A ß-Turn Endocytic Code Is Required for Optimal Internalization of the Growth Hormone Receptor but Not for
-Adaptin Association
Lieve Vleurick,
Alain Pezet,
Eduard R. Kühn,
Eddy Decuypere and
Marc Edery
INSERM U344 (L.V., A.P., M.E.) Faculté de Médecine
Necker F-75730 Paris, Cedex 15, France
Leuven Poultry
Research Group Zoological Institute (L.V., E.R.K.) and
Department of Animal Sciences (E.D.) Katholieke Universiteit
Leuven B-3000 Leuven, Belgium
 |
ABSTRACT
|
---|
Intracellular trafficking of GH and its receptor,
more particularly the chicken GH receptor (cGHR), was examined in COS-7
cells using biochemical and structural studies. Internalization of
radioactive GH by the cGHR is reduced as compared with the rat GHR. On
the contrary, activation of gene transcription through Janus kinase-2
was similar for both species. Secondary structures of the cytoplasmic
domain of chicken and rat GHR were compared, since ß-turns were
reported as internalization signals. The substitution of
Pro335-Asp336, present
in mammalian GH receptors, with
Thr307-Gln308 in the
cGHR leads to the loss of a ß-turn within a conserved cytoplasmic
region. Mutational analysis indicated that the lower rate of
internalization of cGHR, as compared with mammalian GHR, was due to
this motif. Our data further show that
-adaptin, a subunit of
adaptor protein AP-2, associates with the GHR upon hormone stimulation.
The clathrin-coated pit pathway therefore seems to be involved in the
endocytosis of cGHR, as AP-2 is known to intervene in the recruitment
of receptors to these pits. Interaction with
-adaptin may occur
through a common epitope of the chicken and mammalian GHR, since
receptors from both species bind similar amounts of
-adaptin;
alternatively, two different epitopes with similar affinity may be
involved. Therefore, not
-adaptin but an uncharacterized factor,
presumably interacting with the identified ß-turn endocytic code, is
responsible for the difference in internalization kinetics. Finally,
the present study illustrates that functional amino acid motifs of
receptors can be derived from comparative studies.
 |
INTRODUCTION
|
---|
GH is an important regulator of growth and differentiation in a
variety of tissues and species. The receptor mediating GH action (GHR)
belongs to the hematopoietic receptor superfamily (1). In addition to
the transmembrane GHR, a soluble GH-binding protein (GHBP) has been
identified, first in mammals and later in chickens (1, 2). Dimerization
of a single GH molecule with two receptors appears to be the first step
in hormone-receptor interaction (3). Subsequently, a cytoplasmic
tyrosine kinase, Janus kinase 2 (JAK2), associates with each GHR, and
JAK2 and GHR molecules are tyrosyl phosphorylated (4). Further
downstream the signal transduction pathway, proteins that couple ligand
binding to activation of gene transcription, signal transducers and
activators of transcription (STAT proteins), are phosphorylated
(1).
A characteristic of receptors for polypeptide hormones is their ability
for receptor-mediated endocytosis (5). Internalization starts with the
recruitment of hormone-receptor complexes into clathrin-coated pits by
AP-2, a plasma membrane-specific, heterotetrameric adaptor protein (6).
Several cytoplasmic motifs, called endocytic codes or internalization
signals, are required for efficient internalization and/or AP-2
association (7, 8). Studies with rat GHR (rGHR) deletion mutants have
indicated that the first 90 amino acid (aa) residues of the cytoplasmic
domain contain endocytic codes, of which Phe346 has been
identified (9). A Leu-pair and a tetrapeptide predicted to adopt a
ß-turn were suggested to be internalization signals for the short
isoform of the rat PRL receptor (srPRLR) (10). Ligand-mediated
endocytosis, however, appears not to be a prerequisite for GH-induced
gene transcription mediated by the JAK/STAT signal transduction pathway
(9).
Our interest in the internalization of GH and GHR arose from studies in
which hepatic GHR capacity was reduced after injecting GH into
hypophysectomized chickens (11). Fractionation experiments of adult hen
liver indicated that a major portion of GHR was allocated to an
intracellular compartment (12). A detailed in vitro study of
internalization kinetics, the first in a nonmammalian species, became
possible by the cloning of the chicken GHR (cGHR) (13). Moreover,
analysis of the GHR amino acid sequence suggested that an endocytic
code identified in the srPRLR (10) was present in mammalian GH
receptors but not in the cGHR, making the cGHR an interesting model for
the study of receptor-mediated endocytosis.
 |
RESULTS
|
---|
Characterization of Binding to cGHR
COS-7 cells were transiently transfected using the cDNA encoding
the full-length cGHR cloned in pSG5, a eukaryotic expression vector.
Results from specific binding of [125I]cGH and
[125I]hGH to whole cells are presented in Fig. 1
. By Scatchard analysis, properties of
binding were calculated. Since the heterologous human GH (hGH) is a
better competitor of [125I]cGH than cGH,
[125I]hGH was used for further experiments. Dissociation
constant (Kd) for hGH is 0.50 ± 0.06 nM
(n = 4), which is within the range reported for binding to the GHR, in
a similar transfection study or in hepatic microsomal fractions
(14, 15, 16). On the average, 1,000,000 binding sites for hGH are found per
transfected cell.

View larger version (18K):
[in this window]
[in a new window]
|
Figure 1. Characterization of Binding to cGHR
COS-7 cells were transiently transfected with 50 ng cGHR cDNA. Intact
cells were incubated with [125I]GH and varying
concentrations of unlabeled GH. Cells were incubated overnight at 4 C.
