A Short Isoform of the Human Growth Hormone Receptor Functions as a Dominant Negative Inhibitor of the Full-Length Receptor and Generates Large Amounts of Binding Protein
R. J. M. Ross,
N. Esposito,
X. Y. Shen,
S. Von Laue,
S. L. Chew,
P. R. M. Dobson,
M.-C. Postel-Vinay and
J. Finidori
Department of Medicine, Clinical Sciences Centre
(R.J.M.R., X.Y.S., S.V.L.)and Institute of Cancer Studies
(P.R.M.D.) Sheffield University, Sheffield S5 7AU Department of
Endocrinology (S.L.C.) St. Bartholomews Hospital London EC1A
7BE, UK INSERM unite 344 (N.E., M.-C. P.-V., J.F.) Endocrinologie
Moleculaire Faculte de Medecine Necker 75730 Paris, France
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ABSTRACT
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The GH receptor (GHR) is a member of the cytokine
receptor family. Short isoforms resulting from alternative splicing
have been reported for a number of proteins in this family. RT-PCR
experiments, in human liver and cultured IM-9 cells, using primers in
exon 7 and 10 of the GHR, revealed three bands reflecting alternative
splicing of GHR mRNA: the predicted product at 453 bp and two other
products at 427 and 383 bp. The 427-bp product (GHR1-279) utilized an
alternative 3'-acceptor splice site 26 bp downstream in exon 9; the
predicted C-terminal residues are six frameshifted exon 9 codons ending
in an inframe stop codon. The 383-bp product (GHR1-277) resulted from
skipping of exon 9; the predicted C-terminal residues are three
frameshifted exon 10 codons ending in an in-frame stop codon. RNase
protection experiments confirmed the presence of the GHR1-279 variant
in IM-9 cells and human liver. The proportion of alternative splice to
full length was 110% for GHR1-279 and less than 1% for GHR1-277.
The function of GHR1-279 was examined after subcloning in an expression
vector and transient transfection in 293 cells. Scatchard analysis of
competition curves for [125I]-hGH bound to cells
transfected either with GHR full length (GHRfl) or GHR1-279 revealed a
2-fold reduced affinity and 6-fold increased number of
binding sites for GHR1-279. The increased expression of GHR1-279
was confirmed by cross-linking studies. The media of cells transfected
with GHR1-279 contained 20-fold more GH-binding protein (GHBP) than
that found in the media of cells transfected with the full-length
receptor. Immunoprecipitation and Western blotting experiments, using a
combination of antibodies directed against extracellular and
intracellular GHR epitopes, demonstrated that GHRfl and GHR1-279 can
form heterodimers and that the two forms also generate a 60-kDa GHBP
similar in size to the GHBP in human serum. Functional tests using a
reporter gene, containing Stat5-binding elements, confirmed that while
the variant form was inactive by itself, it could inhibit the function
of the full-length receptor. We have demonstrated the presence of a
splice variant of the GHR in human liver encoding a short form of the
receptor similar in size to a protein previously identified in human
liver and choroid plexus. Expression studies in 293 cells support the
hypothesis that while the expression of the splice variant accounts for
only a small proportion of the total GHR transcript, it produces a
short isoform that modulates the function of the full-length receptor,
inhibits signaling, and generates large amounts of GHBP. The
differential expression of GHR receptor short forms may regulate the
production of GHBP, and truncated receptors may act as trans-port
proteins or negative regulators of GHR signaling.
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INTRODUCTION
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The GH receptor (GHR) is a member of the cytokine family of
receptors that possess a single transmembrane domain, are devoid of
intrinsic enzyme activity, and associate with cytoplasmic tyrosine
kinases to form multisubunit receptor complexes. Binding of a single
molecule of GH results in receptor dimerization and activation. A short
isoform of the receptor identical to the extracellular domain of the
receptor circulates as a binding protein. In rodents there is
alternative splicing of mRNA just proximal to the transmembrane domain
with the full-length message (4.5 kb) coding for the GHR and a
truncated message (1.2 kb) coding for the GH-binding protein (GHBP). In
the rat the sequence for the GHBP is identical to that of the GHR up to
three amino acids before the putative transmembrane domain where an
additional 17 amino acids are encoded before a stop codon is
encountered (1). In contrast, in the human no alternative splice is
seen on Northern analysis, and it has been proposed that the GHBP is
derived by proteolytic cleavage of the extracellular domain of the GHR.
