New insights into interactions between the human PTH/PTHrP
receptor and agonist/antagonist binding
Shoichi
Fukayama1,
Miryam
Royo5,
Masahiko
Sugita4,
Amy
Imrich2,
Michael
Chorev5,
Larry J.
Suva5,
Michael
Rosenblatt5, and
Armen H.
Tashjian Jr.1,3
1 Department of Molecular and
Cellular Toxicology; 2 Program in
Physiology, Department of Environmental Health, Harvard School of
Public Health, 3 Department of
Biological Chemistry and Molecular Pharmacology, Harvard Medical
School; 4 Lymphocyte Biology
Section, Division of Rheumatology and Immunology, Brigham and
Women's Hospital, Harvard Medical School, and
5 Division of Bone and Mineral
Metabolism, Harvard-Thorndike and Charles A. Dana Laboratories,
Department of Medicine, Beth Israel Deaconess Medical Center,
Harvard Medical School, Boston, MA 02115
 |
ABSTRACT |
We prepared a
polyclonal antiserum [Ab-(88
97)] against residues
88-97 of the NH2-terminal
tail of the human (h) parathyroid hormone (PTH)/PTH-related protein
(PTHrP) receptor. Ab-(88
97) bound specifically to the receptor, as
assessed by fluorescence-activated cell sorter analysis of HEK C21
cells, which stably express ~400,000 hPTH/PTHrP receptors per cell.
Unlike PTH, Ab-(88
97) binding did not elicit either adenosine
3',5'-cyclic monophosphate or intracellular calcium
concentration signaling responses in these cells. Incubation of C21
cells for 90 min at 4°C with hPTH-(1
34) plus antiserum reduced
the Ab-(88
97) binding to the cells by up to 40-50% of control
values in a PTH concentration-dependent fashion with a half-maximal
effective concentration of ~5 nM. The decrease in Ab-(88
97) binding
caused by hPTH-(1
34) was completely reversed by coincubation with
hPTHrP-(7
34). We conclude that residues 88-97 of the hPTH/PTHrPR
are involved, either directly or indirectly, in agonist but not
antagonist binding to the receptor.
parathyroid hormone/parathyroid hormone-related protein receptor
antibody; agonist; antagonist
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INTRODUCTION |
PARATHYROID HORMONE (PTH) is the most important
hormonal regulator of mineral ion homeostasis in mammals (21).
PTH-related protein (PTHrP) was first identified and cloned from
malignant tumor cells and tissues from patients with the syndrome of
humoral hypercalcemia of malignancy (16, 25, 27). PTHrP-(1
34) and PTH-(1
34), which share a high degree of sequence identity in their
NH2 terminus, act via a single
species of cloned receptor (PTH/PTHrPR) (1, 12, 22, 23), although
recent studies have shown that specific receptors for each of these
peptides may also exist (29, 31).
Structure-function analyses of PTH and PTHrP peptides and the
PTH/PTHrPR have been studied extensively (4, 13, 14, 20). Although
previous deletion, mutation, and chimeric investigations of the
opossum, rat, and human PTH/PTHrPR (13, 14) have suggested that the
NH2-terminal extracellular tail of
the PTH/PTHrPR contains primary determinants for ligand binding,
specific amino acid residues for the ligand-receptor interaction were
not determined from these studies because alterations in the amino acid
sequence of the NH2-terminal tail
of the PTH/PTHrPR often resulted in a dramatic decrease in the
expression of mutant receptors on the cell surface (13, 14). However,
these studies revealed that residues 61-105, encoded by exon E2 in
the receptor gene, were not involved in ligand-receptor affinity (13,
14). More recently, by use of photoaffinity cross-linking, it has been
shown that residues 173-189 of the human (h) PTH/PTHrPR contain a
ligand-receptor contact domain (32).
Antibodies directed against specific epitopes have been used in probing
receptor structure and function in certain G protein-coupled receptors
(3, 5, 28). In the process of studies on regulation of the hPTH/PTHrPR
(6), we have raised polyclonal antisera (Ab) against the
NH2-terminal tail of the
hPTH/PTHrPR. In preliminary experiments in which we used one of these
antisera [Ab-(88
97)] and HEK C21 cells (clone C21) stably
expressing ~400,000 hPTH/PTHrP receptors/cell (18), we unexpectedly
found that PTH-(1
34) attenuated the Ab-(88
97) binding. In this
report, we show that PTH/PTHrPR agonists (but not antagonists) decrease
Ab-(88
97) binding to the receptor and that Ab-(88
97) also alters
125I-labeled PTH-(1
34) binding
to the receptor.
 |
MATERIALS AND METHODS |
Materials.