At the end of incubation, cell-bound radioactivity was recovered by
lysis with 1 N NaOH. a, Competition of
[125I]cGH from the cGHR by a range of unlabeled cGH ()
or hGH ( ) concentrations. b, The data in panel a were used to
perform Scatchard analysis.
|
|
Internalization of GH by cGHR and rGHR
To examine the kinetics of GH uptake, we compared the
internalization of [125I]hGH in COS-7 expressing the cGHR
or rGHR (Fig. 2
). At t = 0 min, cell
surface binding values for rGHR and cGHR are identical, 17% and 20%,
respectively. Internalization by the cGHR is slower as compared with
the rGHR. After 15 min at 37 C, label internalized through the cGHR is
barely detectable, whereas internalization in the rGHR-transfected
cells has already started (P < 0.01). After 1 h
at 37 C, internalization by the cGHR is 40% lower than by the rGHR
(P < 0.0001). When cGHR-transfected cells are left
longer at 37 C, the curve flattens and does not attain rat maximal
levels (results not shown). Labeled hGH and cGH are internalized
equally by the cGHR (results not shown).

View larger version (17K):
[in this window]
[in a new window]
|
Figure 2. Internalization of Surface-Bound GH by cGHR or rGHR
COS-7 cells expressing cGHR () or rGHR ( ) were incubated with
[125I]hGH at 4 C, washed, and subsequently transferred to
37 C to start internalization. At various time points, cells were
washed with acidic buffer to remove surface-bound radioactivity and
lysed to determine intracellular radioactivity. Internalization is
expressed as the percentage of specific bound counts at time 0 and is
corrected for nonspecific internalization. Each point represents the
mean (±SEM) of duplicate measurements in four to six
independent experiments.
|
|
Secondary Structure Prediction of GHR Amino Acid Sequences
To further investigate the mechanism behind the reduced
internalization rate of cGHR as compared with rGHR, the primary and
secondary structures of the first 90 residues from the cytoplasmic
domain of both sequences were compared (Fig. 3
). In the rGHR, seven tetrapeptides are
predicted to adopt a ß-turn configuration by the Chou-Fasman
algorithm (17). In the rGHR, as well as in the cGHR, the first ß-turn
is predicted with the highest probability and ends in two vicinal Leu
(Asp311-Pro312-Asp313-Leu314-Leu315).
The next three ß-turns are positioned between aa 330 and 344 and are
part of one region contiguous with Phe346. However, the
middle ß-turn of these three is not present in chicken due to a
triple mutation. In this ß-turn, a Pro is in second place in the
rGHR, which, of all amino acids, has the strongest ß-turn potential
on this position. Consequently, replacing Pro335 by
Thr307 reduces ß-turn potential of the cGHR. As the third
residue Asp336 is changed to Gln308, a ß-turn
becomes even more improbable. The fourth residue does not affect
ß-turn potential.

View larger version (26K):
[in this window]
[in a new window]
|
Figure 3. Predicted Potential for ß-Turn Formation of the
Cytoplasmic Domain of cGHR and rGHR
Predicted potential for a ß-turn encompassing four residues starting
at position i is calculated from the product of bend frequencies (f):
p(t)= fi x fi+1 x
fi+2 x fi+3. The conformational
parameters Pt, P , and Pß,
express the relative frequency of a specific amino acid to occur in a
ß-turn, -helix, or ß-sheet, respectively, and are averaged over
the four residues ({Pt}, {P } and
{Pß}). A ß-turn is predicted if p(t) >
0.75 x 10-4, {Pt} > 1,
{P } < {Pt} >
{Pß}, and pi-1(t) <
pi(t) > pi+1(t). The signal peptide (16
amino acids) is included when numbering residues. The extracellular
domain of the cGHR is 28 amino acids shorter as compared with mammalian
GHRs due to the absence of the exon 3 homolog of the hGHR (15 ). a,
rGHR. b, cGHR.
|
|
Characterization of Flag-Tagged cGHR Construct
To enable immunoprecipitation and immunodetection of the cGHR with
high efficiency, we added an 8-aa-long Flag epitope to the N terminus
of the mature cGHR (Flag-cGHR). A cDNA fragment encoding the cGHR was
generated by PCR and subcloned into the expression vector pFlag-CMV-1,
immediately after the Flag epitope. For functional studies (activation
of gene transcription, JAK/STAT pathway), cytomegalovirus (CMV)
promoter-containing plasmids are usually transfected in 293 cells,
because they give consistent results in our laboratory (18, 19, 20, 21) as well
as other laboratories (22, 23). On the other hand, since COS cells have
been previously used to study GHR internalization, we used this cell
line for internalization studies. It has been established that
simian virus 40 (SV40) promoter-containing plasmids are usually
transfected in this cell line because it contains the large T antigen.
We did not find a significant difference between the level of GHR
expressed using SV40 or CMV promoters in COS cells as determined by
Scatchard analysis (Table 1
), in
agreement with previous results (20). Signal transduction and
activation of transcription are similar for the wild-type and
Flag-tagged GHR as well. GH-dependent induction of luciferase activity
is used to assay bioactivity. Furthermore, indirect immunofluorescence
studies using Flag-cGHR-transfected cells showed that the
epitope-tagged receptor is internalized to cell compartments that
participate in the endocytic pathway (data not shown). Indeed, previous
studies with N- and C-terminally Flag-tagged PRLR have shown that
epitope tagging does not significantly change bioactivity, binding
properties, or internalization behavior (24).