Support for this concept comes from transfection studies with the GHR
(2, 3) and studies of GHBP in the media from cultured IM-9 cells
(4).
The human GHR and GHBP are products of a single gene (5). Exon 2 codes
for the signal peptide, the extracellular domain is coded by exons 3 to
7, the transmembrane domain is coded by exon 8, while exons 9 and 10
encode the cytoplasmic domain and the 3'-untranslated region (6).
Different isoforms for various members of the cytokine receptor
superfamily have been reported. For the GHR an exon 3 skipped isoform
(exon 3-) is expressed in placenta, liver, and various cultured cells
(7). For the rat PRL receptor, short isoforms with a limited or absent
cytoplasmic domain have been identified (8, 9). For PRL the short and
long isoforms are expressed in a tissue-specific manner and regulated
by estrus (10). Five different human granulocyte-colony stimulating
factor (G-CSF) isoforms, arising from alternative splicing, have been
isolated and are identical in the extracellular domain but differ in
their downstream sequences (11). Three different isoforms of the
-subunit of the granulocyte macrophage (GM)-CSF receptor have been
reported, one of which encodes a soluble receptor (12).
We postulated that there is alternative splicing of the human GHR
around the transmembrane domain but that this may not have been
identified on Northern blotting if all transcripts were of
approximately the same size or if transcripts were of low abundance. To
test this hypothesis we designed primers in exons 7 and 10 and, using a
RT-PCR-based technique, tested for alternative splicing in mRNA from
human liver and cultured cells. In this paper, we show that two
variants could be identified, and functional studies indicate that, in
spite of their low abundance, they could have a physiological role
generating large amounts of GHBP and interacting with the full-length
receptor.
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RESULTS
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Identification of Splice Variants in Human Liver and IM-9 Cells
Using primers PS and PAS in exons 7 and 10 of the human GHR (Fig. 1
) RT-PCR of human liver revealed three products at 453,
427, and 383 bp (Fig. 2
). The individual bands were
extracted from polyacrylamide, cloned into pCRII, and sequenced. The
453-bp product had an identical sequence to that previously published
for the GHR (GHRfl) (5). The 427-bp (GHR1-279) product utilized an
alternative 3'-acceptor splice site in exon 9. Exon 8 was as expected
but spliced to a 3'-splice acceptor site within exon 9, thus omitting
the upstream 26 bp of exon 9. The nucleotide sequence at this
position in exon 9 matches the mammalian consensus for a 3'-acceptor
CAG/TT at position 944 in the Genbank sequence (X06562). The 383-bp
(GHR1-277) product skipped exon 9 with exon 8 splicing to exon 10. The
predicted peptides have been labeled by their amino acid number
according to Leung et al. (5).

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Figure 1. Schematic Representation of the GHR Gene and
Alternative Splice Products
a, Human GHR gene alternative splicing and position of the primers used
for PCR. Exons are shown as boxes, primers as
arrows, and TM indicates transmembrane domain. b, Nucleotide
and translated sequence of the full-length GHR and alternative splices
GHR1-279 and GHR1-277. Exon boundaries are marked by
slashes, deleted sections by periods, in-frame
stop codons underlined, and the end of the putative
transmembrane domain by |. Nucleotide and amino acid residue
homologies are in uppercase.
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Figure 2. Detection of GHR Isoforms in Human Livers
RT-PCR products from five different livers using primers PS and PAS
were analyzed by Southern blot with labeled gel-purified products from
pGHR453. Lanes 13 are markers at 453, 427, and 383 bp generated in a
PCR reaction with clones pGHR453, pGHR1-279, and pGHR1-277 as
templates. Five liver samples (lanes 48) demonstrated products at 453
and 427 bp. Overexposure (lane 8) revealed three bands at 453, 427, and
383 bp although the 427 is partly obscured.
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The predicted C-terminal residues of the GHR1-279 peptide are six
frame-shifted exon 9 codons ending in an in-frame stop codon and for
the GHR1-277 peptide are three frame-shifted exon 10 codons ending in
an in-frame stop codon (Fig. 1
). The translated sequence is the same as
the full-length receptor for the first three to four amino acids of the
cytoplasmic domain of GHR1-279 and GHR1-277, respectively, but the
predicted peptides deviate from each other before the proline-rich box
I region of the receptor that is required for signal transduction (13, 14).
