(Received for publication, January 3, 1995; and in revised form, April 11, 1995)
From the
The functional significance of growth hormone (GH) receptor
(GHR) internalization is unknown; therefore, we have analyzed domains
and individual amino acids in the cytoplasmic region of the rat GHR
required for ligand-mediated receptor internalization, receptor
down-regulation, and transcriptional signaling. When various mutated
GHR cDNAs were transfected stably into Chinese hamster ovary cells or
transiently into monkey kidney (COS-7) cells, internalization of the
GHR was found to be dependent upon a domain located between amino acids
318 and 380. Mutational analysis of aromatic residues in this domain
revealed that phenylalanine 346 is required for internalization.
Receptor down-regulation in transiently transfected COS-7 cells was
also dependent upon the phenylalanine 346 residue of the GHR, since no
GH-induced down-regulation was observed in cells expressing the F346A
GHR mutant. In contrast, the ability to stimulate transcription of the
serine protease inhibitor 2.1 promoter by the GHR was not affected by
the phenylalanine 346 to alanine mutation. These results demonstrate
that phenylalanine 346 is essential for GHR internalization and
down-regulation but not for transcriptional signaling, suggesting that
ligand-mediated endocytosis is not a prerequisite for GH-induced gene
transcription.
The biological action of growth hormone (GH)
The domain(s) in the GHR
involved in GH internalization has not yet been identified. In mutants
of other receptors, those lacking the cytoplasmic domain accumulate at
the cell surface, suggesting that signals required for rapid
internalization are located in their cytoplasmic tails. Aromatic
residues appear to be essential components of the internalization
signals for most receptors(13) , yet the absence of any
sequence similarities in the cytoplasmic region of receptors that are
known to internalize rather suggests that it is a conformational signal
for internalization that is recognized by a common cytoplasmic
receptor(14) . Recent attempts to identify domains involved in
the GHR signaling mechanism have shown that the conserved proline-rich
box close to the trans-membrane region is essential for JAK-2 kinase
activation (15) and mitogen-activated protein kinase activation
as well as for the transcriptional activation of the serine protease
inhibitor (SPI) 2.1 gene(16) . Similarly this region was also
required for the mitogenic activity in GHR-transfected
interleukin-3-dependent cells(17) . In addition to this region,
the C-terminal 184 amino acids were required for expression of both the
SPI 2.1 (16) and insulin (18) genes. Since the domain
of the GHR required for internalization has not been identified, we
have generated a series of mutated GHRs and tested their ability to
internalize, down-regulate, and activate transcription after
transfection.
CHO K1 cells were cultured in
Ham's F-12 medium supplemented with 10% fetal calf serum, 100
units/ml penicillin, 100 µg/ml streptomycin at 37 °C in the
presence of 5% CO
Figure 1:
Schematic
representation of the mutant GHR molecules. Mutants were constructed as
described under ``Experimental Procedures.'' Signal peptide (SP), extracellular, transmembrane (TM), and
intracellular domains are indicated by the hatched and shadedbars. The numbers correspond to amino
acid residues. Shown are wild type (a), which consists of 638
amino acids including the putative signal peptide, mutant
GHR
Figure 2:
GH
binding competition curve in transiently transfected COS-7 cells.
Binding of
Figure 3:
Internalization of surface-bound GH by
COS-7 cells expressing wild type and mutant GHRs. Cells were incubated
as described under ``Experimental Procedures.'' At various
time points, the cells were washed with acidic buffer and solubilized
to determine intracellular radioactivity. Each point represents the
mean of duplicate measurements in 5-10 experiments. The S.D.