GH-Dependent Induction of Phosphorylation of JAK-2 and STAT5
The cGHR activates the JAK/STAT pathway, as indicated by the
GH-dependent induction of the luciferase reporter gene (Table 1
).
Phosphorylation on tyrosine residues of JAK2 and STAT5 was analyzed for
the rat and chicken GHR (Fig. 4
). JAK2- and
STAT5-containing complexes were precipitated from cell lysates using
specific anti-JAK2 or anti-STAT5 antibodies. Tyrosine phosphorylation
was then evaluated by SDS-PAGE in reducing conditions, followed by
probing of the obtained blots with a specific monoclonal
antiphosphotyrosine antibody. Tyrosyl phosphorylation of JAK2
(Mr 130,000) is induced by GH, as early as 15 min after GH
addition. Later on, GH-dependent tyrosyl phosphorylation of JAK2
decreases. Similarly, activation of STAT5 (Mr 95,0000) by
GH is maximal at 15 min. Although internalization of the cGHR is
reduced, this does not inhibit the initial steps of signal transduction
by the JAK/STAT pathway, i.e. activation of tyrosine kinase
JAK2 and of STAT5 during the same time course. On the contrary, tyrosyl
phosphorylation of STAT5 appears prolonged for the cGHR, as compared
with rabbit GHR (rbGHR).

View larger version (28K):
[in this window]
[in a new window]
|
Figure 4. GH-Induced Tyrosine Phosphorylation of JAK2 and
STAT5 by cGHR and rGHR
Cells, transiently expressing GHR, were stimulated or not with 50
nM hGH for 1560 min; whole-cell lysates were prepared; an
anti-JAK2 or anti-STAT5 antibody was used for immunoprecipitation (IP);
precipitates were subsequently analyzed by SDS-PAGE; proteins
transferred to membrane and blots were probed with antiphosphotyrosine
( -PY). In parallel experiments, the antibody used for IP was used
for blotting, identifying the induced protein band and indicating that
similar amounts of JAK2 and STAT5 proteins are present in each lane.
|
|
Involvement of
-Adaptin in GHR Internalization
To establish the involvement of coated pits in endocytosis by the
cGHR, we used Western blot analysis to study the interaction between
cGHR and
-adaptin, a component of adaptor protein AP-2 (Fig. 5
). COS-7 cells expressing either Flag-cGHR
or a mammalian GHR [Flag-tagged rbGHR (Flag-rbGHR)] were analyzed for
their association with
-adaptin upon hormone stimulation. Immunoblot
analysis of Flag-GHR complexes that were purified using
immunoprecipitation with an anti-Flag antibody reveals that a specific
band of apparent Mr of 100,000 corresponds to
-adaptin
and that it displays a GH-dependent association with the GHR. The
amount of
-adaptin associated with the cGHR is at least as large as
that associated with the rbGHR.
Internalization Study of rGHR and cGHR Mutants
The involvement in receptor-mediated endocytosis of the ß-turn
identified in previous experiments with the cGHR and rGHR was further
tested by mutational analysis. A mutant Flag-cGHR was constructed in
which the defective ß-turn was replaced by the respective residues
present in the rGHR (Thr307-Gln308
Pro307-Asp308). Mutation of the potential
endocytic code did not affect binding characteristics of hGH
(Kd, 0.4 nM; 21% specific binding; on the
average 910,000 binding sites for hGH are found per transfected cell).
Notwithstanding the different endocytic behavior of the rat and
wild-type chicken GHR, internalization rates of the mutant cGHR and the
rGHR were similar, confirming the relevance of the mutated ß-turn for
receptor-mediated endocytosis of GH (Fig. 6
).
Activation of gene transcription by the JAK/STAT pathway was identical
(luciferase activity induction factor of 9.1 ± 0.8 for wild-type
cGHR vs. 9.5 ± 0.5 for
Pro307-Asp308 cGHR mutant). The association of
the mutant to
-adaptin was similar to that of wild-type cGHR (data
not shown). Furthermore, the pivotal role of Pro335 for
optimal internalization of the rGHR was corroborated by alanine
scanning of the
Lys334-Pro335-Asp336-Phe337
tetrapeptide in the rGHR. The Pro335
Ala335
rGHR mutant internalized less (t = 60 min, P <
0.01) than Lys334
Ala334,
Asp336
Ala336, and Phe337
Ala337 mutants that are very similar in internalization
compared with the wild-type rGHR (Fig. 7
).

View larger version (16K):
[in this window]
[in a new window]
|
Figure 6. Internalization of Surface-Bound GH by Wild-Type or
P307D308 Mutated cGHR
See Fig. 3 for protocol details. Each point represents the mean
(±SEM) of duplicate measurements in three independent
experiments.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Figure 7. Internalization of Surface-Bound GH by rGHR Mutants
Generated by Alanine Scanning of the Putative ß-Turn Internalization
Motif
See Fig. 3 for protocol details. Each point represents the mean
(±SEM) of duplicate measurements in four independent
experiments.
|
|
 |
DISCUSSION
|
---|
In this report, kinetics and mechanisms of GH internalization by
receptor-mediated endocytosis are described. In the absence of a
conserved ß-turn in the juxtamembranous intracellular domain,
internalization is strongly reduced. Furthermore, a more detailed
understanding of endocytosis was obtained by studying the interaction
of a GHR with
-adaptin, a subunit of the AP-2 complex that initiates
formation of clathrin-coated pits (6). The identified endocytic code
was not involved in ligand binding,
-adaptin association, or
activation of JAK/STAT.
