Constitutive Activation of the Cyclic Adenosine 3',5'-Monophosphate Signaling Pathway by Parathyroid Hormone (PTH)/PTH-Related Peptide Receptors Mutated at the Two Loci for Jansen’s Metaphyseal Chondrodysplasia

E. Schipani, G. S. Jensen, J. Pincus, R. A. Nissenson, T. J. Gardella and H. Jüppner

Endocrine Unit (E.S., G.S.J., J.P., T.J.G., H.J.), Department of Medicine and Children’s Service (H.J.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, Department of Medicine (R.A.N.), Veterans Affairs Medical Center and University of California, San Francisco, California 94121-1545


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Two different activating PTH/PTH-related peptide (PTHrP) receptor mutations, H223R and T410P, were recently identified as the most likely cause of Jansen’s metaphyseal chondrodysplasia. To assess the functional importance of either amino acid position in the human PTH/PTHrP receptor, H223 and T410 were individually replaced by all other amino acids. At position 223, only arginine and lysine led to agonist-independent cAMP accumulation; all other amino acid substitutions resulted in receptor mutants that lacked constitutive activity or were uninformative due to poor cell surface expression. In contrast, most amino acid substitutions at position 410 conferred constitutive cAMP accumulation and affected PTH/PTHrP receptor expression not at all or only mildly. Mutations corresponding to the H223R or T410P exchange in the human PTH/PTHrP receptor also led to constitutive activity when introduced into the opossum receptor homolog, but showed little or no change in basal cAMP accumulation when introduced into the rat PTH/PTHrP receptor. The PTH/PTHrP receptor residues mutated in Jansen’s disease are conserved in all mammalian members of this family of G protein-coupled receptors. However, when the equivalent of either the H223R or the T410P mutation was introduced into several other related receptors, including the PTH2 receptor and the receptors for calcitonin, secretin, GH-releasing hormone, glucagon-like peptide I, and CRH, the resulting mutants failed to induce constitutive activity. These studies suggest that two residues in the human PTH/PTHrP receptor, 223 and 410, have critical roles in signal transduction, but with different sequence constrains.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The receptor for PTH and PTH-related peptide (PTHrP) is a member of the superfamily of receptors coupled to guanyl nucleotide-binding regulatory G proteins; it signals through at least two second messenger systems, adenylate cyclase and phospholipase C, and can be activated with similar or indistinguishable potency and efficacy by two peptides, the amino-terminal fragments of PTH and PTHrP, respectively (1). Together with the receptors for calcitonin (CT) and secretin, it belongs to a distinct family of G protein-coupled receptors, which also includes the PTH2 receptor (2). All members of this secretin/CT/PTH receptor family have seven membrane-spanning helices, a relatively long amino-terminal, extracellular domain with six conserved, functionally important cysteine residues (3), and up to four potential sites for N-linked glycosylation. Approximately 45 amino acids, mostly located in the membrane-embedded region, are strictly conserved in all members of this receptor family and thus are likely to have important functions in ligand binding and/or signal transduction (1).

Two activating PTH/PTHrP receptor mutations, H223R and T410P, were recently identified as the likely cause of Jansen’s metaphyseal chondrodysplasia, a rare genetic form of short-limbed dwarfism that is associated with severe hypercalcemia and hypercalciuria, despite normal or undetectable levels of PTH and PTHrP (4, 5). Compared with COS-7 cells transfected with the complementary DNA (cDNA) encoding the wild-type (WT) human PTH/PTHrP receptor, cells expressing receptors with the H223R or the T410P mutation showed constitutive, ligand-independent basal cAMP accumulation and increased binding affinity for agonists, but not for antagonists (4, 6).

