 |
INTRODUCTION |
Growth hormone (GH)1 has
diverse biological actions, including promoting cellular growth and
differentiation, that are mediated by binding to a specific, high
affinity cell surface receptor (GHR). The GHR is a member of the type I
cytokine family of receptors, which possess in common a single
transmembrane domain and lack intrinsic kinase activity (1). Binding of
a single molecule of GH results in receptor dimerization and signaling
through a phosphorylation cascade that includes the Jak-Stat pathway.
Activation of the Jak-Stat pathway is dependent on an intact
cytoplasmic domain of the receptor forming multisubunit complexes with
associated tyrosine kinases. Recently, we (2) and others (3) identified a truncated form of the receptor, GHR1-279, in normal human liver and
certain human cell lines. This truncated receptor lacks 97% of the
cytoplasmic domain of the receptor and has a dominant negative action
on Jak-Stat signaling (2). In human tissues the truncated receptor is
expressed at a low level compared with the full-length receptor (2, 4).
However, in cell transfection experiments, the truncated receptor
always demonstrates a greater level of receptor expression at the cell
surface compared with the full-length receptor (2). It has been
hypothesized that the truncated receptor lacks essential
internalization signals in the cytoplasmic domain and therefore remains
at the cell surface available to heterodimerize with the full-length
receptor and inhibit signaling (2, 5). In addition, the truncated
receptor generates large amounts of GH-binding protein (GHBP) (2, 3).
Confirmation that these truncated receptors have a dominant negative
action on GHR signaling comes from patients who are heterozygous for a
mutation that encodes a very similar truncated receptor, GHR1-277 (6,
7). These patients have high levels of GHBP and GH insensitivity,
presumably because of increased levels of truncated receptor at the
cell surface, which fails to internalize and heterodimerizes with the full-length receptor to inhibit signaling (6).
The GHR is internalized via clathrin-coated pits (8, 9). This
internalization is dependent upon an intact ubiquitination system, and
GHR ubiquitination is dependent upon an intact endocytic pathway (9).
It has been suggested that a small fraction of GHR is constitutively
ubiquitinated and internalized but that GH-induced dimerization results
in an increase in ubiquitination and internalization (9). The
Phe327 residue within the cytosolic tail of the GHR is
involved in both GHR ubiquitination and ligand-induced receptor
endocytosis (9, 10). In addition, it has been reported that there is an
ubiquitin-independent internalization signal, based on a di-leucine
repeat (Leu347,348), which is inactive in the full-length
receptor but is activated in a truncated receptor, GHR1-349 (11). It
has been suggested that this motif may function in the internalization
of an as yet unidentified GHR isoforms (11).
Internalization is not required for Jak-Stat signal transduction, as
GHR mutated at Phe327 signals but is not internalized (10).
However, there is strong evidence for the importance of receptor
dimerization for signal transduction, which is derived from a number of
experiments. In the presence of a molar excess of GH, which favors the
formation of the monomeric GH-GHR complex, there is inhibition of the
GH signal (12), truncated receptors that lack the cytoplasmic signaling domain block signal transduction by heterodimerization (2), and
receptor dimerization is associated with a conformational change that
is required for receptor signaling (13). The strongest evidence,
however, comes from work with a GH antagonist mutated at GH binding
site 2 (G120R), which blocks GH-stimulated cell proliferation (14) and
does not generate the conformational change associated with receptor
dimerization and Jak-Stat signaling (13). However, the GH antagonist
G120R does appear to dimerize in a (GH)2-(GHR)2
complex and is internalized (15). Another GH antagonist mutated at
binding site 2 (G120K) is currently being successfully investigated for
its potential as a treatment for acromegaly, a disease resulting from
an excess GH secretion from a pituitary tumor (16, 17). This
antagonist, B2036, has 8 additional mutations at GH binding site 1, which increases the affinity of the antagonist for GHBP (18). There are
no publications on the effect of this antagonist on internalization and
cell signaling.
