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Development of a Homologous Radioimmunoassay for Mouse Growth Hormone Receptor¹

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

	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.

	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.

	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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

View larger version (25K): [in this window] [in a new window]   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.

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).

View larger version (20K): [in this window] [in a new window]   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.

View larger version (53K): [in this window] [in a new window]   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.

View larger version (37K): [in this window] [in a new window]   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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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.

	Acknowledgments

 
We thank Dr. J. Southard for initial preparation of antiserum, and Dr. L. Ogren for help in preparing this manuscript.

	Footnotes

 
¹ This work was supported by NIH Grants CA-71590 and GM-08132 (to F.T.).

Received January 8, 1998.

	References

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