Although internalization of the cGHR, as compared with mammalian GH
receptors, was reduced, gene transcription through the JAK/STAT signal
transduction pathway was activated by the cGHR. Transcriptional
activity mediated by STAT5 was evaluated with a reporter gene. The gene
construct contains lactogenic hormone response element (LHRE) coupled
to the thymidine kinase minimal promoter and the luciferase gene. LHRE
is the element of the ß-casein promoter that was used for affinity
purification of STAT5 (25). Moreover, the level and timing of tyrosine
phosphorylation of JAK2 of the cGHR and rGHR did not differ. Tyrosyl
phosphorylation of STAT5 seemed even prolonged. If the cGHR has a
longer half-life on the plasma membrane, then STAT5 has more time to
bind to phosphorylated tyrosines within the GHR and will be more
phosphorylated by JAK2. Distinction of internalization capacity and
signal transduction was previously evaluated at the molecular level;
different cytoplasmic regions of the GHR sequence are responsible for
these processes. Proximal to the membrane, the GHR contains a Pro-rich
region, called Box 1 (aa 297311 in rGHR), that is conserved in the
cytokine/GH/PRL receptor family and is required for signal transduction
by the JAK/STAT pathway (16). On the other hand, the cytoplasmic region
of the rGHR ranging from amino acid residue 318380 is involved in
internalization. More specifically, Phe346 was identified
as being critical for GH-dependent internalization of the rGHR, but not
for activation of gene transcription by JAK/STAT (9).
Sequences of internalization signals were first elucidated for the
low-density lipoprotein receptor, the transferrin receptor, and the
cation-independent mannose-6-phosphate receptor. Although endocytic
codes of these receptors differ in specific sequence, they all share a
common three-dimensional conformation and chemistry and form tight (or
ß-) turns. For the transferrin receptor, it was reported that a
Tyr-containing ß-turn internalization signal could be replaced by
Leu-Leu, which suggests that these two signals are functionally
equivalent (7). Recently it was reported, however, that the di-Leu
motif is solely involved in the internalization of truncated GHR or
PRLR (srPRLR), but not of the full-length GHR (26).
Secondary structure analysis of the juxtamembranous cytoplasmic region
of the GHR with high interspecies homology revealed the presence of
several peptide motifs, predicted to adopt a ß-turn configuration,
the first tight turn having the highest probability found in our
analyzed region. In the region important for efficient internalization
by the rGHR (aa 318380), three contiguous ß-turn tetrapeptides
occur within a highly conserved stretch of 15 aa. The middle ß-turn
of this trio, however, is not present in chicken, as Pro307
and Asp308 are replaced in cGHR by residues with less
ß-turn potential. The fourth residue, Phe309, is not
conserved either, but this mutation does not affect ß-turn
probability. Moreover, by Ala mutation, Phe309 was
previously shown not to be required for internalization (9). Finally,
the rGHR Phe346 identified previously as an internalization
motif (9) is conserved in the cGHR. The reduced internalization
capacity of the cGHR may therefore be due to the different
three-dimensional structure of the receptor molecule, which hinders
endocytosis. The current vision is that these ß-turn internalization
motifs are exposed on the surface of the cytoplasmic tail, enabling
interaction with adaptor molecules (7). Deletion of a similar motif
(Leu-Pro-Gly-Gly) in the srPRLR caused a 50% reduction in
internalization (10), which corresponds to the diminished
internalization of cGHR, as compared with rGHR. The remaining
internalization capacity of the cGHR could then be due to endocytic
codes common to both species.
Little is known about the underlying mechanisms controlling GHR
internalization and trafficking. In general, receptor-mediated
endocytosis involves the concentration of receptors in clathrin-coated
pits with the help of a plasma membrane-associated adaptor protein
AP-2. This protein complex consists of four subunits:
2- and
ß-adaptin of 100 kDa, a µ2-chain of 50 kDa, and a small
2-chain
of 16 kDa (6). Upon ligand stimulation,
-adaptin associated with the
srPRLR (10). Our results demonstrate, for the first time, the
GH-inducible binding of a GHR to
-adaptin.
Furthermore, we compared the interaction of
-adaptin with chicken or
mammalian GHRs, in view of the species-dependent internalization
behavior. As both receptors bind similar amounts of
-adaptin, our
experiments indicate that cytoplasmic motifs required for
internalization and for
-adaptin binding are not necessarily the
same. Further experiments are necessary to determine which region(s) of
the GHR is (are) responsible for
-adaptin association. In
vitro studies showed that Tyr-containing sorting signals interact
with the µ2-subunit of AP-2 (27). The existence of distinct motifs
for internalization and AP-2 association, respectively, was reported
for the receptor of epidermal growth factor. Kinetics of
internalization of a mutant receptor for epidermal growth factor
lacking the Tyr-containing AP-2 binding site were indistinguishable
from those of its wild-type counterpart and were independent of AP-2
(28).
Although there is no doubt that AP-2 is involved in clathrin-dependent
endocytosis, the previous and present studies suggest that
-adaptin
may be necessary, but not sufficient, for a maximal response. The
ß-turn lost in cGHR may interact with ß-arrestin that induced a
concentration of the ß2-adrenergic receptor in
clathrin-coated pits (29). Furthermore, mammalian and chicken GHR may
differ in ubiquitin association. Recently it was shown that GHR
ubiquitination is not only a prerequisite for GHR degradation, but also
for ligand-dependent endocytosis (30). Moreover, the ubiquitin
conjugation and ligand-induced internalization are coupled events,
since they are both disrupted by the F345A mutation (31). Finally, GHBP
may interfere with GHR internalization. The culture medium of cells
transfected with the cGHR contains large amounts of GHBP, since cGHBP
is generated by proteolysis of the full-length GHR (32), contrary to
rGHBP that is not detectable in medium of rGHR-transfected cells (33).
