Endocrinology Vol. 139, No. 8 3585-3589
Copyright © 1998 by The Endocrine Society
Development of a Homologous Radioimmunoassay for Mouse Growth Hormone Receptor1
Ignacio G. Camarillo,
Gudmundur Thordarson,
Yonca N. Ilkbahar and
Frank Talamantes
Department of Biology, University of California, Santa Cruz,
California 95064
Address all correspondence and requests for reprints to: Dr. Frank Talamantes, Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064. E-mail: PRL{at}aol.com
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Abstract
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A RIA for mouse GH receptor (mGHR) was developed. A synthetic peptide
corresponding to the carboxyl-terminal 14 amino acids of the mGHR
(GHR-2 peptide) was used as the antigen for antiserum production. The
synthetic peptide was also used as the standard and radioligand in the
RIA. The ability of the antiserum to recognize the mGHR was
demonstrated by quantitating receptor concentrations in liver and
mammary gland from virgin and 15-day-pregnant mice. Serial dilutions of
these samples yielded displacement curves parallel to the synthetic
peptide. No significant cross-reactivity was seen with serum from
virgin or 15-day-pregnant mice, mGH, recombinant mGH-binding protein
(mGHBP), a synthetic peptide identical to the hydrophilic tail of
mGHBP, or a 14-amino acid synthetic peptide corresponding to amino
acids 338–351 of mGHR (GHR-1 peptide). The concentration range of the
mGHR RIA was 0.5–200 nM, and the intra- and interassay
coefficients of variation were 6.5% and 6.1%, respectively. The
concentration of liver GHR increased significantly during pregnancy
compared with that in virgin mice, from 0.246 ± 0.045 pmol/mg
protein (mean ± SEM; n = 5) in the virgin
animals to 1.015 ± 0.159 pmol/mg protein (n = 5) in pregnant
mice. In contrast, the mGHR concentration in the mammary gland
decreased significantly during pregnancy from 0.606 ± 0.201
pmol/mg protein (mean ± SEM; n = 5) to
0.299 ± 0.027 pmol/mg protein (n = 5). Comparison of the
total number of binding sites in livers from virgin and pregnant mice
using the GH RRA and the combined results of the mGHR and mGHBP RIAs
showed that the two methods gave almost identical results for livers
from virgin animals, or 0.363 ± 0.063 pmol/mg protein (mean
± SEM; n = 3) and 0.371 ± 0.008 pmol/mg protein
(n = 3) for the GH RRA and the mGHR plus mGHBP RIAs, respectively.
However, in livers from pregnant animals, the combined results from the
mGHR and mGHBP RIAs were approximately 1.8 times higher than those
obtained by the GH RRA, or 6.732 ± 0.612 pmol/mg protein
(mean ± SEM; n = 3) and 3.693 ± 0.67
pmol/mg protein (n = 3) for the mGHR plus the mGHBP RIAs and the
GH RRA, respectively. The increase in the total GH binding capacity in
livers from pregnant mice compared with those from virgin animals was
largely due to an increase in the GHBP content. The increase in GHR was
only 2.4-fold, or from 0.153 ± 0.01 pmol/mg protein (mean ±
SEM; n = 3) in virgin mice to 0.364 ± 0.03
pmol/mg protein (n = 3) in the 15-day-pregnant mice, whereas GHBP
increased almost 30-fold during pregnancy, or from 0.218 ± 0.003
pmol/mg protein (mean ± SEM; n = 3) in virgin
animals to 6.369 ± 0.607 pmol/mg protein (n = 3) in pregnant
mice.
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Introduction
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THE BIOLOGICAL actions of GH are mediated
through its interaction with a membrane-bound GH receptor (GHR) (1) and
a GH-binding protein (GHBP) (2). In rodents, GHBP is identical to the
extracellular domain of the GHR except for a 27-amino acid hydrophilic
tail that substitutes for the transmembrane and cytoplasmic domains of
the GHR (3, 4). It has recently been shown that mouse (m) and rat (r)
GHBPs are generated from alternatively spliced messenger RNAs (5, 6, 7, 8).
In contrast, human and rabbit GHBPs probably result from the
proteolytically cleaved extracellular portions of the GHR (3, 9).
The levels of mGHR in different tissues have previously been assessed
by RRA (10, 11, 12). In this assay, cell membrane preparations from
homogenated tissues have been isolated by differential centrifugation,
followed by an incubation of the cell membrane preparation with
125I-labeled GH for measurement of total GH binding.