Culture media and sera were purchased from GIBCO (Grand Island, NY),
and tissue culture plasticware was obtained from Corning (Corning, NY).
Transfection reagents, lipofectamine, and OPTI-MEM were purchased from
GIBCO-BRL (Grand Island, NY). Synthetic human PTH
[hPTH-(1
34)NH2: lots
ZM-080, ZL-216, and ZM-579]; human PTHrP [hPTHrP-(1
34)NH2: lots
ZM-189 and SM-189]; hPTH-(53
84): lots 993C and ZG-027;
hPTH-(44
68): lot QH-418; and hPTHrP-(107
139): lot ZJ-949; and
bPTH-(3
34): lot 585D were purchased from Bachem California (Torrance,
CA). Synthetic hPTH-(39
84): lot 027865 was from Peninsula (Belmont,
CA).
[Leu11,D-Trp12]hPTHrP-(7
34)NH2
was synthesized in the Division of Bone and Mineral Metabolism, Beth
Israel Deaconess Medical Center, as described previously (12, 21).
Fluorescein isothiocyanate (FITC)-conjugated swine antibodies to goat
immunoglobulins were obtained from Biosource International (Camarillo,
CA). All other chemicals were purchased from Sigma Chemical (St. Louis,
MO).
Preparation of antibodies.
After analysis of the hydrophilicity and antigenic indexes of the amino
acid sequence of the cloned hPTH/PTHrPR (22, 23), as well as the
species differences of the corresponding residues among other cloned
PTH/PTHrP receptors (22), we selected amino acid residues 88-97
(NH2 terminus) and residues
477-497 (COOH terminus) of the hPTH/PTHrPR as immunogens. A
cysteine residue was introduced at one end of each peptide unless this
residue was already present (as it was at position 98). These peptides were: 1) C-CPTHR:
[Cys498]hPTHR-(478
498)NH2;
2) C-NPTHR:
hPTHR-(89
98)NH2;
3) N-CPTHR: Ac[Cys477]hPTHR(477
497)NH2; and
4) N-NPTHR:
Ac[Cys88]hPTHR-(88
97)NH2.
These peptides were then cross-linked to keyhole limpet
hemocyanin via their terminal cysteine residues, as
described (15). The conjugates were then sent to Berkeley Antibody
(Richmond, CA) for custom antibody production. Mixtures of conjugates
of peptides 1 plus
2 and
3 plus
4, in incomplete Freund's adjuvant, were each injected into goat 1 and
goat 2, respectively, every 3 wk for
eight immunizations in total. Serum samples were obtained 10 days after
each immunization. Antibodies against the peptide sequences were
monitored by fluorescence-activated cell sorter (FACS) analysis by use
of either intact (for NH2-terminal
receptor sequences) or permeabilized (for COOH-terminal sequences) HEK C21 cells (18).
Cell culture and transfection of DNA.
Wild-type HEK 293 cells and HEK C21 cells stably expressing the cloned
hPTH/PTHrPR were cultured as described previously (18).
Wild-type and mutant hPTH/PTHrPR DNAs (13, 22) were kindly donated by
Drs. E. Schipani and H. Jüppner (Massachusetts General Hospital,
Boston, MA). Subconfluent to confluent HEK 293 cells (lacking
hPTH/PTHrPR), on 15-cm2 plastic
tissue culture dishes, were transfected transiently by the
lipofectamine method (according to the manufacturer's protocol). For
FACS analysis with transiently transfected or untransfected cells,
cells were harvested 72 h after transfection, as described below. For
PTH/PTHrPR binding assays with transiently transfected cells, cells
were trypsinized and plated into 24-well plastic culture dishes 24 h
after transfection. Forty-eight hours after plating (72 h after
transfection), ligand binding assays were performed.
Ligand binding to the PTH/PTHrPR.
Confluent cells in 24-well plastic culture dishes were used for
receptor binding experiments essentially as described previously (24,
30). Binding was determined by incubating cells with 125I-labeled
[Nle8,18,Tyr34]bovine
PTH-(1
34)NH2 in the absence or
presence of various concentrations of hPTH-(1
34) for 6 h at 4°C.