The formation of GH.GHR.GHBP heterotrimers may hinder internalization
of the cGHR.
In conclusion, our study shows that 1) the cGHR is internalized, a
process presumably mediated by the clathrin-coated pits endocytic
pathway, 2) reduced internalization of the cGHR as compared with
mammalian GH receptors is related to the loss of a conserved
cytoplasmic internalization motif predicted to adopt a ß-turn, and 3)
the level of internalization is not correlated to the level of
-adaptin association or activation of gene transcription.
 |
MATERIALS AND METHODS
|
---|
Hormones and Expression Vectors
Recombinant hGH was kindly provided by Serano Ares Laboratories
(Geneva, Switzerland). Recombinant cGH was a generous gift of
Ciba-Geigy (Basel, Switzerland) and natural cGH was a gift
of Dr. Luc Berghman (K.U. Leuven, Belgium) (34). [125I]GH
was prepared as described previously [hGH (35), recombinant cGH (11)]
to a specific activity of 80140 µCi/µg.
Dr. J. Burnside (University of Delaware, Newark, DE) kindly
provided the cDNA encoding the cGHR cloned in the pSG5 expression
vector that is under SV40 transcriptional control (36). The rGHR
expression plasmid pLM108, a pUC8 construct containing the human
metallothionein IIa promoter and SV40 enhancer, was donated by Dr. G.
Norstedt (Center for Biotechnology, Karolinska Institute, NOVUM,
Huddinge, Sweden) (37).
Construction of Flag-Tagged cGHR
To enhance immunoprecipitation and immunodetection, the cGHR was
epitope-tagged at the N terminus with an octapeptide, named Flag
(Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys). Monoclonal antibodies directed
against the Flag-epitope are commercially available (Eastman Kodak Co., Rochester, NY). The cDNA encoding the mature
wild-type cGHR was inserted in the pFlag-CMV-1 expression vector
(Eastman Kodak Co.), after the preprotrypsin signal
peptide sequence that ensures correct membrane-bound expression. By
oligonucleotide-directed mutagenesis using PCR, a cGHR insert was
produced, excluding the original signal peptide (the first 16 aa) and
introducing at the 5'- and 3'-end unique restriction enzyme sites
recognized by NotI and XbaI. An N-terminally
Flag-tagged cGHR construct (pFlag-cGHR) was obtained after enzyme
digestion, ligation, and ampicillin selection in Escheria
coli DH5 cells. The presence of the Flag epitope in the engineered
cGHR was confirmed by dideoxynucleotide sequence analysis (38). The
rbGHR inserted in pFlag-CMV-1 vector (pFlag-rbGHR) was obtained from S.
Moutoussamy (INSERM U344) (22).
Construction of Flag-Tagged cGHR (T307P,Q308D) and rGHR Mutants
(K334A; P335A; D336A; F337A)
Substitutions of Thr307 and Gln308 by
Pro and Asp, respectively, were obtained using the Flag-CMV-1-cGHR
plasmid. A single-stranded DNA was generated by using the origin of
replication of the M13 phage present in the vector, in the
Escheria coli CJ236 strain in the presence of M13K07 helper
phage. This single-stranded DNA was then used as a template for
oligonucleotide-directed mutagenesis with the primer
5'-CATTGTATAGGTCTGGCTTGTAGTTG-3'. A similar strategy was used to
construct K334A, P335A, D336A, and F337A mutants. The modified regions
were verified by sequencing (38).
Cell Culture and Transfection
COS-7 cells were grown as monolayers in DMEM containing 10%
FCS, 2 mM L-glutamine, 50 IU/ml penicillin, and
50 µg/ml streptomycin. Routinely, cells were cultured at 37 C, in a
humid 5% CO2 incubator. At 7080% confluence, cells were
transfected by the diethylaminoethyl-dextran/chloroquine method. Four
hours after addition of DNA precipitates, cells were subjected to
Me2SO shock, washed, and cultured in fresh complete medium
for 48 h (10).
Analysis of GHR Binding
Hormone binding to transfected COS-7 cells was performed in
six-well plates. Before binding, cells were washed with DMEM and kept
in serum-free medium for at least 4 h. Subsequently, plates were
put on ice and washed with ice-cold HEPES binding buffer (HBB: 25
mM HEPES, 124 mM NaCl, 4 mM KCl, 1
mM CaCl2, 1.5 mM MgCl2,
and 2 mM KH2PO4, pH 7.4). In a
final volume of 1 ml HBB containing 1% BSA (Fraction V, Sigma Chemical Co., St. Louis, MO) (HBB-BSA) whole cells were
incubated with 80,000100,000 cpm [125I]GH. At the end
of incubation, unbound label was removed by two HBB washes; 1 ml 1
N NaOH was added, and radioactivity in lysates was measured
using a
-counter. Specific binding was determined by subtracting the
amount of [125I]GH bound in the presence of excess
unlabeled GH (91 nM). For Scatchard analysis, increasing
amounts of unlabeled GH were added to compete with
[125I]GH for binding in saturating conditions (overnight
at 4 C). With LIGAND software (Elsevier-BioSOFT, Cambridge, UK),
Kd and binding capacity were calculated (39). Data are
represented as means ± SEM.