Although this method may give a reasonable assessment of the total
binding capacity for GH in a particular tissue, it does not distinguish
between the binding of GH to the GHR and the GHBP. This distinction is,
however, very important, as growing evidence now indicates that a
substantial portion of the total GH binding capacity in different
tissues is the result of the presence of GHBP (13, 14). Previously, we
developed a RIA for mGHBP (15). The development of a sensitive and
specific RIA for GHR will allow us to accurately measure the levels of
GHR in different tissues and compare them with those of GHBP.
In this report, we describe the development of a GHR RIA using
antiserum generated against a synthetic peptide corresponding to
the carboxyl-terminus of the mGHR, a sequence not present in the
GHBP. The RIA was used to measure liver and mammary gland mGHR
concentrations at different physiological states. In addition, the
values obtained by RIAs for GHR and GHBP were compared with those
obtained by RRA, which assesses total GH binding.
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Materials and Methods
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Timed pregnant Swiss-Webster mice were obtained from Simonsen
Laboratories (Gilroy, CA; plug found = day 0). The mice were
housed in a controlled environment with a 14-h light, 10-h dark
lighting cycle and fed ad libitum. The care and use of
animals in this study were approved by the chancellor’s animal care
committee at the University of California-Santa Cruz.
A synthetic peptide corresponding to amino acids 338–351 (GHR-1
peptide) of the mGHR and a synthetic peptide corresponding to amino
acids 642–655, the carboxyl-terminus of the GHR (GHR-2 peptide) (7),
were synthesized by Chiron Mimotopes U.S. (Emeryville, CA). A synthetic
peptide identical to the hydrophilic tail of the mGHBP (mGHBP-tail
peptide) (15) was synthesized by Multiple Peptide Systems (San Diego,
CA).
mGH and recombinant mGHBP (rmGHBP) were purified as previously
described by this laboratory (16, 17). Recombinant bovine GH was a gift
from Monsanto (St. Louis, MO). GHR-2 peptide and bovine GH were
radiolabeled with Na125I (Amersham, Arlington Heights, IL)
using the Iodogen method (18).
GHR-2 peptide was conjugated to keyhole limpet hemocyanin (KLH; Pierce,
Rockford, IL) as described previously (19). Two rabbits were initially
injected sc with 500 µg of the synthetic mGHR-peptide-KLH conjugate
in Freund’s complete adjuvant in a ratio of 1:2 (conjugate/adjuvant)
in a total volume of 1 ml/animal. One month after the initial
injection, the rabbits received a second sc injection identical to the
first injection. One month after the second injection, the animals were
injected with 100 µg synthetic mGHR-peptide-KLH conjugate in
Freund’s incomplete adjuvant in the same ratio of conjugate to
adjuvant and in the same volume as described above.
Liver, mammary gland, and blood were collected from virgin and
15-day-pregnant mice. Blood was centrifuged at 2,000 x
g for 10 min, and serum was harvested and stored frozen.
Tissues were washed in 0.9% saline, frozen on dry ice, and stored at
-70 C. Tissues were then homogenized in 4 x (wt/vol)
homogenization buffer (0.3 M sucrose, 50 mM
HEPES, 1 mM phenylmethylsulfonylfluoride, 1
µM pepstatin A, 10 mM EDTA, and 1 mg/ml
bacitracin, pH 8.0) for 30 s at 16,000 rpm using a Brinkmann
Polytron homogenizer (Brinkmann Instruments, Westbury, NY). Homogenates
were centrifuged at 20,000 x g for 30 min, after which
the resulting supernatant was centrifuged at 100,000 x
g for 1 h. The pellet was washed in solubilization
buffer (50 mM HEPES, 10 mM EDTA, 1
mM Pefabloc (Boehringer Mannheim, Indianapolis, IN), 1
µM pepstatin A, and 1 mg/ml bacitracin, pH 7.5) and
centrifuged again at 100,000 x g for 1 h. Pellets
were resuspended in RIA buffer containing 2% Triton X-100 and left
shaking on an orbital shaker for 1 h at 4 C. Samples were then
recentrifuged at 100,000 x g, and the supernatant was
harvested. The resulting solubilized membrane proteins were frozen at
-70 C until use in the GHR RIA. The total protein concentration in
each sample was determined using the bicinchoninic acid protein assay
kit (Pierce). A solubilized membrane protein preparation of several
maternal livers from 17-day-pregnant mice was used as an internal
control in each RIA. This preparation was also used to determine the
inter- and intraassay coefficients of variability.