Specific binding was determined as follows. Specific binding (%) = (total binding
nonspecific binding) × 100/(total tracer
added
nonspecific binding), where nonspecific binding was
determined in the presence of
10
6 M hPTH-(1
34).
Measurement of adenosine 3',5'-cyclic monophosphate.
Cells were treated with appropriate agonists (see
RESULTS for experimental protocols) in
the presence of 1 mM 3-isobutyl-1-methylxanthine, and the concentration
of intracellular adenosine 3',5'-cyclic monophosphate
(cAMP) in acid (50 mM HCl) extracts was measured by radioimmunoassay,
as described previously (7).
FACS analysis.
Cells grown on 15-cm2 plastic
dishes were harvested as described previously (10) and aliquoted into
2.0-ml microtubes (1-3 million cells/tube). For permeabilization,
cells were first fixed in 2% paraformaldehyde in
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered salt solution [Hank's balanced salt
solution (HBSS) II, containing (in mM) 118 NaCl, 4.6 KCl, 10 D-glucose, and 20 HEPES, pH
7.4] for 10 min at 4°C. Cells were then permeabilized with
0.1% saponin in HBSS II for 10 min at room temperature. After the
washing with HBSS II, cells were incubated with test serums (1st Ab) at
4°C for 90 min unless otherwise specified. After a triple wash with
HBSS II, cells were then incubated with a second antibody
(FITC-conjugated swine antibodies to goat immunoglobulins) at 4°C
for 90 min, unless otherwise specified, and cellular fluorescence was
measured using either an Ortho 2150 cytofluorograph equipped with an
air-cooled argon laser (488 nm excitation line, 15 mW output) (26) or a
FACSort flow cytometer (Becton-Dickinson, Mountain View, CA). Propidium
iodide (PI) was added before flow analysis as a measure of
cell viability. Live cells that were able to exclude PI were selected
for determination of fluorescence.
 |
RESULTS |
Characterization of antibodies.
Incubation with serum samples from goat
2 elicited a marked increase in fluorescence in intact
HEK C21 cells but not in wild-type HEK cells (data not shown). The
fluorescence intensity increased after each subsequent booster and
reached a plateau after the sixth immunization. Such an increase in
fluorescence was not observed when the cells were incubated with
preimmune serum (data not shown). These results indicate that the
immune serum samples recognize the
NH2-terminal tail of the
hPTH/PTHrPR (residues 88-97). Goat 2 was immunized with both
hPTHR-(477
497)NH2 and
Ac[Cys88]hPTHR-(88
97)NH2.
To examine whether the antiserum also contained antibody against
COOH-terminal residues (477-497) of the hPTH/PTHrPR, we
permeabilized HEK C21 cells, as described in MATERIALS
AND METHODS. Permeabilization of the cells did not
increase the fluorescence further (data not shown), indicating that the
antiserum contains antibodies directed predominantly against the
NH2-terminal tail of the
hPTH/PTHrPR. Significant antibody binding was observed in HEK cells
transiently expressing the wild-type hPTH/PTHrPR but not in cells
transiently expressing a mutant hPTH/PTHrPR in which the E2 domain
(residues 61-105) was deleted (13) (Table 1). The expression level
of the mutant receptor, as assessed by radioligand binding, was ~70%
that of the wild-type hPTH/PTHrPR (Table
1). No significant fluorescence was
observed when HEK BP16 cells, which stably express the PTH2 receptor
(2), were incubated with the same antiserum (data not shown). Moreover, fluorescence was blocked by 15 µg/ml of the peptide antigen N-NPTHR but not by the C-CPTHR peptide (Table 2).
These results demonstrate that the antiserum contains antibodies
against NH2-terminal residues 88-97 of the human PTH/PTHrPR. Therefore, we have used antiserum from the eighth bleeding from goat 2 [Ab-(88
97)] in most of the subsequent experiments.
Significant fluorescence was observed when the antiserum was diluted as
much as 1:25,600 (data not shown). We also found little or no
difference in background fluorescence intensity when HEK C21 cells were
incubated without (control) or with preimmune serum (data not shown).
Therefore, because limited amounts of preimmune serum were available,
cells incubated without the first antibody served as controls in most
subsequent experiments.