Internalization Studies
Internalization was analyzed as described by Allevato et
al. (9). Briefly, cells transfected with 0.11 µg cDNA were
treated as described for binding analysis. Cells were incubated with
70,000100,000 cpm [125I]GH at 4 C to bind cell surface
receptors (hGH for 2.5 h, cGH for 6 h); the low temperature
prevented label internalization. Unbound ligand was removed by washing
twice with ice-cold HBB; culture plates containing 1 ml HBB-BSA were
then transferred to 37 C for various times; surface-bound
[125I]GH was removed by a 3-min exposure to an acid wash
buffer (150 mM NaCl and 50 mM glycine, pH 2.5),
and cells were lysed to recover acid-resistant binding. For each time
point, internalization was expressed as the percentage of specific
intracellular radioactivity toward specific binding at t = 0 min.
Data were statistically analyzed with SAS software (SAS Institute, Inc., Cary, NC) using the general linear model
procedure and are represented as means ± SEM.
Secondary Structure Prediction
The occurrence of ß-turns was predicted using the Chou-Fasman
algorithm (17). ß-Turns are chain-reversal regions consisting of
tetrapeptides. Based on x-ray crystallography data of 29 proteins, the
average overall frequencies to be part of an
-helix
(<f
> = 0.38), a ß-sheet (<fß> =
0.20), or a ß-turn (<ft> = 0.32) were determined.
Conformational parameters Pt, P
, and
Pß were obtained for all 20 amino acids, by expressing
their frequency (f
, fß, and
ft) relative to the respective average frequency. Strong
ß-turn formers are Asn, Gly, and Pro, all with Pt >
1.50. In addition, the bend frequency (f) of each amino acid was
calculated for the four positions of the ß-turn. The probability of a
bend starting at residue i is then p(t) = fi x
fi+1 x fi+2 x fi+3. To
predict whether a tetrapeptide is a ß-turn or rather part of an
-helix coil or ß-sheet, Pt, P
, and
Pß are averaged over the four residues. A ß-turn is
predicted if p(t) > 0.75 x 10-4,
{Pt} > 1 and {P
} <
{Pt} > {Pß}.
Luciferase Bioassay
To test biological activity of Flag-tagged and wild-type
receptors, a 293-cell bioassay was used (40). A 6-well plate was
transfected by the calcium phosphate technique using, per well, 33 ng
of receptor plasmid, 17 ng of a ß-galactosidase expression vector
(pCH110, Pharmacia Biotech, Uppsala, Sweden), and 250 ng
of LHRE-tk-Luc. The latter is a construct that contains a GH-responsive
promoter [thymidine kinase (tk) minimal promoter and 6 repeats of the
LHRE of the ß-casein promoter] fused to the firefly luciferase
reporter gene. One day after transfection, cells were incubated
overnight with 0 or 23 nM hGH serum-free medium. Cell
extracts were prepared and enzyme activities were determined. Activity
of ß-galactosidase was used to normalize luciferase expression levels
for differences in transfection efficiency.
Immunoprecipition and Western Blot Analysis
COS-7 cells were grown on 100-mm dishes, transfected with
Flag-tagged receptor plasmid (1 µg/dish), cultured overnight in
serum-free medium, and stimulated (or not) with 45 nM hGH
(20 min at 37 C). Cellular proteins were extracted in 1 ml of lysis
buffer (50 mM Tris, 2 mM CaCl2, 100
mM NaCl, 8% glycerol, 0.8% Triton X-100, pH 7.6)
containing phosphatase and protease inhibitors (1 mM
o-Na3VO4, 1 mM
phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 2 µg/ml
leupeptin, 1 µg/ml pepstatin). Lysates were incubated overnight at 4
C with agarose beads bearing Flag monoclonal antibodies (M1) for
precipitation of receptor complexes. Immunoprecipitated complexes were
washed with fresh cold lysis buffer, boiled in SDS sample buffer, and
subjected to 7.5% SDS-PAGE. Proteins were then transferred onto a
polyvinylidene difluoride membrane (PVDF, Polyscreen, Dupont NEN, Boston, MA), and blots were incubated for 2 h at room
temperature with monoclonal antibodies directed against either Flag
(M2, 0.5 µg IgG/ml) or
-adaptin (AC1-M11, 1:100, Affinity BioReagents, Inc. Neshanic Station, NJ). Finally, membranes
were incubated for 1 h with alkaline phosphatase-linked goat
antimouse secondary antibody (1:10,000), and proteins were revealed
using the Vistra ECF Western blotting system (Amersham Pharmacia Biotech, Little Chalfont, UK).
To evaluate JAK2 activation by GH, 293 cells were cotransfected with
GHR (4 µg) and human JAK2 (2 µg) cDNA. For the determination of
GH-dependent induction of STAT5 tyrosyl-phosphorylation, cells were
transfected with GHR (4 µg/ml, JAK2 (0.1 µg/ml), and STAT5 (2
µg/ml) cDNA. JAK2 and STAT5 expression vectors were kindly provided
by Drs. J. Ihle and B. Groner, respectively. Cell lysates were
incubated with the designated antibody [anti-JAK2 (1 µg/ml;
Upstate Biotechnology, Inc., Lake Placid, NY) or
anti-STAT5 (1 µg/ml; Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA)] and protein A-Sepharose (50%, vol/vol). Finally,
immunoprecipitated complexes were analyzed by Western blot, using a
monoclonal antiphosphotyrosine (4G10; Upstate Biotechnology, Inc.; 1:4,000), a polyclonal anti-JAK2 antibody (1:5,000), or a
monoclonal anti-STAT5 (1:1,000) antibody. Finally, the membranes were
incubated with an antirabbit or antimouse IgG-conjugated horseradish
peroxidase (1:8,000) and revealed by the enhanced chemiluminesence
(ECL) detection system (Amersham Pharmacia Biotech).
 |
ACKNOWLEDGMENTS
|
---|
The authors are grateful to W. Van Ham for iodinating cGH, Dr.