To date, most studies of GHR trafficking have depended on techniques
that require the use of nonphysiological conditions, including acid
stripping of cell surface receptor (19) and low temperature for ligand
binding (9), and have used either the rat or rabbit GHR (the human GHR
has proved difficult to clone and express (20)). These experiments have
been of great value in defining the components required for
internalization but are limited in their ability to establish the real
time dynamics of receptor trafficking. Fluorescent labeling of ligand
(9) and generation of fusion proteins with green fluorescent protein
have made it possible to study the active transport of cellular
proteins (21) and receptors, including nuclear receptors (22) and G protein-coupled receptors (23, 24). We have used fluorescent confocal
microscopy and a combination of fluorescent-labeled GH and GHR to study
GHR trafficking in real time at the level of the single cell. The
results show: 1) the previously unrecognized, extremely rapid
internalization of GH and of its antagonist, B2036; 2) that this
internalization is independent of signaling; and 3) the failure of the
naturally occurring truncated GHR1-279 to internalize.
 |
MATERIALS AND METHODS |
Plasmids--
The full-length GHR and truncated receptor
GHR1-279 were subcloned into pcDNA1/ampicillin (Invitrogen) as
described previously (2). The LHRE-EGFP reporter construct was
generated by subcloning the Stat5-binding element (LHRE) fused to the
minimal TK promoter (25) into EGFP-p1 (CLONTECH).
The truncated GHR1-317-EGFP chimera was created by subcloning the N
terminus of EGFP to the EcoRI site in the cytoplasmic domain
of the human GHR in the expression vector pcDNA1/ampicillin (Invitrogen).
Stable clones were generated in 293 cells (human kidney embryonal cell
line) by transfection with the calcium phosphate procedure of the GHR
plasmid with pcDNA3/Neo (Invitrogen). Selection was made using G418
(400 µg/ml, Sigma).
Binding Assays--
24 h after plating, cells were serum-starved
for 12 h. Cells were then washed with phosphate-buffered saline
containing 1% bovine serum albumin and incubated with
125I-hGH (1 × 105 cpm/well) for 3 h
at room temperature in the absence or presence of various
concentrations of unlabeled hGH. The cells were then washed in the same
buffer and solubilized in 1 ml of 1 N NaOH for counting radiation.
The recombinant human GH used was Genotrophin (Pharmacia and Upjohn,
Milton Keynes, UK). The GH antagonist, B2036, is mutated at GH
binding site 2 with G120K. In addition, to the G120K mutation there are 8 additional mutations at site 1 that are thought to increase
the affinity of the receptor (18). The GH antagonist (B2036) was kindly
supplied by William Bennett of Sensus Drug Development Corp.(Austin, Texas).
Texas Red labeling was performed using Texas Red-X succinimidyl ester
(Molecular Probes, Leiden, Netherlands). GH or antagonist were dialyzed
overnight against borate-buffered saline (pH 8.5). 100 µg of GH or
antagonist were incubated with Texas Red at a 1:1 molar ratio in a
final volume of 200 µl for 1 h at room temperature and away from
light. The reaction mixture was separated on a 1-ml-bed volume Bio-Gel
P10 5-kDa polyacrylamide gel.
Transcription assays were performed in 293 cells expressing the GHR
using either a reporter gene containing a Stat5-binding element (LHRE)
fused to a minimal TK promoter and luciferase or the LHRE-TK fused to
EGFP. Luciferase activity was measured as previously reported (2). In
studies using the LHRE-TK·EGFP, 293 cells were plated on 4-well
coverglass slides, precoated with fibronectin at a density of 3 × 104 cells per well (ml), transfected by the calcium
phosphate method with LHRE-TK·EGFP (550 ng/slide), and incubated
overnight in complete medium before transfer into serum-free medium
containing either 500 ng/ml GH and 100 ng/ml dexamethasone (stimulated)
or dexamethasone alone (unstimulated). Fluorescence was detected
24 h later on the stage incubator of the confocal
microscope, an analysis of images was performed using the
public domain NIH Image (http://rsb.info.nih.gov/nih.image).