In the course of experiments on the regulation of the hPTH/PTHrPR, we
observed that coincubation of HEK C21 cells with Ab-(88
97) plus
hPTH-(1
34) at room temperature resulted in reduced Ab-(88
97) binding. However, it was not clear whether the decrease in Ab-(88
97) binding was due to direct competition of hPTH-(1
34) and Ab-(88
97) for a common site on the receptor or to some step after PTH binding such as a conformational change in the receptor induced by
hPTH-(1
34). Therefore, we next examined effects of coincubation with
various PTH/PTHrPR peptides on Ab-(88
97) binding at 4°C, a
condition under which agonist-induced major conformational changes in
the PTH/PTHrPR are less likely to occur than at 37°C (4).
Effects of PTH/PTHrPR agonist/antagonist peptides on Ab-(88
97)
binding.
Incubation of C21 cells for 90 min at 4°C with
10
7 M hPTH-(1
34) plus
Ab-(88
97) reduced Ab-(88
97) binding by up to 40-50% of
control values (Table 3). When the cells
were preincubated with 10
7
M hPTH-(1
34) for 15 min before addition of Ab-(88
97) at 4°C, somewhat more inhibition of Ab-(88
97) binding was observed (Table 4). When the cells were incubated with
10
7 M hPTH-(1
34) plus
Ab-(88
97) at 37°C for 90 min, there was only a minimal decrease
in Ab-(88
97) binding (Table 3). Moreover, Ab-(88
97) binding at
37°C was significantly lower than that at 4°C (Table 3). These
results argue against the hypothesis that conformational change(s) in
the hPTH/PTHrPR induced by hPTH-(1
34) plays a major role in the
decrease in Ab-(88
97) binding.
Inhibition of Ab-(88
97) binding by coincubation with hPTH-(1
34) was
observed in a concentration-dependent fashion with a half-maximally
effective concentration (EC50)
of 4-5 nM (Fig. 1). It has been shown
that NH2-terminal PTH-(1
34) and
PTHrP-(1
34) bind to and activate the PTH/PTHrPR in an
indistinguishable manner in various experimental systems, including in
HEK C21 cells (18, 19). Therefore, we examined the effect of
hPTHrP-(1
34) on Ab-(88
97) binding. Unexpectedly, we found that
significantly more hPTHrP-(1
34) than hPTH-(1
34) was required to
inhibit Ab-(88
97) binding (EC50 of 15-20 nM; Fig. 1).

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Fig. 1.
Dose-response curves for parathyroid hormone (PTH)- and PTH-related
protein (PTHrP)-induced inhibition of polyclonal antibody
[Ab-(88 97)] binding. HEK C21 cells were incubated with
Ab-(88 97) (1:100) in the absence (zero value for peptide
concentration) or presence of various concentrations of
hPTH-(1 34)/hPTHrP-(1 34) at 4°C for 90 min. Cells were then
incubated with a 2nd antibody (1:100) at 4°C for 90 min. Mean
fluorescence intensity (MFI) values are expressed as a percentage of
control value (100 ± 2.1%). Values are means ± SE of 4 separate experiments, each performed in triplicate. Half-maximal
effective concentration (EC50)
values (dashed lines) were estimated to be 4-5 nM and 15-20
nM for hPTH-(1 34) and hPTHrP-(1 34), respectively, which were
significantly different (P < 0.01).
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Incubation of HEK C21 cells with antiserum plus
10
6 M
[Leu11,D-Trp12]hPTHrP-(7
34),
a potent antagonist of the PTH/PTHrPR (4), for 90 min at 4°C did
not alter Ab-(88
97) binding (Tables 2 and 4). Likewise, coincubation
with 10
6 M bovine
PTH-(3
34) [bPTH-(3
34)], another antagonist of the PTH/PTHrPR, was also without effect on Ab-(88
97) binding (Table 4).
Incubations of HEK C21 cells with
10
6 M hPTH-(39
84),
hPTH-(53
84), or hPTHrP-(107
139), none of which bind to or activate
the hPTH/PTHrPR (18, 19), were without effect on Ab-(88
97) binding
(Table 4), arguing against nonspecific interactions between PTH/PTHrP
peptides and the Ab-(88
97) or cells.
It has been shown in various cell systems including HEK C21 cells that
[Leu11,D-Trp12]hPTHrP-(7
34)
is a potent, specific antagonist of the hPTH/PTHrPR and blocks the
cAMP/protein kinase A (PKA) and
Ca2+/protein kinase C signaling
pathways elicited by hPTH-(1
34) or hPTHrP-(1
34) (2, 11, 17).