H. Buteau for plentiful advice, and S. Kotanen for help in manuscript
preparation.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Lieve Vleurick, 13 Avenue Maréchal Juin, B-3000 Gembloux, Belgium.
This work was supported by the Fund for Scientific Research-Flanders
(Belgium) (F.W.O. G.0235.97).
Received for publication October 19, 1998.
Revision received July 3, 1999.
Accepted for publication July 26, 1999.
 |
REFERENCES
|
---|
-
Finidori J, Kelly PA 1995 Cytokine receptor signalling
through two novel families of transducer molecules: Janus kinases, and
signal transducers and activators of transcription. J Endocrinol 147:1123[Medline]
-
Vasilatos-Younken R, Andersen BJ, Rosebrough RW, McMurtry JP,
Bacon WL 1991 Identification of circulating growth hormone-binding
proteins in domestic poultry: an initial characterization. J Endocrinol 130:115122[Abstract]
-
Cunningham BC, Ultsch M, de Vos AM, Mulkerrin MG, Clauser KR,
Wells JA 1991 Dimerization of the extracellular domain of the human
growth hormone receptor by a single hormone molecule. Science 254:821825[Medline]
-
Argetsinger LS, Campbell GS, Yang X, Witthuhn BA,
Silvennoinen O, Ihle JN, Carter-Su C 1993 Identification of JAK2 as a
growth hormone receptor-associated tyrosine kinase. Cell 74:237244[Medline]
-
Posner BI, Bergeron JJM, Josefsberg Z, Khan MN, Khan RJ,
Patel BA, Sikstrom RA, Verma AK 1981 Polypeptide hormones:
intracellular receptors and internalization. Recent Prog Horm Res 37:539582[Medline]
-
Robinson MS 1994 The role of clathrin, adaptors and dynamin
in endocytosis. Curr Opin Cell Biol 6:538544[Medline]
-
Trowbridge IS, Collawn JF, Hopkins CR 1993 Signal-dependent
membrane protein trafficking in the endocytic pathway. Annu Rev Cell
Biol 9:129161[CrossRef]
-
Kirchhausen T, Bonifacino JS, Riezman H 1997 Linking cargo to
vesicle formation: receptor tail interactions with coat proteins. Curr
Opin Cell Biol 9:488495[CrossRef][Medline]
-
Allevato G, Billestrup N, Goujon L, Galsgaard ED, Norstedt G,
Postel Vinay M-C, Kelly PA, Nielsen JH 1995 Identification of
phenylalanine 346 in the rat growth hormone receptor as being critical
for ligand-mediated internalization and down-regulation. J Biol
Chem 270:1721017214[Abstract/Free Full Text]
-
Vincent V, Goffin V, Rozakis-Adcock M, Mornon J-P, Kelly PA 1997 Identification of cytoplasmatic motifs required for short
prolactin receptor internalization. J Biol Chem 272:70627068[Abstract/Free Full Text]
-
Vanderpooten A, Darras VM, Huybrechts LM, Rudas P, Decuypere
E, Kühn ER 1991 Effect of hypophysectomy and acute administration
of growth hormone (GH) on GH-receptor binding in chick liver membranes.
J Endocrinol 129:275281[Abstract]
-
Vleurick L, Van Veldhoven P, Decuypere E, Kühn ER 1998 Intracellular growth hormone receptors in chicken liver. In: Vaudry H,
Tonon M-C, Roubos EW, De Loof A (eds) Trends in Comparative
Endocrinology and Neurobiology. Ann NY Acad Sci 839:538540[Free Full Text]
-
Burnside J, Liou SS, Cogburn LA 1991 Molecular cloning of the
chicken growth hormone receptor complementary deoxyribonucleic acid:
mutation of the gene in sex-linked dwarf chickens. Endocrinology 128:31833192[Abstract]
-
Kühn ER, Huybrechts LM, Vanderpooten A, Berghman L 1989 A decreased capacity of hepatic growth hormone (GH) receptors and
failure of thyrotrophin-releasing hormone to stimulate the peripheral
conversion of thyroxine into triiodothyronine in sex-linked dwarf
broiler hens. Reprod Nutr Dev 29:461467[Medline]
-
Burnside J, Cogburn LA 1993 Molecular biology of the chicken
growth hormone receptor. In: Sharp PJ (ed) Avian Endocrinology. Journal
of Endocrinology Ltd, Bristol, UK, pp 161176
-
Goujon L, Allevato G, Simonin G, Paquereau L, Le Cam A, Clark
J, Nielsen JH, Djiane J, Postel-Vinay M-C, Edery M, Kelly PA 1994 Cytoplasmatic sequences of the growth hormone receptor necessary for
signal transduction. Proc Natl Acad Sci USA 91:957961[Abstract]
-
Chou PY, Fasman GD 1978 Emperical predictions of protein
conformation. Annu Rev Biochem 47:251276[CrossRef][Medline]
-
Martini J-F, Pezet A, Guezennec CY, Edery M, Postel-Vinay M-C,
Kelly PA 1997 Monkey growth hormone (GH) receptor gene expression:
evidence for two mechanisms for the generation of the GH binding
protein. J Biol Chem 272:18511858
-
Pezet A, Buteau H, Kelly PA, Edery M 1997 The last proline of
Box 1 is essential for association with JAK2 and functional activation
of the prolactin receptor. Mol Cell Endocrinol 129:199208[CrossRef][Medline]
-
Buteau H, Pezet A, Ferrag F, Perrot-Applanat M, Kelly PA,
Edery M 1998 N-Glycosylation of the prolactin receptor is not required
for activation of gene transcription but is crucial for its cell
surface targeting. Mol Endocrinol 12:544555[Abstract/Free Full Text]
-
Moutoussamy S, Renaudie F, Lago F, Kelly PA, Finidori J 1998 Grb10 identified as a potential regulator of growth hormone (GH)
signaling by cloning of GH receptor target protein. J Biol Chem 273:1590615912[Abstract/Free Full Text]
-
Endo TA, Masuhara M, Yokouchi M, Suzuki R, Sakamoto H, Mitsui
K, Matsumoto A, Tanimura S, Ohtsubo M, Misawa H, Miyazaki T, Leonor N,
Taniguchi T, Fujita T, Kanakura Y, Komiya S, Yoshimura A 1997 A new
protein containing an SH2 domain that inhibits JAK kinases. Nature 387:921924[CrossRef][Medline]
-
Ali S 1998 Prolactin receptor regulates STAT5 tyrosine
phosphorylation and nuclear translocation by two separate pathways.