Fluorescence Imaging--
For experiments on living cells,
fluorescence was detected using a Molecular Dynamics CLSM2010 confocal
fluorescent microscope equipped with dual fluorescence and transmission
detection. Images were transferred from the Silicon Graphics INDY on
the CSLM 2010, converted to Macintosh 8-bit Tagged Image File Format,
and analyzed with NIH Image, and the data were exported to EXCEL for
further analysis. All experiments were performed at 37 °C using a
stage incubator enclosure fitted to the microscope. For the detection of Texas Red, cells were excited at 568 nm, and detection was through a
610 nm long pass filter (red channel). For EGFP, cells were excited at
488 nm and detection was through a 530 nm band pass filter (green
channel). In all experiments used for quantitative analysis, the laser
power was kept below the level of saturation for the fluor and the
photomultiplier voltage set so that the brightest pixels were <250
8-bit units. In the fluorescence energy transfer (FRET) experiments
cells were excited with the 488 nm line, and data were collected on the
red channel.
Calculation of Internalization--
Using NIH Image the
fluorescence within the cell membrane was divided by the total
fluorescence of the cell after subtracting the background. This number
was multiplied by 100 to give a percentage, and the mean of two
measurements for each cell was used in the analysis.
Western Ligand Blotting--
293 cells were starved overnight in
serum-free medium and then stimulated for 15 min at 37 °C with GH or
antagonist. Cells were lysed in PBS-TDS (phosphate-buffered saline, 1%
Triton X-100, 12 mM sodium deoxycholate, 3.5 mM
SDS, and 4.7 nM sodium orthovandate), and 1 mg of protein
was precipitated with Jak2 antibody (Upstate Biotechnology, Buckingham,
UK) at 1:250 dilution with 20 µl of protein A-agarose (Sigma).
Precipitated proteins were separated on a 10% SDS-polyacrylamide gel
and after blotting onto polyvinylidene difluoride, phosphorylation was
detected with an antiphosphotyrosine antibody (1:2500) (4G10, Upstate
Biotechnology, Buckingham, UK) and the ECL system (Amersham Pharmacia Biotech).
 |
RESULTS |
To compare internalization of the truncated GHR1-279 with the
full-length human GHR, we generated stable clones in a human kidney
cell line, 293 cells. Cells were transfected with either receptor and
selected under G418. Clones were picked that expressed a high level of
GH binding. In our experimental conditions (125I-hGH,
1 × 105 cpm, on 3 × 105 cells), the
selected full-length human GHR expressing clone showed 16% specific
binding, and the GHR1-279 clone showed 28% specific binding.
Characterization of Stable Clone 293 Cells Expressing the
Full-length and Truncated GHR and Affinity of the GH
Antagonist--
In cells expressing the full-length receptor,
competition studies with 125I-hGH and
125I-B2036 showed a single class of receptors (Fig.
1). The antagonist B2036 has 8 mutations
in the GH binding site 1, which would be expected to increase its
affinity for the extracellular domain of the GHR (18). In an assay
based on GHBP immobilized on an enzyme-linked immunosorbent assay
plate, the antagonist had an affinity five times greater than native
GH.2 We compared the binding
of GH and the antagonist, B2036, on intact cells expressing the
full-length GHR. In displacement studies using either labeled GH or
antagonist, the apparent affinities calculated by Scatchard analysis
for GH (Ka = 1.00 × 109
M
1) and B2036 (Ka = 0.96 × 109 M
1) were
comparable (Fig. 1). Thus, in intact cells, the antagonist appears to
have a similar affinity to native GH. Functional activity of the
full-length receptor was measured in transient transfections with Stat5
reporter constructs, consisting of Stat5 binding sites (LHRE) fused to
either EGFP or luciferase. In the luciferase assay test, GH stimulation
induced a bell-shaped curve with maximal stimulation (5-fold) occurring
between 50 and 500 ng/ml and returning to basal values for GH levels
>10 µg/ml. These results confirm that excess of GH favors the
formation of GH-GHR monomers and blocks signal transduction (12). In
the EGFP test, stimulation with 500 ng/ml GH also resulted in a 5-fold
induction of fluorescence compared with unstimulated cells (Fig.