G protein-coupled receptors are thought to exist, in the absence of an agonist, in an equilibrium between inactive and active conformational states (R and R*, respectively). Agonist ligands bind to R* with higher affinity than to R, while pure antagonists show similar binding affinities for either receptor conformation (7, 8). Besides the findings in Jansen’s disease (4, 5, 9), various mutations that lead to constitutive activation of different receptors have been identified as the molecular basis of several human diseases (10). Such mutations can potentially provide important insights into the fundamental mechanisms involved in ligand binding and receptor activation, and they are an important tool to study the mechanism(s) by which inverse agonists restore the R conformation of the receptor (6). To further characterize the roles of positions 223 and 410 in activation of the cAMP pathway by the human PTH/PTHrP receptor, the native residues at these sites, histidine and threonine, respectively, were replaced individually by all 19 natural amino acids. Because both residues are conserved in all mammalian members of the secretin/CT/PTH receptor family, the equivalent of either the H223R or the T410P mutation was also introduced into several other members of this receptor family. The aim of the study was 2-fold: 1) to identify specific residues at the two positions that are important for receptor activation, and 2) to evaluate the potential generality of the activating mechanism of the cAMP pathway within this distinct family of G protein-coupled receptors.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Residues H223 and T410 in the human PTH/PTHrP receptor were individually changed by mutagenesis to each of the other 19 natural amino acids (Fig. 1Go). Cell surface expression and maximal specific radioligand binding of all mutated receptors were then assessed. For this purpose, COS-7 cells were transiently transfected with comparable amounts of plasmid DNAs (100–200 ng/well) and tested with the anti-H2 antibody that recognizes the amino-terminal, extracellular domain of the human PTH/PTHrP receptor (11). Radioligand binding with radiolabeled PTH-(1–34) was performed as described in Materials and Methods. All amino acid substitutions at position 223, with the exception of H223N, H223V, and H223T, resulted in mutant receptors with levels of surface expression less than 50% of those observed with the WT receptor (Fig. 2AGo). The average maximal specific radioligand binding of these mutant receptors, calculated as a percentage of the maximal specific radioligand binding to the WT receptor, was 58 ± 9.7% (mean ± SE). In contrast, H223N, H223V, and H223T showed higher radioligand binding of 109 ± 8.4% (mean ± SE), as predicted by their higher levels of expression. Consistent with these findings, the replacement of H220 in the opossum PTH/PTHrP receptor (which is equivalent to H223 in the human PTH/PTHrP receptor) by either aspartate or alanine resulted in mutant receptors with reduced or undetectable levels of cell surface expression (12).



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Figure 1. Scheme of the Human PTH/PTHrP Receptor

The positions of residues H223 and T410 are indicated. Partial amino acid sequences of other members of this family of G protein-coupled receptors are aligned.

 


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Figure 2. COS-7 Cells Expressing Human PTH/PTHrP Receptors with Amino Acid Substitutions at Position 223 Were Assessed for Cell Surface Expression and cAMP Accumulation

COS-7 cells were transiently transfected with comparable amount of plasmid cDNAs (100–200 ng/well) encoding WT ({blacksquare}), the H223R mutant ({square}), or other mutant receptors (), as described in Materials and Methods, and were then evaluated for receptor expression levels, and basal and PTH-stimulated cAMP accumulation. A, Anti-H2 antibody binding. Data are presented as percentage of the expression levels of WT receptor and are the mean ± SE of at least two independent experiments, each performed in duplicate. B, Basal cAMP accumulation. Data are expressed as fold increase over basal cAMP levels obtained with cells transfected with the WT receptor and represent the mean ± SE of at least two independent experiments, each performed in duplicate. C, cAMP accumulation after stimulation with PTH-(1–34) (70 nM). Data are expressed as fold increase over basal cAMP levels obtained in cells transfected with the WT receptor and represent the mean ± SE of at least two independent experiments, each performed in duplicate.

 
A different pattern of expression was observed for mutations at position 410; most receptors with mutations at this site, with the exception of T410I, T410P, and T410V, had levels of surface expression that reached at least 50% those of the WT receptor (Fig. 3AGo). The average maximal specific radioligand binding of all mutant receptors at position 410 was 115 ± 8% (mean ± SE). These data suggested that residue 223 is important for proper folding and/or expression of the receptor, whereas residue 410 is less critical for these functions.