The alternative splice variants of the GHR were also identified in
cultured IM-9 cells. Since all three splice variants compete for the
same primers and PCR reagents in a common reaction, the relative
optical densities of the three bands were assumed to reflect the
proportion of the variants in the template cDNA. The 453 bp and 427 bp
products are clearly seen in Fig. 3a
. The 383 product
was seen only on overexposed autoradiographs (data not shown).
Quantification on appropriately exposed autoradiographs by an image
densitometer revealed that the proportions of the alternative splice
products were similar in all experiments. Thus, GHR1-279 was 110% of
GHRfl, and GHR1-277 was consistently less than 1%. RNase protection
(Fig. 3b
) confirmed the presence of the GHR1-279 alternative splice
product in human liver and IM-9 cells in similar proportions to the
full-length receptor as seen by RT-PCR. The GHR1-277 splice variant was
not identified by RNase protection.

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Figure 3. Alternative Splicing for GHR in IM-9 Cells
a, GHR gene expression detected in four separate cultures of IM-9 cells
(lanes 47) by RT-PCR and Southern blotting. Lanes 13 are markers at
453, 427, and 383 bp. b, RNase protection for the GHR using a GHR1-279
probe. Lane 1 markers: 2, undigested probe; 3, IM-9 cells; 4, human
liver; 5, untransfected HepG2 cells; 6, HepG2 cells transfected with
GHRfl. The alternative splice for GHR1-279 is seen at 296 bp in human
liver (lane 4) and IM-9 cells (lane 3). The full-length receptor is
detected at 217 bp in IM-9 cells, human liver, and transfected HepG2
cells (lane 6).
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Expression of GHR1-279 in 293 Cells
293 cells were transiently transfected with GHRfl or GHR1-279
cDNAs. Expression of the two receptor forms was analyzed in binding
experiments of [125I]-hGH to intact cells. Scatchard
analysis of the competition experiments indicated a 2-fold reduced
affinity for GHR1-279 compared to GHRfl; association constant
(Ka) for GHRfl = 1.2 x 109
M-1 and GHR1-279 = 0.6 x
109 M-1. The level of expression
was high, as expected in 293 cells. The calculated number of binding
sites per cell was about 1.2 x 105 when 1 µg GHRfl
cDNA was transfected into 3 x 106 cells, and 7
x 105 sites per cell for 1 µg GHR1-279 cDNA
transfected.
Cross-linking studies with [125I]-hGH on cells
transiently transfected with GHRfl or GHR1-279 cDNAs are shown in Fig. 4
. In both cases specific complexes, displaced in the
presence of an excess of native hormone, were observed. In cells
transfected with GHRfl, the apparent sizes of the radioactive bands
(
140 and
260 kDa) were those expected for complexes of one or two
molecules of receptor and one hormone molecule, respectively. In cells
transfected with GHR1-279 cDNA, the amount of radioactivity in both
complexes is much higher than that observed with the full-length
receptor. This is consistent with the data from binding experiments
indicating that the number of receptors in the cell membrane is greater
after transfection with the GHR1-279 cDNA. The apparent sizes of the
two complexes are approximately 75 kDa and 150 kDa and correspond to
the sizes expected for one or two molecules of GHR1-279 bound to one
molecule of hormone.

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Figure 4. [125I]-hGH Cross-Linking to the GHRfl
and GHR1-279 Receptor Forms in Transfected 293 Cells
The cross-linked receptors were analyzed by SDS gel electrophoresis as
described in Materials and Methods. Cross-linking was
performed without (-) or with (+) 3 µg unlabeled hGH.
Numbers indicate the size of protein standards in
kilodaltons.
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Soluble forms of the receptor generated by GHRfl or GHR1-279 in the
media of transfected cells were studied by HPLC gel filtration. The
elution profiles of [125I]-hGH incubated with
concentrated media (20-fold) of cells transfected with GHRfl and
GHR1-279 revealed two peaks (Fig. 5
). The first peak was
completely displaced by an excess of native hGH, and its elution time
corresponded to that previously described for GHBP in human serum (15)
or media of human hepatoma cells (16). The second peak corresponds to
the free [125I]-hGH. Sixty percent of radioactivity was
in the first peak when 100 µl of culture media from cells transfected
with GHRfl were analyzed. A similar binding activity, corresponding to
GHBP, was found when only 5 µl GHR1-279 media were used,
demonstrating a 20-fold increase of GHBP in media from cells
transfected with GHR1-279.

