. Kroeger, A. C. Hanyaloglu, R. M. Seeber, L. E. C. Miles, and K. A. Eidne Constitutive and Agonist-dependent Homo-oligomerization of the Thyrotropin-releasing Hormone Receptor. DETECTION IN LIVING CELLS USING BIOLUMINESCENCE RESONANCE ENERGY TRANSFER J. Biol. Chem., April 13, 2001; 276(16): 12736 - 12743. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