J Biol Chem 273:77097716[Abstract/Free Full Text]
-
Perrot-Applanat M, Gualillo O, Buteau H, Edery M, Kelly PA 1997 Internalization of prolactin receptor and prolactin in transfected
cells does not involve nuclear translocation. J Cell Sci 110:11231132[Abstract/Free Full Text]
-
Wakao H, Gouilleux F, Groner B 1994 Mammary gland factor (MGF)
is a novel member of the cytokine regulated transcription factor gene
family and confers the prolactin response. EMBO J 13:21822191[Abstract]
-
Govers R, van Kerkhof P, Schwartz AL, Strous GJ 1998 Di-leucine-mediated internalization of ligand by a truncated growth
hormone receptor is independent of the ubiquitin conjugated system.
J Biol Chem 273:1642016433
-
Ohno H, Stewart J, Fournier MC, Bosshart H, Rhee I, Miyatake
S, Saito T, Gallusser A, Kirchhausen T, Bonifacino JS 1995 Interaction
of tyrosine-based sorting signals with clathrin-associated proteins.
Science 269:18721875[Medline]
-
Sorkin A, Mazzotti M, Sorkina T, Scotto L, Beguinot L 1996 Epidermal growth factor receptor interaction with clathrin adaptors is
mediated by the Tyr974-containing internalization motif.
J Biol Chem 271:1337713384[Abstract/Free Full Text]
-
Goodman Jr OB, Krupnick JG, Santini F, Gurevich VV, Penn RB,
Gagnon AW, Keen JH, Benovic JL 1996 ß-arrestin acts as a clathrin
adaptor in endocytosis of the ß2-adrenergic receptor.
Nature 383:447450[CrossRef][Medline]
-
Strous GJ, van Kerkhof P, Govers R, Ciechanover A, Schwartz AL 1996 The ubiquitin conjugation system is required for ligand-induced
endocytosis and degradation of the growth hormone receptor. EMBO J 15:38063812[Abstract]
-
Govers R, van Kerkhof P, Schwartz AL, Strous GJ 1997 Linkage
of the ubiquitin-conjugating system and the endocytic-pathway in
ligand-induced internalization of the growth hormone receptor. EMBO J 16:48514858[Abstract/Free Full Text]
-
Vleurick L, Kühn ER, Decuypere E, Burnside J, Pezet A,
Edery M 1999 Generation of chicken growth hormone-binding proteins by
proteolysis. Gen Comp Endocrinol 113:283289[CrossRef][Medline]
-
Sotiropoulos A, Goujon L, Simonin G, Kelly PA, Postel-Vinay
M-C, Finidori J 1993 Evidence for generation of the growth
hormone-binding protein through proteolysis of growth hormone membrane
receptor. Endocrinology 132:18631865[Abstract]
-
Berghman LR, Van Beeumen J, Decuypere E, Kühn ER,
Vandesande F 1988 One-step purification of chicken growth hormone from
a crude pituitary extract by use of a monoclonal immunoadsorbent. J
Endocrinol 118:381387[Abstract]
-
Kelly PA, Leblanc G, Dijane J 1979 Estimation of total
prolactin-binding sites after in vitro desaturation.
Endocrinology 104:16311638[Medline]
-
Huang N, Cogburn LA, Agarwal SK, Marks HL, Burnside J 1993 Overexpression of a truncated growth hormone receptor in the sex-linked
dwarf chicken: evidence for a splice mutation. Mol Endocrinol 7:13911398[Abstract]
-
Billestrup N, Moldrup A, Serup P, Mathews LS, Norstedt G,
Moldrup A, Nielsen JH 1990 Introduction of exogenous growth hormone
receptors augments growth hormone-responsive insulin biosynthesis in
rat insulinoma cells. Proc Natl Acad Sci USA 87:72107214[Abstract]
-
Sanger F, Nicklen S, Coulsen AR 1977 DNA sequencing with chain
terminating inhibitors. Proc Natl Acad Sci USA 74:54635467[Abstract]
-
Munson PJ, Rodbard D 1980 LIGAND: a versatile computerized
approach for characterization of ligand-binding systems. Anal Biochem 107:220239[Medline]
-
Sotiropoulos A, Moutoussamy S, Renaudie F, Clauss M, Kayser C,
Gouilleux F, Kelly PA, Finidori J 1996 Differential activation of Stat3
and Stat5 by distinct regions of the growth hormone receptor. Mol
Endocrinol 10:9981009[Abstract]