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Figure 5. Elution Profiles of [125I]-hGH
Incubated with Medium from Transfected 293 Cells
Cells were transfected with 5 µg human GHRfl or GHR1-279. Medium was
recovered and analyzed as described in Materials and
Methods. Incubation was performed in the absence (total binding,
solid line) and presence (nonspecific binding, thin
line) of 2 µg native hGH; 100 µl of media were analyzed for
GHRfl (A) and 5 µl GHR1-279 (B).
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Immunoprecipitation and Western blot analysis were used to assess the
sizes of the membrane-bound and soluble forms of GHR in cells
transfected with GHRfl and GHR1-279 (Fig. 6
). The
expression of GHRfl was detected by immunoprecipitation using a
polyclonal antibody (
GHR-intra) raised against an epitope in the
intracellular domain of human GHR (not present in GHR1-279) (2). To
purify the receptor in cells transfected with GHR1-279 alone, we
incubated the cells with biotinylated hGH and purified the complexes
with streptavidin beads after lysis. The soluble forms in media were
also purified with biotinylated hGH and streptavidin beads. In order to
detect a possible heterodimerization between GHRfl and GHR1-279, cells
were cotransfected with 5 µg of each cDNA and stimulated with hGH (in
order to induce dimerization) before lysis. The complexes formed were
then immunoprecipitated with the
GHR-intra antibody. Western blots
were performed with mAb263, which recognizes all membrane and soluble
forms of the GHR. The results are shown in Fig. 6
. GHRfl
appeared as a single band around 120 kDa (lane 1). GHR1-279 consisted
in two bands; the size of the major band was around 55-60 kDa; the
minor band, around 46 kDa, was likely a degradation product (lane 3).
Soluble receptors in the supernatant of cells transfected with either
GHRfl (lane 5) or GHR1-279 (lane 6) were single bands of apparently 60
kDa, similar in size to the upper band identified in normal serum (lane
7). In cells cotransfected with GHRfl and GHR1-279, three bands were
detected with the mAb263 (lane 2). The upper band migrated as the GHRfl
(lane 1), and the two other bands migrated as GHR1-279 (lane 3). No
band could be detected when lysates of cells transfected with GHR1-279
alone were immunoprecipitated with the
GHR-intra antibody (lane 4).
These results demonstrate that GHRfl and GHR1-279 can heterodimerize on
GH stimulation.
Functional Studies of GHR1-279
The function of the wild type and variant form of the GHR
was examined in 293 cells transfected with a reporter gene containing a
Stat5-binding element (LHRE) fused to a minimal TK promoter and
luciferase. Such a test allows analysis of GH-dependent Stat5
transactivation (17). As box 1 is not present in the short cytoplasmic
domain of the variant receptor, it would not be expected to activate
Stat5, but we could speculate that when coexpressed with wild type
receptor it could competitively inhibit receptor signaling. To test
this hypothesis, functional tests were performed. Cells were
transfected with 0.1 µg GHRfl cDNA per six-well plates (to give
2 x 103 receptor sites per cell) and increasing
amounts of GHR1-279 cDNA. In conditions providing maximal stimulation
of the reporter gene (20 nM hGH), we observed a 12-fold
stimulation of luciferase activity in cells transfected with GHRfl
alone. No stimulation of luciferase was observed, as expected in cells
transfected with GHR1-279 alone. When increasing amounts of GHR1-279
cDNA (from 0.01 to 1.0 µg) were cotransfected with 0.1 µg of GHRfl
cDNA, a dose-dependent inhibition was observed (Fig. 7
).
For a ratio similar to that observed in vivo (GHR1-279
vs. GHRfl = 10%), the inhibition of luciferase
activity was 5% when 20 nM hGH was used in the functional
test but 30% when a more physiological level, 1 nM hGH,
was used. This physiological hormone concentration induced a 3-fold
stimulation of the reporter gene in cells transfected with GHRfl. As
the degree of inhibition appears dependent on the concentration of hGH
in the medium, this suggests that the inhibition of GHRfl signaling by
the short form could be also due to a competition between GHRfl and
GHR1-279 for binding of GH.

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Figure 7. Inhibition of STAT5-Dependent Transcriptional
Activity Mediated by GHR in the Presence of the Short Form GHR1-279
293 cells were transiently cotransfected with plasmids containing cDNAs
encoding GHRfl and increasing amounts of the GHR1-279 cDNA together
with the reporter gene LHRE/TK/luciferase. After transfection the cells
were incubated in the presence or the absence of 20 nM (A)
or 1 nM (B) hGH for 16 h. One representative of
several experiments is shown: the fold induction represents the ratio
of luciferase activity in the presence and in absence of hGH.