values were <3%. Values are expressed as the percentage of initial
bound counts at time 0 and corrected for nonspecific internalization
at each time point. GHR P300,301,303,305A,
GHR
Figure 4:
Down-regulation of GHRs in COS-7 cells
expressing wild-type and mutated GHRs. Transiently transfected COS-7
cells were incubated with 100 ng/ml of hGH for various time points (A) or overnight with the indicated concentration of hGH (B), and GHR down-regulation was measured as described under
``Experimental Procedures.'' Data from cells expressing the
wild-type GHR (
Figure 5:
Transcriptional activity of mutant GHRs in
the SPI/CAT assay. CHO cells were transiently co-transfected with
plasmids containing cDNAs encoding either the wild type
(GHR
The present results indicate that internalization and
transcriptional signaling by the GHR are independent events. Results
obtained using CHO (data not shown) and COS-7 cells confirm and extend
our previous data from RIN cells(18) , which demonstrated that
a C-terminal truncation of the GHR at position 454 results in loss of
GH-stimulated insulin expression but not the ability to internalize. In
addition, truncation at position 294 resulted in loss of both
activities. These data indicate that the domain required for
internalization is located between residues 295 and 455 and that
receptor internalization itself cannot initiate GH actions. The
paradoxical finding that deletion of box 1 had no effect on
internalization whereas mutation of the four proline residues reduced
internalization suggests that the profound distortion of the
conformation by changing all four proline residues prevents the domain
required for internalization from interacting with the putative
cytoplasmic receptor recognizing internalization signals(14) .
Since the GHR
Aromatic
residues have been shown to be important for the internalization of
other receptors. For the transferrin receptor, the internalization
sequence is YXRF(31) , and for the LDL and insulin
receptors it is NPXY(32, 33) ; both these
sequences are localized in the cytoplasmic domain close to the
transmembrane region, but except for the aromatic residue, there are no
other additional sequence similarities. Conformational studies have led
to the conclusion that an aromatic residue present in the context of a
tight turn appears to be a common structural motif necessary for
internalization signals(34, 35, 36) . Our
results fit this general concept and further support a direct role of
phenylalanine 346 as being important for the internalization sequence
in the GHR.
Other biological effects of GH also do not require the
C-terminal domain of the receptor, as demonstrated in recent studies
where GHR
We thank Dr. Povl Nilsson and Charlotte
Bj(Novo Nordisk A/S, Gentofte, Denmark) for the
preparation of iodinated hGH. We thank Tina Kisbye and Jannie Rosendahl
Christensen for excellent technical assistance. We thank Drs. Erica
Nishimura and Annette MPerregaard for critical review of
the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)is initiated by its binding to specific receptors on
the plasma membrane of target cells. The GH receptor (GHR) is a single
trans-membrane protein consisting of an extracellular hormone binding
domain with homology to other members of the cytokine/GH/prolactin
receptor superfamily (1) and a cytoplasmic domain with little
homology to the other members of the family and without any known
catalytic activity. However, GH-induced tyrosine phosphorylation of the
GHR has been demonstrated(2) , and this is probably dependent
on the activation of a tyrosine kinase, JAK-2, which is associated with
the receptor(3) . Recently it has been shown that one GH
molecule binds to the extracellular region of two GHR molecules,
indicating that receptor dimerization is necessary for biological
activity(4) . Furthermore, receptor aggregation and endocytosis
appear to play an essential role in transmembrane signaling of other
growth factor receptors(5, 6) . Internalization itself
has been suggested to be essential for signal transduction by the
epidermal growth factor and other
receptors(7, 8, 9) . The GHR is known to
internalize in various cell types. In cultured rat adipocytes there
appears to be constitutive internalization of the GHR, which is
accelerated by the presence of GH(10) , whereas in IM-9
lymphocytes and mouse fibroblasts (11) the GHRs redistribute
and aggregate in response to the addition of GH with no evidence for
constitutive internalization. Interestingly, it has been suggested that
part of the GHR itself exerts a biological effect after internalization
based on the demonstration of GH binding protein and GHR in the nucleus
of GH-stimulated cells(12) .
Mutant Construction
The expression plasmid,
pLM108, containing the full-length rat GH receptor cDNA under the
transcriptional control of the human metallothionein IIa promoter and
the simian virus 40 enhancer was constructed as described previously (19) . The cDNAs encoding GHR and
GHR
were generated from a BamHI/EcoRI fragment of the pLM108 plasmid, which was
subcloned into M13 mp19 by primer-directed in vitro mutagenesis as described previously(18) . The remaining
mutant cDNAs were constructed by using the polymerase chain reaction to
splice out or alter regions as described(20) . Oligonucleotides
carrying the different mutations were synthesized and used as primers
in the polymerase chain reactions in order to introduce stop codons
(GHR
, GHR
), deletions
(GHR
), and point mutations
(GHR
, GHR
,
GHR
, and GHR
) in
the rat GHR cDNA. The introduced mutations were confirmed by DNA
sequence analysis.