For the mGHR RIA, 100-µl aliquots of serial dilutions of uncoupled
mGHR-2 peptide (standard) in RIA buffer [10 mM
Na2HPO4 (pH 7.5), 10 mM EDTA, 150
mM NaCl, 0.1% (wt/vol) RIA grade BSA, 0.01% (wt/vol)
thimerosal, and 2% Triton X-100] were mixed with 20,000 cpm
[125I]iodo-GHR-2 peptide in 100 µl RIA buffer and 100
µl of a 1:2000 dilution of GHR-2 peptide antiserum in RIA buffer
containing 3% nonimmune rabbit serum. After a 16-h incubation at 23 C,
100 µl goat antirabbit IgG diluted 1:10 in RIA buffer were added to
each tube and incubated for 30 min at 23 C, followed by the addition of
100 µl 30% polyethylene glycol. The tubes were immediately vortexed
and centrifuged at 9000 x g for 20 min at 4 C. The
supernatants were aspirated, and the pellets were counted for
radioactivity in a 
-counter. Nonspecific binding was determined by
substitution of 100 µl RIA buffer containing 3% nonimmune rabbit
serum for the antipeptide antiserum. For determination of GHR
concentrations in tissue samples, 100 µl solubilized membrane
proteins, serial diluted several times in RIA buffer containing 2%
Triton X-100, were used. All samples were assayed in triplicate.
Interference of endogenous mGH with the mGHR RIA was tested by assaying
identical protein preparations in the absence or presence of 5 µg/ml
mGH.
To measure the GH binding capacity, liver microsomal membranes from
virgin or 15-day-pregnant mice prepared as outlined above were
MgCl2 stripped as described by Gerasimo et al.
(20), and the protein concentration was determined. Membranes from
virgin or pregnant mice were diluted in RRA buffer (50 nM
HEPES, 0.1% BSA, 10 mM MgCl2, and 0.01%
thimerosal, pH 8.0) at 4 and 2 mg/ml, respectively. The resulting
membrane preparations were used in a RRA according to methods described
previously by our laboratory (12). Subsamples from the same tissue
homogenates used for the RRA were detergent solubilized in RIA buffer
and assayed with the RIA for the mGHR and with a RIA for mGHBP
previously developed in our laboratory (15). This allowed a direct
comparison of the values for the GHR and GHBP obtained using the RRA
with those obtained by the RIAs for GHR and GHBP. It should be noted
that the calculated values obtained using the RRA represent picomoles
of bound GH per mg protein, whereas values from the RIAs represent
picomoles of GHR or GHBP per mg protein.
Statistics
The slopes of the RIA displacement curves were compared by
linear regression analysis. The concentrations of GHR in maternal liver
and mammary gland of virgin and 15-day-pregnant mice were compared by
ANOVA followed by Fisher’s protected least difference test. Analysis
of competitive binding assays was performed using the method of
Scatchard (21). In all cases, differences between mean concentrations
were considered significant at P < 0.05.
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Results
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Dilution of the GHR-2 peptide primary antiserum to 1:2000 resulted
in approximately 55% total binding of the [125I]GHR-2
peptide. Nonspecific binding ranged between 3–5% (n = 8). The
mGHR RIA standard curve was compared with displacement curves generated
from increasing concentrations of solubilized membrane proteins from
liver and mammary gland of virgin female and 15-day-pregnant mice (Fig. 1
). The usable range of the standard
curve was from 0.5–200 nM. Displacement curves for liver
and mammary gland from both virgin and 15-day-pregnant mice were
parallel to the standard curve.
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Figure 1. Displacement curves for the GHR-2 peptide, liver
and mammary gland membrane samples, and serum from virgin and
15-day-pregnant mice using [125I]iodo-GHR-2 peptide as a
tracer and antiserum generated against the GHR-2 peptide. Each dilution
was assayed in triplicate. VG-MG, Mammary gland membrane sample from
virgin mice; PG-MG, mammary gland membrane sample from pregnant mice;
VG-LV, liver membrane sample from virgin mice; PG-LV, liver membrane
sample from pregnant mice; VG-SER, serum sample from virgin mice;
PG-SER, serum sample from pregnant mice.
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The intraassay coefficient of variance was 6.5% (n = 30), as
assessed for a liver sample pool from 17-day-pregnant mice. The
interassay coefficient of variance for the same liver sample was 6.1%
(n = 6).
The antiserum specificity was tested by generating
displacement curves using increasing concentrations of various
peptides and serum from virgin and 15-day-pregnant mice. No
cross-reactivity of the antiserum with GHR-1 peptide, mGH, rmGHBP,
mGHBP tail peptide, or sera from virgin and pregnant mice was observed
(Figs. 1
and 2). To assess possible
interference of endogenous GH in the RIA, identical liver membrane
preparations from virgin and 15-day-pregnant mice were assayed with the
GHR RIA in either the absence or presence of 5 µg/ml mGH. No
significant difference in GHR concentration was found in the tissue
preparations regardless of whether they were assayed in the presence or
absence of excess mGH (data not shown).