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DISCUSSION
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The studies presented here demonstrate alternative splicing in the
cytoplasmic domain of the human GHR. We have sequenced two clones
derived from human liver that code for short isoforms of the GHR with
partial or complete skipping of exon 9. Based on RT-PCR and RNase
protection experiments, these alternative splices represent a small
proportion of the total GHR transcript. We detected transcripts with an
alternative 3'-acceptor site in exon 9 (GHR1-279) and transcripts with
complete deletion of exon 9 (GHR1-277) in human liver and IM-9 cells.
The GHR1-279 represented 110% and the GHR1-277 less than 1% of the
total transcript. GHR1-279 and GHR1-277 encode short isoforms of the
GHR with very short cytoplasmic domains of nine and seven residues,
respectively, which differ from the full-length receptor three to four
residues after the transmembrane domain and before the proline-rich box
region that is required for signal transduction (13, 14).
Following transfection of the short form, GHR1-279, in mammalian
cells we identified a
75-kDa protein in cell membranes by
cross-linking with GH. A similar size protein had been previously
identified in human liver (18). At that time, two molecular forms of
the hormone-receptor complex were observed in human liver with
estimated sizes of 124 and 75 kDa (18). It was not clear whether the
smaller form was a degradation product of the expected 124-kDa receptor
complex. Taking into account our current results it is possible it
could be generated from the splice variant that we detected in human
liver. In human choroid plexus, cross-linking studies identified only
one hormone-bound complex again at
75 kDa (19). In the rat there is
evidence for a short form of the receptor bound in the membrane of
adipocytes (20), and it has been speculated that similar forms will be
found in the human (21).
This paper investigated the functional significance of the most
abundant alternative splice variant reported, GHR1-279. This short
isoform of the receptor, which retains the transmembrane domain but is
divergent in the cytoplasmic domain, was subcloned into an expression
vector and transfected into 293 cells. Binding assays with entire cells
indicated that in spite of its short cytoplasmic domain, GHR1-279 is
held in the cell membrane. However, the GHR1-279 had a reduced affinity
and increased binding capacity compared to the full-length receptor.
The binding affinity (Ka) for GHRfl was 1.2 x
109 M-1 similar to that previously
published for the human liver GHR (18). GHR1-279 had a 2-fold lower
affinity of 0.6 x 109 M-1
similar to the GHBP in human serum (22), which has a 5-fold lower
affinity compared to the human liver GHR (23). A possible explanation
for these results is that the length of the cytoplasmic domain could
affect the general structure of the receptor and its ligand affinity.
The differences in the binding capacity for expressed GHR1-279
vs. GHRfl is consistent with that previously observed
for truncated GHRs. In CHO clones stably expressing a truncated form of
the rabbit GHR (which retains only 46 amino acids in its cytoplasmic
domain) the number of binding sites for this mutant was 8 times higher
than that for CHO clones stably expressing GHRfl (14). We have observed
that the increase in binding sites correlates with an impaired
internalization of the receptor (L. S. Moutoussamy et al.,
manuscript in preparation). A similar finding has been reported with
short isoforms of the rat GHR (24). Critical residues for
internalization of the receptor have been mapped for the rat GHR (24),
which are located in a cytoplasmic domain that is absent in the
truncated rabbit GHR or in GHR1-279. Recently the involvement of the
ubiquitin system has been demonstrated in GHR internalization and the
putative amino acid sequence for ubiquitination present in the GHR
sequence is absent in GHR1-279 as well as in the short forms of rat and
rabbit receptor mentioned above (25). However, when coexpressed in
cells with the GHRfl, a proportion of GHR1-279 could be internalized
through heterodimerization with GHRfl. The extent of this phenomenon
remains to be established.
GHBP in the media of cells was studied by HPLC gel filtration and
Western blotting after affinity purification. By HPLC, a GHBP with
similar characteristics to human serum GHBP was identified in the media
of cells transfected with GHR1-279. There was an increased amount of
GHBP in the medium of cells transfected with GHR1-279 as compared with
that measured in the media of cells transfected with GHRfl, when
similar amounts of cDNA were transfected. This could be related to
increased levels of the short isoform at the cell surface and/or
reduced internalization allowing more receptor to be available for
proteolysis. On Western blotting human serum revealed two bands of the
expected sizes for the GHBP (60 and 55 kDa) (22, 23, 26). Media from
cells transfected with GHRfl and GHR1-279 demonstrated a protein of 60
kDa similar to the predominant protein in human serum. We can postulate
that, in spite of its low level of mRNA expression, the spliced variant
could generate a proportion of the circulating GHBP.