Cell Culture, Transfection, and Chloramphenicol
Acetyltransferase (CAT) Assay
COS-7 cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100
µg/ml streptomycin at 37 °C in a humidified atmosphere
containing 5% CO in air. Cells were grown to 50% confluence
in 100-mm cell culture dishes (Nunc, Roskilde, Denmark) and transiently
transfected by a modified DEAE-dextran/chloroquine method (21, 22) with 5 µg of GHR expression plasmid
containing the cDNA of either the wild type GHR or mutated GHR.
Forty-eight hours after transfection the cells were tested for GH
binding and internalization.
. Cells were grown to 50% confluence in
60-mm dishes and were transiently transfected by the calcium phosphate
procedure (23) with 3 µg of pCH110 (
-galactosidase
expression vector from Pharmacia Biotech Inc.), 1.5 µg of the
construct containing the bacterial CAT coding sequence linked to the
sequence -175/+59 of the serine protease inhibitor (SPI 2.1)
promoter (24, 25) and 3 µg of the different
mutated GHR plasmids. Cells were cultured for 48 h in the absence or
presence of 400 ng/ml hGH, and cell extracts were normalized for
-galactosidase activity and then assayed for CAT activity.
GH Receptor Binding, Down-regulation, and
Internalization
COS-7 cells transiently transfected with the
various GHR mutants were plated in 6-well dishes (Nunc) 24 h after
transfection and cultured in Dulbecco's modified Eagle's
medium supplemented with 10% fetal calf serum for a further 24 h. The
monolayers were carefully washed in Hepes binding buffer (10 mM Hepes, 124 mM NaCl, 4 mM CaCl, 1.5
mM MgCl
, pH 7.4) and then incubated for 90 min at
room temperature in binding medium (Hepes binding buffer, 1% human
serum albumin) containing
I-hGH (100,000 cpm). After
incubation, the cells were washed four times in Hepes binding buffer
and solubilized with 1 ml of 1 M NaOH. Radioactivity was
counted in a
-counter. Results are presented as the average of
triplicate determinations from at least six experiments.
Internalization and down-regulation were measured in COS-7 cells, which
were plated in 6-well dishes and cultured as described above. For
internalization measurements, monolayers were washed with Hepes binding
buffer and kept on ice for 3 h in 1 ml of binding buffer containing
100,000 cpm
I-hGH. Medium was then removed, and cells
were rapidly warmed and incubated at 37 °C for the indicated time.
Cells were again placed on ice, incubation medium was removed, and
monolayers were washed four times with ice-cold Hepes binding buffer.
Surface-associated (noninternalized) GH was determined by acid wash
(0.15 M NaCl, 0.05 M glycine, pH 2.5)(26) ,
and internalized GH was determined by measuring the cell-associated
radioactivity after solubilization with 1 M NaOH.
Down-regulation was measured by incubating cells with 100 ng/ml of hGH
for different time periods or overnight with different hGH
concentrations. Free and surface bound hGH was removed by an acid wash,
and residual GHRs at the cell surface were measured by
I-hGH binding for 90 min at room temperature. After
binding, the cells were washed four times in binding buffer,
solubilized in 1 M NaOH, and counted in a
-counter.
Growth Hormone Internalization in Cell Lines Expressing
Truncated GH Receptors
A schematic representation of all the GHR
mutants used in this study is shown in Fig. 1. A series of
truncated GHRs were constructed by site-directed mutagenesis in order
to identify regions in the intracellular part of the GHR involved in
receptor internalization (Fig. 1, a-e). Deletion
and point mutations were introduced in the conserved box 1 region,
which previously has been shown to be required for JAK-2 binding and
signal transduction (Fig. 1, f-g). Finally,
aromatic residues in the region found to be required for
internalization were mutated to alanines (Fig. 1, h-i). The binding of I-hGH to the various
mutated GHRs was measured in COS-7 cells transiently transfected with
each mutant GHR cDNA. All GHR constructs tested exhibited an IC
of between 8.6 and 17.5 ng/ml of GH; no significant difference in
IC
between the various GHRs was observed, except
GHR
, which binds GH with a lowered affinity
similar to that of the GH binding protein (27) (Fig. 2).