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Figure 2. Test of cross-reactivity of mGHR-2 peptide
antiserum with various peptides. Increasing concentrations of GHR-1
peptide, mGH, rmGHBP, and GHBP (tail peptide) were tested in the mGHR
RIA as described in Materials and Methods. Each dilution
was assayed in triplicate.
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Subsequently, the mGHR RIA was used to assess changes in GHR
concentrations in tissues in different physiological states. The
concentration of liver GHR increased significantly during pregnancy
compared with that in virgin mice, or from 0.246 ± 0.045 pmol/mg
protein (mean ± SEM; n = 5) in virgins to
1.015 ± 0.159 pmol/mg protein (n = 5) in pregnant mice (Fig. 3). Conversely, the mGHR concentration in
the mammary gland decreased significantly during pregnancy from
0.606 ± 0.201 pmol/mg protein in virgins (mean ±
SEM; n = 5) to 0.299 ± 0.027 pmol/mg protein
(n = 5) in the pregnant mice (Fig. 3).
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Figure 3. Concentrations of GHR in maternal liver and
mammary gland microsomal membrane fractions of virgin and
15-day-pregnant mice. Concentrations of mGHR were determined by RIA as
described in Materials and Methods. Each
bar represents the mean ± SEM (n
= 5). Upper- and lowercase letters indicate significant differences
(P < 0.05) in GHR concentrations between samples
within the same grouping.
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Comparison of the total number of binding sites in livers from virgin
and pregnant mice using the GH RRA and the combined results from the
mGHR and mGHBP RIAs showed that the two methods gave almost identical
results for livers from virgin animals, or 0.363 ± 0.063 pmol/mg
protein (mean ± SEM; n = 3) and 0.371 ±
0.008 pmol/mg protein (n = 3) for the GH RRA and the mGHR
plus mGHBP RIAs, respectively. However, in livers from pregnant
animals, the combined results from the mGHR and mGHBP RIAs were
approximately 1.8 times higher than those obtained by the GH RRA, or
6.732 ± 0.612 pmol/mg protein (mean ± SEM;
n = 3) and 3.693 ± 0.67 pmol/mg protein (n = 3) for the
mGHR plus the mGHBP RIAs and the GH RRA, respectively (Fig. 4).
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Figure 4. Concentrations of GHR and GHBP in liver microsomal
membrane fractions as determined by RRA and RIA. Concentrations of mGHR
in liver membranes from virgin or 15-day-pregnant mice were determined
by RIA and RRA as described in Materials and Methods. In
addition, the concentration of mGHBP was determined by RIA in the same
samples. Each bar represents the mean ±
SEM (n = 3). Upper- and lowercase letters indicate
significant differences (P < 0.05) in GHR
concentrations between samples within the same grouping.
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The increase in the total GH binding capacity in livers from pregnant
mice compared with that in virgin animals was largely due to an
increase in the content of the GHBP. The increase in GHR was only
2.4-fold, or from 0.153 ± 0.01 pmol/mg protein (mean ±
SEM; n = 3) in virgin mice to 0.364 ± 0.03
pmol/mg protein (n = 3) in the 15-day-pregnant mice, whereas the
GHBP increased almost 30-fold during pregnancy, or from 0.218 ±
0.003 pmol/mg protein (mean ± SEM; n = 3) in
virgin animals to 6.369 ± 0.607 pmol/mg protein (n = 3) in
pregnant mice (Fig. 4).
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Discussion
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Previously, GHR in mice has been measured by evaluating GHR
messenger RNA (mRNA) expression and the ability of
125I-labeled GH to bind to membrane samples (10, 11, 12).
Although each of these techniques gives valuable information regarding
relative GHR gene expression, neither specifically quantitates mGHR at
the protein level. The characterization of GHR mRNA concentrations is
necessary, but may not always correspond with concentrations of the
translated product. Binding studies require large quantities of samples
and are tedious and time consuming. Additionally, a serious problem
with [125I]GH binding is that it does not distinguish
between GHR or other similar proteins, such as GHBP, interacting with
the radioactive ligand. Another problem with using a binding assay for
measuring GHR concentrations is the interference from binding of
endogenous GH.