Immunoprecipitation experiments demonstrated that heterodimers could be
formed between GHRfl and GHR1-279. Western blot analysis of the
complexes immunoprecipitated with a cytoplasmic domain antibody,
GHR-intra, and probed with an extracellular domain antibody, mAb263,
indicated that GHR1-279 could only be immunoprecipitated when complexed
with GHRfl. The identity of GHR1-279 in the heterodimeric complex was
assessed by its comigration with the very large band detected when
GHR1-279 was precipitated with streptavidin after binding to
biotinylated hGH. The finding of heterodimerization suggests that
GHR1-279 may act in a dominant negative fashion to inhibit receptor
signaling in addition to competitively binding GH.
GHR signaling involves GH-dependent receptor dimerization,
activation of the tyrosine kinase JAK2, and the subsequent recruitment
and tyrosine phosphorylation of various Stat proteins including Stat1,
Stat3, and Stat5 (27). JAK2 activation is dependent on its association
with the receptor that is mediated by the juxtamembranous region of the
cytoplasmic domain, including the proline-rich region box1 (14, 28).
Functional tests using a reporter gene containing the Stat5-binding
element were performed to test the possibility that GHR1-279 could act
as a negative regulator of the full-length receptor. We observed such
an effect even when the ratio of the cDNAs transfected was 1:10 for
GHR1-279 to GHRfl. As the degree of inhibition was dependent on the
concentration of hGH, it suggests that inhibition was also related to a
decreased availability of the ligand for the active GHRfl dimer when
the short form receptor is expressed. Preliminary data have been
reported suggesting a dominant negative effect for other GHR mutants
(29), and a dominant negative effect was seen with the PRL receptor
when similar proportions of cDNA encoding a short form were transfected
(30). In addition, there is a report of a patient heterozygous for a
GHR mutation resulting in the expression of a protein identical to
GHR1-277 (31). This patient presented with short stature, and the
authors suggest the short form of the receptor may act in a dominant
negative fashion. Our data support this hypothesis. Studies with the
G-CSF receptor have shown that expression of the various isoforms was
tissue specific; aberrations in the expression of these various
isoforms have been reported and postulated to play a role in the
pathogenesis of disorders of granulopoiesis (11). Thus, it is possible
that differential expression of GHR isoforms could play a role in the
physiological regulation of receptor function in some genetic or
acquired GH disorders.
The truncated receptor GHR1-279 has recently been identified in rabbit
as well as in human tissues (32). Using PCR analysis there was
differential expression of the truncated receptor with low abundance of
the alternative splice in liver, kidney, and fibroblasts but similar
expression to the full-length receptor in mammary gland and adipose
tissue. The differential tissue expression of alternative splicing may
regulate the production of GHBP. In addition, the truncated receptor
may act to inhibit GH signaling in some tissues or act as a transport
protein, as has been suggested for the truncated form of leptin
receptor expressed in the choroid plexus (33).
Our results indicate that while only a single mRNA species is detected
by Northern blotting, alternative forms of mRNA for the GHR are
transcribed. These splice variants encode short forms of the receptor
that could play a role in the generation of a GHBP in the human and can
modulate the function of the full-length receptor.
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MATERIALS AND METHODS
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Tissues
All samples were human tissues removed at operations, frozen
immediately in liquid nitrogen, and stored at -80 C. Local Ethical
Committee approval and informed consent or assent from patients or
relatives were obtained. Normal liver samples (n = 5 subjects)
were from brain-dead liver transplant donors or patients undergoing
resection of a single lesion, metastasis, or hydatid cyst.
RNA Extraction and RT-PCR
This was performed as described (34). Total RNA was
extracted using an acid phenol/guanidinium isothiocyanate method
(RNAzol B, Biogenesis, Poole, U.K.). RNA quantification was performed
spectrophotometrically and quality assessed by agarose-formaldehyde gel
electrophoresis. Complementary DNA was made using 5 µg total RNA with
200 U MMLV reverse transcriptase (BRL, Gaithersburg, MD), 5 µg random
hexamer primers (Boehringer Mannheim, Indianapolis, IN), and 200
µM final concentration of deoxynucleosidetriphosphate in
a 50-µl reaction. The reaction was performed at 24 C for 10 min, 37 C
for 60 min, and 92 C for 10 min. PCR amplification of GHR transcripts
was performed using 5 µl cDNA (equivalent to 0.5 µg total RNA) in a
50-µl volume with: 200 µM final concentration of
deoxynucleosidetriphosphate; 2.5 mM final concentration
MgCl2, 2 µM final concentration of each
primer, and 1 U Taq DNA polymerase. After heating to 94 C for 3 min, 30
cycles were performed at 94 C for 30 sec, 56 C for 1 min, and 72 C for
2 min, before a final step of 72 C for 10 min. All PCR reactions
included negative controls with water.