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Figure 3. COS-7 Cells Expressing Human PTH/PTHrP Receptors with Amino Acid Substitutions at Position 410 Were Assessed for Cell Surface Expression and cAMP Accumulation

COS-7 cells were transiently transfected with comparable amounts of plasmid cDNAs (100–200 ng/well) encoding WT ({blacksquare}), the T410P mutant ({square}), or other mutant receptors () as described in Materials and Methods, and were then evaluated for receptor expression levels, and basal and PTH-stimulated cAMP accumulation. A, Anti-H2 antibody binding. Data are presented as percentage of the expression levels of WT receptor and are the mean ± SE of at least two independent experiments, each performed in triplicate. B, Basal cAMP accumulation. Data are expressed as fold increase over basal cAMP levels obtained with cells transfected with the WT receptor and represent the mean ± SE of at least two independent experiments, each performed in duplicate. C, cAMP accumulation after stimulation with PTH-(1–34) (70 nM). Data are expressed as fold increase over basal cAMP levels obtained in cells transfected with the WT receptor and represent the mean ± SE of at least two independent experiments, each performed in duplicate.

 
We next examined which amino acid substitutions, in addition to those identified in patients with Jansen’s metaphyseal chondrodysplasia, resulted in altered cAMP signaling. As previously reported, COS-7 cells expressing the WT human PTH/PTHrP receptor showed, depending on the experimental conditions, no or only a small increase in basal cAMP accumulation (4, 5, 6). At position 223, only the substitution of histidine by lysine (H223K) led to constitutive receptor activation (Fig. 2BGo). Therefore, as expected, COS-7 cells transfected with increasing concentrations of the plasmid DNA (12.5–400 ng/well) encoding the H223K mutant showed increasing constitutive activity (Fig. 4Go). However, basal cAMP accumulation by cells expressing the H223K mutant was considerably lower than that observed with the H223R mutant despite similar levels of surface expression (Fig. 2Go, A and B). Among the other receptors with mutations at position 223, some were very poorly expressed and thus uninformative. Most of the other receptors had expression levels comparable to those of the H223R mutant, yet no constitutive activation was detected (Fig. 2Go, A and B). Therefore, only the substitution of a positively charged residue at position 223, with arginine substantially favored, led to ligand-independent cAMP accumulation. However, we cannot rule out that some receptors with mutations at position 223, other than R and K, would display a certain degree of constitutive activity, if expressed at higher levels.



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Figure 4. Basal cAMP Accumulation in COS-7 Cells Transfected with Increasing Amounts of Plasmid DNAs Encoding the WT Human PTH/PTHrP Receptor and the H223K Mutant

COS-7 cells were transiently transfected with increasing amounts of plasmid DNA encoding WT ({square}) or mutant ({blacksquare}) receptors and were then evaluated for basal cAMP accumulation. Data are expressed as picomoles per well/15 min and are the mean ± SD of at least two independent experiments, each performed in duplicate.

 
A different signaling profile was seen with mutations at position 410. Most of the amino acid substitutions at this site resulted in mutant receptors with some degree of constitutive activity, which ranged from 2-fold (D410) to 7.5-fold (C410) above the basal levels of WT receptors (Fig. 3BGo).

Basal cAMP accumulation of cells expressing mutant receptors reached up to 27% of the levels obtained with WT human PTH/PTHrP receptors that had been stimulated with a maximal concentration of PTH (70 nM). Interestingly, this level of constitutive cAMP formation was comparable to that typically observed when COS-7 cells expressing the WT receptor were stimulated with 0.1 nM PTH-(1–34) (4, 5). This agonist concentration is well within the range of the circulating levels of intact PTH in states of parathyroid overactivity (13), and thus could explain why patients with Jan-sen’s disease and nonprogressive hyperparathyroidism have similar changes in mineral ion homeostasis and bone turnover (14).

For the different mutations at position 410, no clear pattern was discernible between the degree of constitutive activity and the characteristics of the side-chain of the introduced amino acid. However, most substitutions at residue 410 resulted in constitutive activity, suggesting that threonine at this position functions to constrain the receptor in an inactive conformation that does not interact with Gs{alpha}. Interestingly, in the {alpha}1B-adrenergic receptor all substitutions of alanine 293, an amino acid predicted to be at a position similar to that of residue T410 in the human PTH/PTHrP receptor, also resulted in constitutive receptor activation (15). As with position 410 of the PTH/PTHrP receptor, there was no obvious correlation between the basal activities of these mutated receptors and the chemical properties of the substituted side-chains. Constitutively active mutants were also reported for the {alpha}2-adrenergic receptor (16) and for the receptors for TSH, FSH, and LH/CG (17, 18, 19, 20) when residues at the cytoplasmatic end of the sixth transmembrane domain were mutated to other amino acid residues.