2). This induction of fluorescence was
seen across the whole range of cell sizes (Fig. 2C). In
contrast, the stable clone expressing the truncated GHR1-279, despite
a high level of GH binding, showed no induction of fluorescence when
transfected with the LHRE-EGFP reporter and no induction of luciferase
activity (data not shown).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 1.
Displacement curves of hGH and B2036 for the
GHR. 293 cells expressing the full-length GHR were incubated with
125I-hGH (A) or 125I-B2036
(B) with increasing amounts of hGH or B2036. The
experimental conditions are described under "Material and
Methods."
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Stimulation of the LHRE-TK·EGFP reporter
gene by hGH. Confocal images (×20) are shown of 293 cells stably
expressing the full-length human GHR and transiently transfected with a
Stat5 reporter construct with the LHRE-TK fused to EGFP. A,
cells incubated in the absence of GH showing occasional cells with
background fluorescence. B, cells incubated with GH at 500 ng showing induction of fluorescence. C, graph showing
intensity of fluorescence plotted against cell size for cells
stimulated with GH or unstimulated. The images analyzed were all under
the level of saturation for the detector. However, to provide a clear
representation and show the backgrounds, the contrast has been enhanced
in both A and B.
|
|
Labeling of GH and Antagonist with Texas Red--
For
internalization studies, GH and the antagonist B2036 were labeled with
Texas Red. We first examined the effect of labeling on GH binding to
its receptor and then incubated Texas Red with GH at different molar
ratios from 1:1 to 1:10 (GH:Texas Red). A ratio greater than 1:3
resulted in a GH molecule with a reduced affinity for the GHR.
Therefore, for all of the studies we elected to label GH and antagonist
with a 1:1 molar ratio of protein to Texas Red.
Internalization of Texas Red-labeled GH in 293 Cells Expressing the
Full-length and Truncated GHR--
To study internalization, 293 cells
were plated on coverglass and incubated overnight in serum-free medium.
Analysis was performed by confocal microscopy at 37 °C. 10 nM Texas Red-labeled GH was added in new starvation medium,
and the cells were incubated for 5 min at 37 °C before washing and
the addition of phenol red-free medium. Confocal images were recorded
sequentially over time. Images were analyzed with NIH Image, and
internalization was assessed by measuring the fluorescence within the
cell membrane expressed as a percentage of the total fluorescence
associated with the cell. No binding or internalization was seen in 293 cells not transfected with the GHR, and in all experiments binding and
internalization were specifically displaced by the simultaneous
addition of 2 µg of unlabeled GH.
For cells expressing the full-length GHR, internalization of GH was
rapid with more than 80% of labeled GH internalized at 5 min (Fig.
3A), and membrane binding was
difficult to detect at 20 min (Fig. 3B). After 15 min, most
GH was internalized (mean ± S.E., 92 ± 1.3%;
n = 8 cells). In contrast, cells expressing the
truncated GHR1-279, which lacks 97% of the cytoplasmic domain of the
receptor, showed little internalization. At 5 min there was well
defined surface binding with <5% internalization of Texas Red-labeled
GH (Fig. 3C), and at 20 min there was <30% internalization (Fig. 3D). After 15 min, internalization of Texas
Red-labeled GH was 6.7 ± 1.8% (mean ± S.E.;
n = 14 cells). The difference in internalization for
the full-length and truncated receptor over time is shown in Fig.
3E.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 3.
Internalization of Texas Red-labeled GH by
293 cells stably expressing the full-length GHR or truncated receptor
GHR1-279. Cells expressing full-length GHR (A and
B) or the truncated GHR1-279 (C and
D) were incubated with 10 nM Texas Red-labeled
GH for 5 min. Cells were observed immediately after washing
(A and C) or 20 min after initial exposure to GH
(B and D). E, percentage of Texas
Red-labeled GH internalized in individual cells against time after
initial exposure to Texas Red-labeled GH. Analysis was performed using
NIH image. The fluorescence inside the cell is expressed as a
percentage of the total fluorescence of the cell.
|
|
Previous publications (19) had reported inhibition of internalization
by cooling cells to 4 °C. We attempted to repeat these studies.