(b), GHR
(c), GHR
(d), and
GHR
(e) in which lysine 455, alanine
381, lysine 319, or lysine 295, respectively, was mutated into a stop
codon, GHR
(f) in which 45 base
pairs encoding amino acids 297-311 were deleted,
GHR
(P300,301,303,305A) (g) in which proline residues 300, 301, 303, and 305 were
changed to alanine residues, GHR
(YFY333,337,338A) (h) in which tyrosine
residues 333 and 338 and phenylalanine residue 337 were changed to
alanine residues, and mutant GHR
(i), in which
phenylalanine 346 was substituted with
alanine.
I-hGH to COS-7 cells transiently transfected
with various GH receptor encoding plasmids is shown. COS-7 cells were
cultured for 48 h after transfection, and binding of
I-hGH in the presence of the indicated concentration of
unlabeled hGH was measured. The IC
values were calculated
from the competition curves and represent the mean of four experiments
± S.D. GHR P300,301,303,305A,
GHR
; GHR
YFY333,337,338A,
GHR
.
To examine the kinetics of GH uptake, we measured the
internalization of GH in stably transfected CHO cells (data not shown)
as well as in transiently transfected COS-7 cells. COS-7 cells
expressing GHR, GHR
, and
GHR
showed internalization of
I-hGH, which reached a maximum after 1 h at 37 °C (Fig. 3). Approximately 60% of the specifically bound GH was
internalized at this time point. As early as 15 min after raising the
temperature to 37 °C, 25% of the specifically bound
I-hGH was inside the cells. In contrast, in COS-7 cells
expressing GHR
internalization was greatly
reduced, with only 15% of the specifically bound GH internalized after
1 h. GHR
showed approximately the same
internalization rate as GHR
. These data indicate
that a domain required for internalization of the GHR is located
between residues 318 and 380. Similar results were observed in stably
transfected CHO cells (data not shown).
; GHR YFY333,337, 338A,
GHR
.
Localization of the Amino Acid Residues Involved in GHR
Internalization
To evaluate the contribution of the proline-rich
region in ligand-mediated endocytosis we constructed two GHR mutants:
one with this portion deleted, GHR, and
the other in which the four proline residues present in this region
were changed into alanines, GHR
(Fig. 1). Plasmids encoding either mutated receptor were
transfected into CHO and COS-7 cells. As shown in Fig. 3,
GHR
demonstrated internalization of GH in
COS cells similar to the wild-type GHR, whereas internalization was
reduced in cells expressing GHR
. In
view of the identification in many other transmembrane receptors of
potential internalization domains in which aromatic residues are
important, we examined the role of aromatic residues present in the
cytoplasmic domain of the GHR. In the region between 318 and 380 only
four aromatic residues are present: tyrosine 333, phenylalanine 337,
tyrosine 338, and phenylalanine 346. We therefore constructed a GHR
mutant in which tyrosines 333 and 338 and phenylalanine 337 were
mutated into alanines, GHR
, and a mutant in
which phenylalanine 346 was mutated into alanine, GHR
(Fig. 1). As shown in Fig. 3,
GHR
internalized GH as effectively as the
wild type receptor. In contrast, GHR
only internalized
to the same extent as mutant GHR
, which lacks all
but five amino acids of the cytoplasmic region.
Role of Phenylalanine 346 in GHR
Down-regulation
In order to compare the domain of the GHR
required for internalization with the domain required for
down-regulation of the GHR, we examined GH-induced GHR down-regulation
in transiently transfected COS-7 cells. Cells expressing the wild-type
GHR showed a dose- and time-dependent down-regulation of the GHR (Fig. 4). After a 5-min exposure to 100 ng/ml GH, 60% of
specific GHRs were down-regulated, and after 10 min 80% of GHRs were
down-regulated (Fig. 4A). This level of down-regulation
remained constant for up to 18 h. In contrast, COS-7 cells expressing
either the GHR or the GHR
mutant
did not exhibit GH-induced receptor down-regulation. An initial
reduction in binding of about 25% was observed after a 5-10-min
exposure to 100 ng/ml of GH; however, this apparent down-regulation was
transient since binding returned to control levels after 60 min of
exposure to GH. This rapid loss of GH binding sites observed in cells
expressing the GHR
or the GHR
mutant might be caused by membrane turnover, and longer exposure
to GH might allow for the insertion of newly synthesized GHRs into the
plasma membrane. A dose-dependent down-regulation of the wild-type GHR
but not of the mutated GHRs was observed after an 18-h exposure to GH (Fig. 4B). These data show that phenylalanine 346 is
required for both internalization and down-regulation and suggests that
receptor internalization is the cause of down-regulation.