In this study, we report the development of a specific and sensitive
homologous RIA for mGHR. The synthetic peptide (GHR-2 peptide), which
corresponds to the carboxyl-terminal region of the mGHR, was used to
generate antiserum and as a tracer and standard in the RIA. The ability
of the antiserum to recognize native mGHR was shown by using the RIA to
specifically measure mGHR in mouse liver and mammary gland samples of
increasing protein concentrations. Serial dilutions of serum samples
served as controls in which no mGHR was detectable. Solubilized liver
and mammary gland extracts, but not serum, from virgin and pregnant
mice displaced mGHR peptide tracer from antibodies in a parallel
fashion to unlabeled mGHR peptide. This parallelism among displacement
curves demonstrated that the assay accurately measures differences in
GHR concentrations between membrane preparations. Further tests of the
accuracy of the RIA were made by assessing mGHR concentrations in
maternal liver and mammary gland from virgin and 15-day-pregnant mice.
The differences between virgin and pregnant mGHR levels in liver and
mammary gland, as determined by RIA, correlated well with the changes
in GHR mRNA levels (22). In addition, the ratio of GHBP/GHR proteins
and mRNAs in the mouse liver both increased during pregnancy, although
this increase was more pronounced for the ratio of the proteins. This
could be an indication of a differential regulation of the stability of
the two mRNAs or a difference in the translational rates of the two
proteins associated with pregnancy. The specificity of the RIA was
established by generating displacement curves using GHR-2 peptide
antiserum in the presence of other peptides (GHR-1 peptide, mGH,
rmGHBP, and mGHBP tail peptide), which showed no detectable antiserum
cross-reactivity. Furthermore, occupation of the GHR by its ligand had
no influence on the efficiency of the RIA, as no significant difference
was found in GHR levels regardless of whether membrane protein samples
had been previously saturated with mGH or were untreated.
To further evaluate whether the antipeptide antiserum used in this GH
RIA might cross-react with other known proteins, a search of homologous
sequences in databanks (GenBank and EMBL) was performed. This search
revealed no homology of the synthetic peptide to any known sequence
except for the rGHR 14-amino acid carboxyl-terminal. This was not
unexpected, as we chose a sequence within the mGHR that had 100%
homology with the rGHR. This will enable us to use this assay for
samples from both mice and rats.
The total GH binding capacity in the liver, as estimated with the
traditional GH RRA, agreed well with the combined value obtained from
the GHR and GHBP RIAs. This is particularly apparent for liver samples
from virgin mice, where the values obtained from these two methods were
identical. An approximately 1.8-fold higher level of total GH binding
capacity in the pregnant mouse was seen for the combined results from
the two RIAs compared with those obtained using the RRA. We do not know
as yet why these two assay methods assessed the GH binding capacity in
the liver of pregnant mice differently. It should be noted, however,
that it appears as if the GH RRA detects the GHBP less effectively than
the GHBP RIA. For example, we showed in this study that most of the
increase in the GH binding capacity that occurs during pregnancy is
caused by increase in GHBP, and it is during pregnancy that the
discrepancy between the two assay methods becomes apparent. There could
be several reasons why the GH RRA is less efficient in measuring GHBP
than GHR. To mention only two possibilities, it has been shown that GH
forms complexes of one ligand to two receptors or binding proteins
(23). Dimerization could be favorable in an environment of high binding
protein concentration or where a larger total number of GH binding
sites exist, when the ligand concentration is kept constant. High
levels of dimerization would register in the RRA as lower levels of
binding sites. It should also be kept in mind that we do not know how
tightly the GHBP is associated with the membrane. It is possible that
some of the ligand-binding protein complexes may solubilize during the
assay procedure and, therefore, become lost upon separation of free and
bound [125I]GH. In addition, we know from the
present study that each tissue sample contains a mixture of GHR and
GHBP. We also know that mGHR has 10-fold higher affinity for GH than
mGHBP (12). A difference in the ratio of GHR/GHBP in each sample,
therefore, could skew the results in the RRA.
In summary, we have developed a sensitive and specific RIA for mGHR.
This combined with our previously developed RIA for the mGHBP (15)
makes us well equipped to elucidate the distribution and ratio of these
two forms of the GHR in different tissues at different physiological
stages. As a first step in this effort, we showed here that
approximately 97% of the increase in the total GH binding capacity of
the liver during pregnancy in mice is caused by an increase in the
binding protein, not by a substantial increase in the level of the GHR
as was previously speculated.
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Acknowledgments
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We thank Dr. J. Southard for initial preparation of
antiserum, and Dr. L. Ogren for help in preparing this manuscript.
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Footnotes
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1 This work was supported by NIH Grants CA-71590 and GM-08132 (to
F.T.). 
Received January 8, 1998.
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Top
Abstract
Introduction