However, cells incubated for 10 min at 4 °C on coverglass behaved
abnormally, showing delayed internalization and frequently separating
from the coverglass.
Studies with GHR-EGFP Fusion Proteins--
To further study the
trafficking of the GHR, we generated fusion proteins with EGFP.
Transient expression of the EGFP expression vector alone (pEGFP-N2,
CLONTECH) in 293 cells resulted in a diffuse distribution of fluorescence throughout the cytoplasm as well as in the
nucleus of some cells. In contrast, transient and stable transfection
of the truncated GHR1-317 fused to EGFP resulted in a cell surface
distribution as shown by radioligand binding and laser scanning
confocal microscopy (Fig. 4). In
addition, the GHR1-317-EGFP was distributed within the cell consistent
with an endoplasmic reticulum (ER) localization as well as in a large juxtanuclear fluorescent area representing the Golgi complex (Fig. 4A). The Golgi localization of the GHR represented a large
proportion of the total fluorescent GHR in the cell. Control
experiments with classical immunofluorescence on fixed cells were
performed to verify that the presence of the GFP moiety did not
significantly alter the distribution of the receptor. Labeling was done
on 293 cells expressing either the full-length or the 279 truncated
receptor permeabilized or not with Triton X-100 (Fig.
5). Labeling was present both at the cell
surface and in intracellular compartments with a juxtanuclear
accumulation in the Golgi apparatus. Such distribution has been
described previously in COS-7 cells (26) and 293 cells (27) transfected
with the GHR. In cells expressing the GHR1-317-EGFP, the fluorescence
resulting from the antibody was superimposed on that from the GFP, so
that even in the absence of Triton X-100, some intracellular labeling
appeared because of the GFP. We analyzed the labeling distribution with
confocal microscopy. Two slices, corresponding respectively to the top of the cell or the middle of the cell, confirm that the fusion receptor
was present both at the cell surface and in intracellular compartments.
This distribution is comparable with that seen for the truncated
receptor GHR 1-279.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 4.
Visualization of the truncated GHR1-317-EGFP
chimera expressed in 293 cells. A, fluorescence
micrograph (×60) showing accumulation of GHR in the Golgi apparatus
(arrow). B, fluorescence micrograph (×60) of
cell demonstrating vesicles generated in the endoplasmic reticulum
(arrow). C, confocal image (×100) demonstrating
fusion of vesicles with the cell surface membrane
(arrow).
|
|

View larger version (81K):
[in this window]
[in a new window]
|
Fig. 5.
Immunofluorescence for GHR in fixed cells
expressing full-length and truncated receptor. 293 cells
expressing full-length GHR (GHR FL, A and
D), GHR1-279 (B and E), or
GHR1-317-EGFP (C and F) were grown on
coverslips. Cells were fixed with 3% paraformaldehyde and
permeabilized (D-F) or not
(A-C) with 0.5% Triton X-100. Cells were then
labeled with 5 mg/ml anti-GHR antibody (monoclonal antibody 263), of
which the epitope is in the extracellular domain of the receptor, and
anti-mouse fluorescein isothiocyanate. Cells were observed under a
Zeiss Axiovert confocal microscope (LSM 510). C and
F show two different slices of the same cell focused,
respectively, at the top of the cell (C) and in
the middle of the cell (F). In this cell, the
fluorescence of monoclonal antibody 263 and GFP are superimposed.
