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 Jansens 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 Childrens 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
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ABSTRACT
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Two different activating PTH/PTH-related peptide
(PTHrP) receptor mutations, H223R and T410P, were recently identified
as the most likely cause of Jansens 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 Jansens 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.
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INTRODUCTION
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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 Jansens 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 Jansens 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.
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RESULTS AND DISCUSSION
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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. 1
). 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 (100200 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-(134) 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. 2A
). 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 (100200 ng/well) encoding WT ( ), the H223R mutant
( ), 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-(134)
(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.
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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. 3A
). 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 (100200 ng/well) encoding WT ( ), the T410P mutant
( ), 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-(134) (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.
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We next examined which amino acid substitutions, in addition to those
identified in patients with Jansens 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. 2B
). Therefore, as expected, COS-7 cells transfected with
increasing concentrations of the plasmid DNA (12.5400 ng/well)
encoding the H223K mutant showed increasing constitutive activity (Fig. 4
). 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. 2
, 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. 2
, 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 ( ) or mutant ( ) 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.
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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. 3B
).
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-(134) (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-sens 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
. Interestingly, in the
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
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-(134) (Figs. 2C
and 3C
), 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. 5
). 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.
We previously reported that cells expressing the naturally occurring
receptor mutants, H223R and T410P, bind the agonist PTH-(134) 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-(734) (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-(134) (IC50 = 5 ± 0.5 and 10 ± 0.8
nM, respectively; n = 5), but not for PTH-(734)
(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-(134) (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 1
). 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 1
).
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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
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Mutations equivalent to H223R or T410P in other receptors, including
the porcine and human CT receptors, failed to cause ligand-independent
cAMP accumulation (Table 1
) (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 Jansens 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.
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MATERIALS AND METHODS
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Materials
[Nle8,18,Tyr34]Bovine PTH-(134)amide
[PTH-(134)] and [Nle8,18,Tyr34]bovine
PTH-(734)amide [PTH-(734)] were synthesized as previously
described (26). Human GRF-(129) was purchased from Peninsula
(Belmont, CA), glucagon-like peptide I-(737), rat CRF, human
secretin-(127), and salmon CT-(132)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 8090% confluency was achieved. The
cells were then transfected by the diethylaminoethyl-dextran method
with 100200 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-(134)
(200,000 cpm/well), and the indicated concentrations of unlabeled
PTH-(134) or PTH-(734). 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
-irradiation. Nonspecific and total binding of radioligand were
determined for each plate from wells containing 1 µM
unlabeled PTH-(134) 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 90106 of the human PTH/PTHrP receptors
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 (0.30.6 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.
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ACKNOWLEDGMENTS
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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.
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FOOTNOTES
|
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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-5070801 (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.
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