All PTH/PTHrP receptors with mutations at position 223 and 410 were activated by PTH-(1–34) (Figs. 2CGo and 3CGo), with the exception of H223W and several other receptors with mutations at position 223 that were poorly expressed. For each mutant receptor, maximal PTH-induced cAMP accumulation correlated with the level of cell surface expression (Fig. 5Go). However, when stimulated cAMP levels were considered as the relative increase above basal levels, all constitutively active PTH/PTHrP receptors mediated the agonist-induced biological response less efficiently than the WT receptor. Our data thus confirm the observation that some constitutively active mutant receptors display, in response to the respective ligand, a less efficient response in second messenger formation than native receptors. This finding is consistent with the hypothesis that constitutively active receptors are chronically desensitized and/or down-regulated (15, 21, 22); however, it could also be related to a specific defect of the receptor mutants in transmitting a signal after ligand binding.



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Figure 5. Correlation between PTH-Stimulated cAMP Accumulation and Cell Surface Expression for Human PTH/PTHrP Receptors with Mutations at Position 223 or 410

COS-7 cells were transiently transfected with plasmid cDNAs (100–200 ng/well) encoding WT or mutant receptors, as described in Materials and Methods, and were then evaluated for cell surface expression and PTH-stimulated cAMP accumulation (data replotted from Figs. 2Go and 3Go). On the y-axis, PTH-stimulated cAMP accumulation is shown as picomoles per well; on the x-axis, anti-H2 antibody binding is shown as a percentage of the expression levels of the WT receptors ( ). {square} and {blacksquare} represent the receptors with mutations at position 223 and 410, respectively; receptors with the H223R ({square}) or the T410P (|p%) mutation are indicated. The data are the mean ± SE of at least two independent experiments, each performed in duplicate.

 
We previously reported that cells expressing the naturally occurring receptor mutants, H223R and T410P, bind the agonist PTH-(1–34) with higher affinity than cells expressing the WT receptor, whereas little or much less of a change in binding affinity was observed for the antagonist PTH-(7–34) (4, 5, 6). Similar to these findings, competition binding studies with the mutant receptor H223K showed, compared with that of the WT receptor, an increased apparent binding affinity for PTH-(1–34) (IC50 = 5 ± 0.5 and 10 ± 0.8 nM, respectively; n = 5), but not for PTH-(7–34) (IC50 = 100 ± 10 and 100 ± 7 nM, respectively; n = 3). However, four receptors with mutations at residue 410 that led to different degrees of ligand-independent cAMP formation, T410C, T410E, T410G, and T410K, did not display an increase in the apparent binding affinity for PTH-(1–34) (data not shown).

Models of G protein-coupled receptor function predict that receptor activation leads to increased binding affinity for agonists (7). Therefore, these findings suggest that some constitutively active PTH/PTHrP receptors do not completely mimic the active conformation of the WT receptor. However, further work is necessary to fully understand the effect of activating mutations in the PTH/PTHrP receptor on ligand binding.

The equivalents of residues H223 and T410 in the PTH/PTHrP receptor are conserved in all mammalian members of the CT/secretin/PTH receptor family (1). We, therefore, tested whether changing these residues in other family members would alter basal and agonist-stimulated cAMP formation. Opossum PTH/PTHrP receptors (OKO), carrying the H220R or the T407P mutation, exhibited ligand-independent constitutive cAMP accumulation (Table 1Go). Interestingly, these mutant receptors demonstrated enhanced, rather than reduced, agonist-stimulated cAMP accumulation relative to that of the WT receptor. This finding would support the previously formulated hypothesis that constitutively active receptors may have a modified signal transduction efficiency. Rat PTH/PTHrP receptors (R15B) with either the H223R or the T407P mutation were poorly expressed (22 ± 3.8% and 29 ± 3.9% of R15B, respectively; n = 2), and only the H223R mutant showed some degree of constitutive activation (Table 1Go).