|
|
Monitoring GH/GHR Trafficking Using GHR1-317-EGFP Fusion
Protein--
We have shown that Texas Red-labeled GH can be used to
monitor GH internalization by confocal microscopy and that
internalization is much more rapid than previously reported in
experiments in which cells were first incubated at 4 °C, washed and
transferred at 37 °C and in which the measure of the acid-stripping
resistant radioactivity was the criterion of internalization. Using the receptor fusion protein and the labeled ligand, we exploited FRET to
monitor the trafficking of the ligand, the receptor, and the ligand-receptor complexes simultaneously. Thus GHR-EGFP is detected using the 488 nm line to excite and a 530 nm band pass filter for
emission (blue to green), the Texas Red-GH is detected using the 568 nm
line to excite and a 610 long pass filter for emission (orange to red),
and the GHR-EGFP·Texas Red-GH complexes are detected using the 488 nm
line to excite and a 610 nm long pass filter for emission (blue to
red). A 488 nm scan images the receptor (green) and ligand-receptor
complexes (red), and a 568 nm scan images the ligand, whether
receptor-bound or not. Ratio images (red/green) of GHR-EGFP alone,
using only 488 nm excitation, were used to set the instrument so that
there was no detectable spillover of EGFP fluorescence onto the red channel.
The truncated GHR-EGFP fusion would not be expected to
internalize based on previous reports (10) suggesting that F327 is required for this process. Cells were incubated with Texas Red-GH as
described above and imaged as described previously (Fig.
6). After 30 min at 37 °C, 24% of the
cell-associated Texas Red-GH was internalized, whereas 85% of the
truncated receptor was within the cell. The FRET image shows that 21%
of the GH-GHR complexes were internalized and that the blue to red and
the orange to red images are superimposed, confirming that ligand
associated with the cell is receptor-bound and internalization of the
truncated receptor-ligand complexes is very inefficient compared with
that seen with full-length GHR.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 6.
FRET in cells expressing the truncated
GHR1-317-EGFP chimera and incubated with Texas Red-labeled GH.
Images were made 30 min after a 5-min exposure to Texas Red-labeled GH.
A, image of a cell stimulated with 568 nm light (absorbance
of Texas Red), recorded on the red channel and showing the distribution
of Texas Red-labeled GH. B, the same cell stimulated with
488 nm light (absorbance of EGFP). The recording was made on the green
channel and shows the distribution of GHR1-317-EGFP chimera.
C, the same cell stimulated with 488 nm light (absorbance of
EGFP) and recorded on the red channel, showing FRET, which confirms the
colocalization of GH with the receptor mainly at the cell
surface.
|
|
Visualization of GHR1-317-EGFP Trafficking--
The EGFP fusion
provided us with the opportunity for real time analysis of protein
trafficking events in individual cells (Fig. 4). Packaging and movement
of the receptor in vesicles was easily visualized. Budding of vesicles
and there transport to the Golgi was rapid, occurring within seconds,
and at this stage a large proportion of the GHR remained within the
Golgi. Vesicles budding from the Golgi apparatus appeared to move in a
random fashion toward the cell surface membrane and fused with the
membrane (Fig. 4). A small proportion of the vesicles were moving from the cell surface membrane into the cell, presumably representing membrane turnover.
Studies on the GH Antagonist--
The GH antagonist, B2036,
inhibited GH stimulation of the Stat5 reporter construct in a
dose-dependent manner with complete inhibition at a 1:5
molar ratio of GH to antagonist (Fig.
7A). Western blotting
confirmed that the antagonist fails to induce Jak2 phosphorylation
(Fig. 7B). Internalization of the antagonist was studied in
a manner similar to native GH. Texas Red-labeled antagonist was
incubated with 293 cells stably expressing the full-length GHR for 5 min and then washed; the cells were then imaged by confocal microscopy.
The Texas Red-labeled antagonist showed identical internalization
dynamics to the native GH with the majority of antagonist internalized
by 5 min after exposure to the antagonist (Fig. 7C).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Antagonist effect of B2036. A, the
induction of luciferase was measured in lysates from 293 cells stably
expressing the full-length GHR and transfected with the Stat5 reporter
construct LHRE-TK· luciferase. The cells were either not stimulated
(Control) or were stimulated with GH alone (500 ng/ml) or GH
plus GH antagonist B2036 (500 ng/ml each or 500 + 2500 ng/ml,
respectively). B, lysates from cells stimulated under the
same conditions as described above were immunoprecipitated with
anti-Jak2 antibody and analyzed by Western blot with
antiphosphotyrosine antibodies (lane 1 shows markers).