Primers (Fig. 1
)
Primers were manufactured by Genosys (Cambridge, U.K.). For the
GHR, primer PS is in exon 7 starting at nucleotide 709
(5'-GGATAAGGAATATGAAGTGC-3') and was used with primer PAS in exon 10
starting at nucleotide 1161 (5'-GATTTCTCATGGTCACTGC-3') predicting a
product of 453 bp.
Cloning, Sequencing, and Southern Blotting
PCR products were cloned into the pCRII vector (Invitrogen, San
Diego, CA) according to the manufacturers protocol, and dideoxy
sequencing was with the Sequenase 2.0 kit according to the
manufacturers instructions (USB, Cleveland, OH). RT-PCR products were
transferred from polyacrylamide gels (612%) onto a nylon membrane
(Hybond N+, Amersham, Little Chalfont, U.K.) by electroblot (Bio-Rad,
Hemel Hempstead, U.K.). GHR cDNA probes were prepared by PCR using 0.1
µg of the GHR453 plasmid as a template; products were run on 1%
agarose, excised, purified (Geneclean, Bio 101) and random primer
labeled with 30 µCi [32P]dCTP (Oligolabeling kit,
Pharmacia, Piscataway, NJ). The probes were separated by a Sephadex G50
column. Prehybridization (4 h) and hybridization (18 h) were performed
at 42 C in 50% formamide. Posthybridization washes were twice in 2x
sodium citrate chloride/0.2% SDS for 30 min at room temperature, then
twice in 0.1 x sodium citrate chloride/0.1% SDS at 55 C for 30
min each. The membranes were exposed to Kodak XAR film.
Quantification
Autoradiographs were scanned by an image densitometer (GS 670,
Bio-Rad) and optical densities analyzed by Molecular Analyst software
(Bio-Rad). A standard PCR reaction was performed on normal liver cDNA
with product being sampled after every tenth cycle for 40 cycles to
assess the kinetics of the reaction.
IM-9 Cell Cultures
Human IM-9 lymphocytes were grown in RPMI 1640 medium (all
reagents, from Sigma, St. Louis, MO), containing 10% (vol/vol) FCS,
100 U/ml penicillin, and 100 µg/ml streptomycin, 2 nM
L-glutamine at 37 C in 5% CO2. The cells were
cultured to stationary phase, counted with a hemocytometer, centrifuged
at 200 x g, washed three times in RPMI 1640, and
resuspended in RPMI 1640 and 0.1% wt/vol BSA. After 24 h cells
were harvested and total RNA extracted as described above. HepG2 and
293 cells were cultured and transfected by the CaPO4 method
as previously described (17, 34).
RNase Protection
The GHR riboprobe was constructed using the GHR1-279
plasmid in pCRII (Invitrogen). The plasmid was linearized by digestion
with Ncol, and in vitro transcription by T7 RNA
polymerase produced an antisense probe of 385 bp, which was gel
purified. RNase protection was performed on 25 µg total RNA from
yeast (negative control), IM-9 cells, human liver, HepG2 cells, and
HepG2 cells stably transfected with the GHRfl (this stably transfected
cell line was used as a positive control for the full-length GHR
message). Total RNA was hybridized with labeled GHR antisense probe
(GHR1-279) overnight at 45 C in 80% (vol/vol) formamide, 40
mM piperazine N,N'-bis (2-ethanesulfonic acid, pH 6.4), 400
mM sodium chloride, and 1 mM EDTA. After
hybridization, 8 µg/ml RNase A and 0.4 µg/ml RNase T1 (Sigma,
Dorset, UK) were added and incubated for 1 h at 30 C to digest
nonhybridized RNA. Protected hybrids were isolated by ethanol
precipitation and separated on a 6% polyacrylamide/7 M
urea denaturing sequencing gel. The dried gel was exposed to x-ray
film. The expected protected fragments were 296 bp for the GHR1-279
alternate splice and 217 bp for the GHRfl (the smaller hybrid for the
full length as GHR1-279 was used as the probe).