Therefore, we examined the effect of
[Leu11,D-Trp12]hPTHrP-(7
34)
on the effect of hPTH-(1
34). The decrease in Ab-(88
97) binding
elicited by 10
8 M
hPTH-(1
34) was completely reversed by coincubation with
10
6 M
[Leu11,D-Trp12]hPTHrP-(7
34)
(Table 2), consistent with the conclusion that inhibition of
Ab-(88
97) binding by hPTH-(1
34) is mediated via agonist binding to
the hPTH/PTHrPR.
Effects of Ab-(88
97) on PTH binding.
We next examined whether incubation of HEK C21 cells with Ab-(88
97)
inhibited 125I-hPTH-(1
34)
binding. Incubation of HEK C21 cells with Ab-(88
97) plus
125I-PTH-(1
34) for 6 h at
4°C reduced labeled tracer binding by up to 30% without changing
affinity (~1-3 nM). Inhibition of
125I-PTH-(1
34) binding by
Ab-(88
97) was blocked by preincubation of the antiserum with the
specific peptide antigen (data not shown).
Effects of Ab-(88
97) on signal transduction pathways mediated via
the PTH/PTHrPR.
Because our data suggest that the recognition epitope(s) of Ab-(88
97)
might overlap with the agonist-receptor interaction domain(s) on the
PTH/PTHrPR, we examined whether Ab-(88
97) alone could stimulate cAMP
production or whether Ab-(88
97) could block PTH-stimulated cAMP
production. As shown in Fig. 2, Ab-(88
97) did not stimulate cAMP production, nor did it block cAMP production stimulated by 10
8 M
hPTH-(1
34). Similarly, addition of 1:100 Ab-(88
97) did not induce
intracellular calcium concentration
([Ca2+]i)
transients, nor did it block the rise in
[Ca2+]i
stimulated by 10
8 M
hPTH-(1
34) in HEK C21 cells (data not shown).

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Fig. 2.
Lack of effect of Ab-(88 97) on cAMP production stimulated by
hPTH-(1 34) in HEK C21 cells. Cells were incubated without (control)
or with 1:400 Ab-(88 97) (Ab),
10 8 M hPTH-(1 34) (PTH),
or with both for 15 min at 37°C. Cellular cAMP concentrations were
determined as described in MATERIALS AND
METHODS. Values are means ± SE for four cultures.
Similar results were obtained in 2 additional experiments of similar
design. ** P < 0.01 vs.
control.
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However, prolonged pretreatment (
12 h) of HEK C21 cells with
Ab-(88
97) (1:400) at 37°C significantly decreased subsequent cAMP
production stimulated by
10
8 M hPTH-(1
34) to
40-50% of control values, whereas pretreatment with
10
7 M hPTH-(1
34) for 12 h
at 37°C induced homologous cAMP desensitization to 20-30% of
control (Fig. 3). As reported previously
(9), pretreatment with hPTH-(1
34) dramatically enhanced the
subsequent cAMP response to
10
5 M forskolin (Fsk) (Fig.
3). Preincubation with Ab-(88
97) was without effect on Fsk-stimulated
cAMP production (Fig. 3), indicating that the action of Ab-(88
97) was
mediated via the PTH/PTHrPR. Preincubation with preimmune serum had no
effect on cAMP production stimulated by either
10
8 M hPTH-(1
34) or
10
5 M Fsk (Fig. 3).

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Fig. 3.
Effects of preincubation with PTH, preimmune serum, and Ab-(88 97) on
cAMP production stimulated by PTH or forskolin (Fsk). HEK C21 cells
were preincubated with 10 7
M hPTH-(1 34), 1:400 preimmune serum, or 1:400 Ab-(88 97) for 12 h at
37°C. After washing, cells were then incubated without (control) or
with 10 8 M hPTH-(1 34) or
10 5 M Fsk for 30 min at
37°C. Cellular cAMP concentrations were determined as described in
MATERIALS AND METHODS. Values are
means ± SE for 4 cultures. Similar results were obtained in 2 additional experiments of similar design. Dashed horizontal line,
cellular cAMP concentration in response to
10 8 M hPTH-(1 34) in
control cells. * P < 0.05 and
** P < 0.01 vs. pretreatment
control values.