C, graph from confocal microscopy showing percentages of
native GH and GH antagonist internalized with time.
|
|
 |
DISCUSSION |
The studies reported here demonstrate that internalization of GH
with its receptor is a much more rapid process than previously appreciated. Studies using iodinated GH have generally been performed at 4 °C to prevent internalization and allow equilibrium of bound and unbound GH to occur before warming the cells and studying internalization by acid stripping the surface bound ligand (9, 15, 19).
In these studies internalization occurred over a period of 1 h
with 30 (9) or 65% (15) internalized at 30 min and 85% at 60 min
(15). In our experiments using confocal microscopy and Texas
Red-labeled GH, surface binding could be demonstrated by briefly
incubating the cells at 4 °C and then scanning. However, when cells
were incubated with fluorescent GH at 37 °C and immediately scanned,
the majority of labeled GH was internalized by 5 min, and after this
time point it became difficult to see the cell surface binding as
almost all GH bound to its receptor was internalized. It is likely that
the previously reported slower course for internalization, as well as
the lack of complete internalization, was due to the chilling of cells, as we were able to delay internalization by this procedure. However, in
our experiments the chilling of cells was also associated with other
changes. In particular, the cells tended to lift from the coverglass
after 10 min at 4 °C, and it is probable that these nonphysiological
conditions may cause other changes in cell function.
We studied the internalization of a truncated GHR1-279 that is found
in normal human tissues (2, 3). This truncated GHR, lacking the major
part of the cytoplasmic domain of the receptor, acts as a dominant
negative inhibitor of GHR signaling (2) and generates large amounts of
GH-binding protein (2, 3). Using confocal microscopy and Texas
Red-labeled GH, there was a great difference between internalization of
the full-length and of the truncated receptor. At 5 min almost all
labeled GH was internalized by the full-length GHR, whereas for the
truncated receptor <5% was internalized. A similar but less dramatic
difference in internalization between the full-length and truncated
receptor (70 versus 10% at 1 h) has been shown by acid
stripping (5). This failure of internalization of the truncated human
receptor could be predicted from previous studies with the rat and
rabbit receptor. In these studies, Phe327, which is deleted
in the truncated receptor, proved to be essential for normal
ubiquitination and internalization of the receptor (9, 10). In our
experiments we also show a great difference in the kinetics of receptor
internalization. After 1 h of exposure, we observed some
internalization of the truncated receptor (between 5 and 30%). This
degree of receptor internalization is similar to that reported for
other truncated receptors (5, 9, 10). This internalization may
represent cell surface membrane turnover or an uncharacterized
internalization signal such as the di-leucine-mediated internalization
reported for another truncated GHR (11). Experiments using acid
stripping to monitor the cell surface bound GH after 1 h of
exposure also reported similar differences in internalization between
the full-length and truncated receptor (70 versus 10%) (5).
The demonstration that the truncated receptor fails to internalize
confirms the hypothesis that a lack of internalization underlies the
mechanism for its dominant negative action (2, 6).
Transfection of 293 cells with the EGFP expression vector alone showed
diffuse fluorescence throughout the cell including the nucleus,
demonstrating that the EGFP protein alone moves between all
compartments of the cell. In contrast, the truncated GHR1-317-EGFP fusion protein showed a specific cellular localization, similar to that
seen by immunofluorescence in cells expressing full-length and
truncated receptor. The GHR-EGFP fusion was visualized within vesicles
generated at the ER and transported to the Golgi. The fluorescence seen
within the cell represents packaging of the GHR and translocation to
the cell surface, which could be observed in real time and was a rapid
process. The truncated receptor emerged from the ER at multiple
peripheral sites resulting in the accumulation of chimeric protein in
numerous peripheral membrane structures. These units, once established,
appeared to rapidly (within seconds) translocate to the Golgi apparatus
by a process resembling a random walk. From the Golgi apparatus
vesicles budded off to then transfer and fuse with the cell membrane.