Construction of GHRfl and GHR1-279 Expression Vectors
The full-length human GHR has proved difficult to assemble and
propagate in E. coli. This problem has been
overcome by changing 24 nucleotides largely in the transmembrane domain
while maintaining the native amino acid sequence (35). This construct,
kindly provided by Genetech, was subcloned into the expression vector
pcDNAI/Amp (Invitrogen) using the BamHI and SnaBI
restriction sites to produce the GHRfl. This construct contains a
unique BstBI site engineered at the end of exon 8, which is
not found in the native sequence. The expression vector GHR1-279 was
constructed by introducing a BstBI restriction site by PCR
amplification of GHR1-279, subcloning back into the pCRII vector
(Invitrogen), then digesting with BstBI and NotI,
and ligating into the GHRfl at the same sites. The construct was then
sequenced to confirm the modified sequence.
Binding Assays
Twenty four hours after transfection, the cells were serum
starved for 12 h. The culture media was removed and concentrated
(20x) to be analyzed for the presence of GHBP using gel filtration and
HPLC (15). Cells were then washed with PBS containing 1% BSA and
incubated with [125I]-hGH (5 x 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 NaOH 1 N for
counting radiation.
Cross-Linking
Cells were grown in six-well plates and transfected with 5 µg
cDNA; 5 x 105 cpm/well of [125I]-hGH
were added to the dishes in the absence or presence of 5 µg/ml of
cold hGH, and incubated for 30 min at 37 C. The cells were then washed
with PBS, and 2 ml PBS were added to each well. Dissuccinimidylsuberate
(0.5 mM in dimethyl sulfoxide, Pierce, Rockford, IL) was
added to the cells, mixed, and incubated for 20 min at room
temperature. The reaction was stopped with 2 ml Tris, 50
mM, NaCl, 150 mM, and cells were lysed in 50
µl SDS-sample buffer. Electrophoresis was performed on a 8%
SDS-polyacrylamide gel. The gel was dried and exposed to x-ray
film.
Immunoprecipitations and Western Blotting
Human GH (Genotropin, Pharmacia) was biotinylated using
the Boeringher kit at a ratio of 1:5 molar. For immunoprecipitations,
5 x 106 cells were transfected with 10 µg of GHRfl
or GHR1-279 cDNA alone or with a combination of 5 µg of each cDNA.
Twenty four hours after transfection, cells were incubated in
starvation media for 16 h and then stimulated with either 2
µg/ml of hGH or biotinylated hGH for 5 min at 37 C. One milliliter of
normal human serum was also incubated with 2 µg biotinylated hGH.
Cell lysates were then incubated overnight at 4 C with 4 µg/ml
affinity-purified
GHR-intra antibody (2) and protein A Sepharose or
with 30 µl of streptavidin beads. Supernatants (10 ml) and serum were
incubated with 30 µl of streptavidin beads. Purified proteins were
applied to a 10% polyacrylamide-SDS gel, transferred to a
nitrocellulose membrane, and probed with 5 µg/ml of the GHR antibody
mAb 263 (Biogenesis). Detection was performed with the chemiluminescent
detection system (Amersham).
Transcription Assays
293 cells were plated in six-well plates at 0.4 x
106 cells per well before being transfected with 0.1 µg
of the pcDNA1 expression vector containing the GHRfl and/or 0 to 1 µg
GHR1-279, 1.5 µg LHRE/TK-luciferase reporter gene (17), and 3 µg
pCH110 (fl-galactosidase expression vector, Pharmacia). Cells were then
incubated for 24 h in serum-free medium with or without 20 or 1
nM hGH. Cells were lyzed and luciferase and galactosidase
activities measured. Luciferase activity was normalized to the
galactosidase activity. All experiments were performed in
triplicate.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful for the support and advice of Professor GM
Besser and Professor PA Kelly.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. R. J. M. Ross, Department of Medicine, Clinical Sciences, Northern General Hospital, Sheffield. S5 7AU, UK.
S.L.C. is supported by a Wellcome Trust Clinician Scientist Fellowship,
and S.V.L. by a grant from the YCRC. The work was supported by grants
from the Clinical Endocrinology Trust, Royal Society, Northern General
Hospital Research Committee, and Serono Laboratories, UK.
Received for publication April 11, 1996.
Revision received December 19, 1996.
Accepted for publication December 20, 1996.
 |
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