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 |
DISCUSSION |
Previous analysis of opossum, rat, and human PTH/PTHrPRs transiently
expressed in COS cells has revealed that deletion of residues
61-105, encoded by exon E2 in the receptor gene, and insertion of
an epitope derived from Haemophilus influenza hemagglutinin into that
sequence did not affect the receptor function, as assessed by
ligand-receptor affinity and cAMP production (13, 14). Deletion of
amino acids of the NH2-terminal
tail of rat or opossum PTH/PTHrP receptors encoded by exon 1 (r
E1,
residues 26-60), exon 3 (r
E3, residues 106-141), or exon G
(r
G, residues 142-181) resulted in almost complete loss of
surface expression and radioligand binding as assessed by use of
antibody to the HA-tagged receptor, whereas deletion of amino acids
encoded by exon 2 (r
E2, residues 61-105) did not alter
ligand-receptor affinity (14). Similarly, deletion of amino acids
corresponding to exon 2 of the hPTH/PTHrPR did not affect
ligand-receptor affinity when transiently expressed in COS cells (13).
Moreover, amino acid sequences of exon 2-corresponding residues of
PTH/PTHrPRs are not well conserved among species from which PTH/PTHrPRs
have been cloned (22). On the basis of these findings, it has been
concluded that the exon 2-corresponding domain of PTH/PTHrPRs is not
involved in ligand-receptor binding (13, 14), although these studies
did not determine the number of receptors expressed on the cell
surface. More recently, photoaffinity cross-linking studies have
directly identified residues 173-189 of the hPTH/PTHrP receptor as
the contact domain to the amino acid residue in position 13 of a
bioactive PTH-(1
34)-derived ligand (32). In that study, a recombinant
hPTH/PTHrP receptor stably expressed in HEK C21 cells (~400,000
receptors/cell) was photoconjugated to a radioactive, photoreactive,
bioactive ligand, [Nle8,18,Lys13(
-3-l-pBz2),2-Nal23,Arg26,27,Tyr34]bPTH-(1
34)NH2.
This 2113-Da cross-linked domain, located at the distal end of the
NH2-terminal tail of the PTH/PTHrP
receptor, is directly involved in ligand-receptor interaction.
Antibodies against specific epitopes have been used in probing the
structure and function of several membrane receptors (3, 5, 28). For
example, by use of antibodies against the cloned glucagon receptor, it
has been proposed that the principal ligand binding/activation domains
of the receptor are located in residues 126-137 of the
NH2-terminal tail and residues
206-219 in the first extracellular loop (28).
In the present study, we have prepared an antiserum that recognizes
amino acid residues 88-97 of the
NH2-terminal tail of the
hPTH/PTHrPR. Using Ab-(88
97), we have shown that incubation of HEK
C21 cells with this antiserum plus hPTH-(1
34) reduced Ab-(88
97)
binding to the cells, and that Ab-(88
97) also reduced 125I-PTH-(1
34) binding.
Interestingly, inhibition of Ab-(88
97) binding was not observed when
the cells were incubated with
10
6 M
[Leu11,D-Trp12]hPTHrP-(7
34)
or bPTH-(3
34), two PTH/PTHrP antagonists. Because inhibition of
Ab-(88
97) binding by 10
8
M hPTH-(1
34) was reversed completely by coincubation with
10
6 M
[Leu11,D-Trp12]hPTHrP-(7
34),
the decrease in Ab-(88
97) binding induced by hPTH-(1
34) appears to
be mediated via the PTH/PTHrPR. These findings can be interpreted in at
least two ways.
First, the recognition epitope of Ab-(88
97) may overlap with the
receptor's hormone binding domain(s), suggesting that residues 88-97 in the receptor contain agonist, but not antagonist
interaction sites; thus Ab-(88
97) and PTH/PTHrP-(1
34) agonists
partially compete for receptor binding sites. It should be noted,
however, that PTH/PTHrP agonists inhibited Ab-(88
97) binding only
modestly (~40-50%), suggesting that residues 88-97 of the
hPTH/PTHrPR contribute, if at all, only to low-affinity interaction
site(s). Inhibition of
125I-PTH-(1
34) binding by
Ab-(88
97) could be explained by direct competition between agonists
and Ab-(88
97) within residues 88-97 of the
NH2-terminal tail.
Alternatively, it is possible that the binding of agonist, but not
antagonist, alters the conformation of the 88-97 domain of the
hPTH/PTHrPR to result secondarily in a decrease in Ab-(88
97) binding.