), GHR
(
) and
GHR
(
) are shown. Each point represents the mean
of duplicate measurements from three
experiments.
Role of GH Internalization in Signal
Transduction
The biological activity of the GHR mutants was
evaluated by a transient co-transfection assay in which the ability of
GH to stimulate the expression of the SPI 2.1 promoter/CAT gene
construct in CHO cells was tested. As shown in Fig. 5, deletion
of only 184 amino acid residues in the C-terminal region of the GHR
resulted in loss of this activity. Furthermore, deletion of the
proline-rich box(297-311) or mutation of the four proline
residues (300, 301, 303 and 305) to alanines abolished transcriptional
activity as described previously (16) . In contrast, mutation
of the aromatic residues 333, 337, and 338 to alanine did not cause any
reduction in activity. In addition, substitution of phenylalanine 346
with alanine in the GHR, despite being deficient in internalization and
down-regulation, was fully capable of stimulating SPI 2.1 promoter
transcriptional activity.
) or the indicated mutated GHR cDNAs
together with the fusion gene SPI/CAT. After transfection the cells
were incubated in the presence or absence of 400 ng/ml hGH 48 h. The
-fold induction was calculated as percentage of chloramphenicol
conversion in the presence of GH divided by the percentage of
conversion in the absence of GH. Results represent the mean ±
S.D. of 5-10 independent experiments. GHR
P300,301,303,305A, GHR
; GHR
YFY333,337,338A,
GHR
.
was able to internalize to the same
extent as the full-length GH receptor, while GHR
or GHR
exhibited impaired internalization,
we concluded that the domain required for internalization was located
between amino acid residues 318 and 380. In this region only four
aromatic residues are present; therefore, we analyzed their role in
internalization. From the present study it appears that an aromatic
residue at position 346, but not in position 333, 337, or 338, is
essential for internalization, since the substitution of phenylalanine
346 with alanine inhibited internalization but not transcriptional
activity. Whether the phenylalanine 346 is directly involved in the
internalization process or it is simply required for the overall
conformation of a region involved in internalization cannot be
determined by this study. It is, however, unlikely that the tertiary
structure of the GHR is changed dramatically by the alanine
substitution, since the transcriptional activation by the GHR
mutant is unchanged. Furthermore alanine substitutions eliminate
side chains beyond the
-carbon and do not impose steric or
electrostatic effects. Substitutions by alanine have been used
frequently by others and shown not to change the tertiary structure of
many proteins(28, 29, 30) .
, although unable to stimulate SPI 2.1
transcription, was still found to be able to transmit the signal for
protein biosynthesis, mitogen-activated protein kinase, and JAK-2
kinase activation and mitotic activity in CHO
cells(15, 37) . Furthermore, in the
interleukin-3-dependent promyeloid cell line FDC-P1, the human GHR
truncated at position 325 (corresponding to 344 in the present
numbering of the rat GHR) was able to transmit a mitogenic signal,
indicating that this activity exclusively resides in box 1, although
the effect was augmented when box 2 was retained(17) . Since
both GH and several other growth factors and cytokines are reported to
activate JAK-2 kinase (38, 39) it may be speculated
that biological responses common to these growth factors such as
protein biosynthesis, mitogen-activated protein kinase activation, and
mitotic activity are due to binding and activation of JAK-2 kinase by
box 1, which then interacts with box 2 in a yet unknown manner.
However, activities attributed to specific members of this receptor
family rely on domains in the C-terminal region. Since nuclear
localization of both GH and GHR has been demonstrated, it has been
proposed that GH, GHR, or GH binding protein exerts biological
functions after being internalized (12) . The present finding
of a GHR mutant that is not internalized but retains full
transcriptional activity of the SPI 2.1 promoter indicates, however,
that such mechanisms are probably not involved in this activity, but
GHR internalization might be required for other biological actions. In
conclusion, these results support the hypothesis that different domains
in the cytoplasmic region of the GHR are responsible for transducing
signals for separate biological activities of GH.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.