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Table 1. Basal and Agonist-Stimulated cAMP Accumulation in COS-7 Cells Expressing Wild-Type and Mutant Receptors of the Secretin/CT/PTH Receptor Family

 
Mutations equivalent to H223R or T410P in other receptors, including the porcine and human CT receptors, failed to cause ligand-independent cAMP accumulation (Table 1Go) (23, 24). Upon challenge with the appropriate agonist, the cAMP response of some of these mutant receptors was greatly impaired, which suggested that they were poorly expressed. With the exception of glucagon-like peptide receptor with the H180R mutation, which appears to be well expressed (24), cell surface expression has not been assessed for other mutants of this receptor family. Nevertheless, our results indicate that the chemical properties of the natural residues at positions 223 and 410 in the PTH/PTHrP receptor and at equivalent positions in other members of this receptor family are not important per se, but only in the context of each receptor type. Comparable data were recently reported for the LH receptor family. In this study, several mutations in transmembrane helices 5 and 6, which cause constitutive activity when introduced into the LH receptor, do not determine ligand-independent cAMP accumulation in the highly homologous FSH receptor unless surrounding portions of the receptors are mutated as well (25).

In summary, our in vitro experiments show that two residues in the human PTH/PTHrP receptor, 223 and 410, play a critical role in signal transduction, and thus extend our previous findings, which indicate that Jansen’s metaphyseal chondrodysplasia is caused by mutations at these sites. Our new data suggest that H223 and T410 modulate different components of the mechanisms involved in receptor activation. Residue 223 is important for efficient receptor expression, and a positive charge at this site appears to be required for agonist-independent receptor activation. The natural threonine at position 410 seems to constrain the receptor in an inactive conformation; this constraint can be relieved by either agonist binding or mutation to almost any other amino acid. Lastly, mutations even of conserved residues can generate different phenotypes in the context of different, but closely related, receptors.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
[Nle8,18,Tyr34]Bovine PTH-(1–34)amide [PTH-(1–34)] and [Nle8,18,Tyr34]bovine PTH-(7–34)amide [PTH-(7–34)] were synthesized as previously described (26). Human GRF-(1–29) was purchased from Peninsula (Belmont, CA), glucagon-like peptide I-(7–37), rat CRF, human secretin-(1–27), and salmon CT-(1–32)amide were purchased from Bachem (Torrance, CA). Na125I (SA, 2000 Ci/mmol) for peptide and monosuccinyl-cAMP tyrosylmethylester iodination, and goat antirabbit [125I]IgG for receptor antibody binding studies were purchased from DuPont-New England Nuclear (Boston, MA). DMEM was obtained from Mediatech (Washington DC), EDTA/trypsin and penicillin/streptomycin were purchased from Life Technologies (Grand Island, NY), and FBS was obtained from Sigma Chemical Co. (St. Louis, MO). COS-7 cells were provided by B. Seed, Laboratory of Molecular Biology, Massachusetts General Hospital (Boston, MA). Oligonucleotide primers were synthesized using an Applied Biosystems 380B DNA Synthesizer (Foster City, CA). DNA sequencing was performed by the dideoxy chain termination method, using the Sequenase version 2 sequencing kit (U.S. Biochemical Corp., Cleveland, OH). Restriction enzymes, T4 polynucleotide kinase, and T4 DNA ligase were obtained from New England Biolabs (Beverly, MA). Calf alkaline phosphatase was purchased from Boehringer Mannheim (Mannheim, Germany). All other reagents were of the highest purity available.

Receptor Mutagenesis
The molecular cloning of complementary DNAs encoding human (HKrk), rat (R15B), and opossum (OKO) PTH/PTHrP receptors was previously reported (26, 27, 28). The plasmids encoding rat secretin receptor (29), porcine CT receptor (30), mouse CRF receptor (31), human PTH2 receptor (2), human GRF receptor (32), and rat glucagon like-peptide 1 receptor (33) were kindly provided by the respective investigators. Single point mutations were introduced by oligonucleotide-directed site-specific mutagenesis as previously described (34); mutations were verified by nucleotide sequence analysis of double strand plasmid DNA (35).