The dynamics of this vesicular transport were very similar to that
shown for ER-to-Golgi transport elucidated with a viral glycoprotein
tagged to GFP (21). To date, there has been no report analyzing the
cellular distribution and dynamic trafficking of a cytokine receptor
linked to GFP. The truncated receptor that we studied allowed us mainly
to visualize translocation to the cell surface membrane, as the
receptor lacked the essential internalization domain (9). The lack of
internalization was confirmed by the studies with FRET. However, a
proportion of fluorescence was internalized and was presumed to be the
result of nonspecific membrane turnover as was the case for the other truncated receptors. Previous receptor studies have reported
visualization of the glucocorticoid receptor (22) and the G
protein-coupled cholecystokinin and
2-adrenergic
receptors (23, 24). The internalization of the
2-adrenergic receptor is slower than we have
demonstrated with the GHR, with only 30% internalized 20 min after
exposure to ligand in HeLa cells (24). However, in 293 cells a
2-adrenergic·GFP fusion protein was at least partially internalized within 5 min of exposure to agonist (28). The kinetics of
internalization are different for these G protein-coupled receptors, which are recycled, and for the cholecystokinin receptor, for which the
recycling time is 20 to 60 min (23).
B2036 is an analogue of human GH in which 8 amino acids in binding site
1 are mutated to increase the binding affinity to the receptor, and 1 amino acid in binding site 2 is mutated to prevent binding to a second
receptor. Preliminary reports in normal subjects and acromegalic
patients suggest that B2036 can reduce IGF-I levels (16, 17). However,
to date there are no reports on the effect of this antagonist at the
level of receptor internalization and signaling. The mutations selected
for site 1 were based on observations made using the extracellular
domain of the receptor to select for high affinity variants of hGH
(18). Using an assay based on displacement of hGH from immobilized
hGHBP, B2036 had five times the affinity to hGH.2 However,
in our assay, based on full-length GHR in intact cells, B2036 had an
affinity comparable to hGH. This assay presumably measures the affinity
of GH for the GHR dimer. It may be that as the GH antagonist (GHa) is
thought to dimerize in the complex GHR-GHa-GHa-GHR (15), this could
give an affinity similar to GH, which forms a dimer in the complex
GHR-GH-GHR. The GH antagonist G120K (B2036) has site 2 for binding GHR
mutated, similar to the previously reported G120R, which blocks
GH-stimulated cell proliferation (14). Our functional studies
demonstrated that the antagonist G120K completely blocked GHR signaling
through Stat5 at a 1:5 molar ratio. This was associated with inhibition
of Jak2 phosphorylation. Our studies confirm and extend the previous
reports that the GH antagonist G120R inhibits signaling (15) by
demonstrating that G120K has a similar action, blocks Jak2
phosphorylation, and also completely abolishes Stat5 signaling. The GH
antagonist G120R has previously been shown to internalize, suggesting
that the abilities of GH to stimulate tyrosine phosphorylation and
internalization are separate functions (15). The time course for the
internalization of the antagonist was similar to that reported for
native GH with approximately 75% internalized within 40 min. We
considered the possibility that the time course for internalization may
differ between the antagonist and native hormone, but this time lag may have been missed in previous experiments. Under physiological conditions we found an identical level of internalization for the
antagonist and native GH, with the major part of both of them internalized within 5 min of exposure to labeled hormone. These results
demonstrate the potent antagonist action of G120K on Jak-Stat signaling, indicating that this antagonist action is unrelated to any
change in internalization.
Together our results demonstrate that GHR trafficking can be studied by
dual-fluorescent confocal microscopy. Using chimeric receptors fused to
EGFP, we have visualized the translocation of the GHR from the ER to
the cell surface. The studies of truncated GHR, which acts as an
antagonist to GHR signaling, confirm that these receptors are unable to
internalize rapidly, which explains their dominant negative action and
also accounts for the accumulation to high levels of such mutants, a
process that would enhance their dominant negative activity. The GH
antagonist G120K behaves in a manner similar to G120R, blocking GHR
signaling through Jak2-Stat5. Fluorescent labeling of the antagonist
G120K confirms that its antagonist action does not effect
internalization, which occurs despite inhibition of receptor signaling.