Transient Expression of WT and Mutant Receptors in COS-7 Cells
COS-7 cells were plated (100,000 cells/well) in 24-well plates in DMEM, 10% heat-inactivated FBS, 200 U/ml penicillin, and 20 µg/ml streptomycin and were grown until 80–90% confluency was achieved. The cells were then transfected by the diethylaminoethyl-dextran method with 100–200 ng/well of plasmid DNA encoding native or mutant receptors (34); these DNA concentrations were previously shown to be saturating for the WT PTH/PTHrP receptor (4). After continuing culture for 72 h, binding of radiolabeled peptide, hormone-induced cAMP accumulation, and cell surface expression were assessed.

RRA of COS-7 Cells Transiently Expressing WT and Mutant PTH/PTHrP Receptors
RRAs were performed with minor modifications, as previously described (36). In brief, binding reactions (final volume, 500 µl/well) contained binding buffer [BB; 50 mM Tris-HCl (pH 7.7), 100 mM NaCl, 5 mM KCl, 2 mM CaCl2, 5% heat-inactivated horse serum and 0.5% heat-inactivated FBS], 125I-labeled PTH-(1–34) (200,000 cpm/well), and the indicated concentrations of unlabeled PTH-(1–34) or PTH-(7–34). After 4 h at 4 C, the binding mixture was removed, and the cells were rinsed twice with 0.75 ml cold BB and lysed with 1.25 ml 1 M NaOH. The entire lysate was counted for {gamma}-irradiation. Nonspecific and total binding of radioligand were determined for each plate from wells containing 1 µM unlabeled PTH-(1–34) or BB, respectively.

cAMP Accumulation in COS-7 Cells Transiently Expressing WT and Mutant Receptors
Cyclic AMP accumulation was assessed as previously described (34). Briefly, transfected COS-7 cells were chilled on ice and rinsed with 500 µl PBS before the addition of 500 µl cAMP assay buffer (DMEM containing 2 mM 3-isobutyl-1-methylxanthine, 0.1% BSA, and 20 mM HEPES, pH 7.4) and 5 µl BB containing increasing concentrations or a maximal dose of the appropriate peptide. After incubation for 15 min at 37 C, the buffer was aspirated, and the cells were placed on powdered dry ice before adding 1 ml 50 mM HCl. The cAMP content in the thawed lysates was quantified by RIA. Data were analyzed using the assayzap software (Elsevier, Cambridge, UK).

Assessment of Cell Surface Expression of Transiently Expressed WT and Mutant PTH/PTHrP Receptors
Cell surface expression was assessed using an affinity-purified polyclonal antibody (anti-H2), which was developed in a rabbit against the synthetic peptide C-E-S-E-E-D-K-E-A-P-T-G-S-R-Y-R-G-R, corresponding to residues 90–106 of the human PTH/PTHrP receptor’s extracellular amino-terminal region. The cysteine at the N-terminus of the peptide was added to facilitate conjugation to keyhole limpet hemocyanin (11). COS-7 cells expressing either WT or mutant receptors were rinsed twice with BB and then incubated at room temperature for 4 h in BB in the presence of anti-H2 (0.3–0.6 µg/250 µl·well; 1:200 dilution). Cells were then rinsed twice with BB and incubated for an additional 2 h at room temperature with 300,000-400,000 cpm/250 µl·well 125I-labeled goat antirabbit Ig. After rinsing the cells three times with BB, they were solubilized in 1 N NaOH, and the radioactivity of the entire lysate was counted. Nonspecific binding was determined with mock-transfected COS-7 cells. The antibody G48 was used as previously described (37) to assess surface receptor expression of the WT and mutant rat PTH/PTHrP receptors.


    ACKNOWLEDGMENTS
 
We thank Drs. Henry Kronenberg, Abdul Abou-Samra, and John Potts, Jr., of the Massachusetts General Hospital Endocrine Unit for helpful discussion and comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dr. H. Jüppner, Endocrine Unit, Massachusetts General Hospital, Wellman 5, Boston, Massachusetts 02114.

This work was supported by NIH Grant DK-50708–01 (to H.J.) and a National Osteoporosis Foundation Fellowship (to E.S.).

Received for publication December 20, 1996. Revision received February 20, 1997. Accepted for publication February 26, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
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