In this case, inhibition of
125I-PTH-(1
34) binding can be
explained by secondary or steric effects of the antibody at a distance
on agonist binding domain(s). However, antibody- or agonist-induced
conformational changes of the hPTH/PTHrPR are considered to be
diminished because our experiments were performed at 4°C, where
physiological conformational changes are less likely to occur (4).
Additional evidence against a major contribution of conformational
changes in the hPTH/PTHrPR to the inhibition of Ab-(88
97) binding is
that addition of hPTH-(1
34) before the incubation with Ab-(88
97) at
4°C resulted in increased inhibition of Ab-(88
97) binding greater
than when hPTH-(1
34) and Ab-(88
97) were added together. Moreover,
only a minimal, if any, decrease in Ab-(88
97) binding was observed at
37°C, a temperature at which conformational changes of the
hPTH/PTHrPR would be more likely. Inhibition of Ab-(88
97) binding by
PTH at 37°C could be explained by internalization of the PTH/PTHrPR
after PTH (and possibly Ab) binding to the receptor. Unlike previous
findings with thyroid-stimulating hormone (5) and glucagon (3, 28)
receptors, in which specific receptor antibodies blocked both ligand
binding and agonist-stimulated cAMP production, we found no inhibition
by Ab-(88
97) of cAMP production stimulated by hPTH-(1
34),
suggesting that residues 88-97 of the hPTH/PTHrPR play little or
no role in activating the cAMP signaling pathway by hPTH-(1
34).
However, we have shown that prolonged (
12-h) preincubation with
Ab-(88
97) induced a significant decrease in the cAMP response to
subsequent stimulation with hPTH-(1
34). Because Ab-(88
97) alone did
not stimulate cAMP production in HEK C21 cells, Ab-(88
97)-induced
cAMP desensitization of the PTH/PTHrPR would appear to be mediated via
a cAMP-independent mechanism. It has been shown that PTH-induced
homologous cAMP desensitization involves both cAMP/PKA-dependent and
-independent mechanisms (8, 9). More recently, we have shown that
activation of
-adrenergic receptor kinase-1 is a critical component
of the acute phase (
2 h) of PTH-induced homologous downregulation and desensitization of the PTH/PTHrPR in human osteoblast-like SaOS-2 cells
(6). It is not likely, however, that analogous mechanisms are involved
in Ab-(88
97)-induced cAMP desensitization of the PTH/PTHrPR, because
acute (
1-h) preincubation with Ab-(88
97) was without effect on
subsequent cAMP response to PTH. One possible explanation is that
prolonged preincubation with Ab-(88
97) induces downregulation of the
PTH/PTHrPR, which, in turn, results in a decrease in the cAMP response
to PTH.
In the present study, we found that hPTHrP-(1
34) is less potent than
hPTH-(1
34) in reducing Ab-(88
97) binding. It has been accepted that
NH2-terminal PTHrP-(1
34) and
PTH-(1
34) can bind to and activate the PTH/PTHrPR in an
indistinguishable manner (12, 18, 21). This is also the finding for HEK
C21 cells that stably express the cloned hPTH/PTHrPR (2, 18). Taken together, our findings suggest subtle differences between PTH-(1
34) and PTHrP-(1
34) in the mode of binding to and/or activating
the common receptor.
Finally, in the present study we found that PTH/PTHrP antagonists such
as hPTHrP-(7
34) or bPTH-(3
34) do not inhibit Ab-(88
97) binding,
results consistent with previous findings that deletion of the
E2-corresponding domain (residues 61-105) of the PTH/PTHrPR did
not alter the apparent PTH-(7
34) binding affinity (13); however, we
have shown that PTH/PTHrP agonists such as hPTH-(1
34) or
hPTHrP-(1
34) reduce Ab-(88
97) binding. As discussed above, we do
not know whether the different effects induced by PTH/PTHrP agonists
and antagonists are due to differences in their binding characteristics
to the receptor or to induced conformational changes of the PTH/PTHrPR.
 |
ACKNOWLEDGEMENTS |
We are grateful to Drs. Sunil Shaw and Chi-Chang Shieh (Lymphocyte
Biology Section, Div. of Rheumatology and Immunology, Brigham and
Women's Hospital) for help in the choice of antigenic peptides and
technical assistance in the initial phase of antibody characterization by FACS analysis, respectively. We also thank Jean Foley for excellent assistance in the preparation of this manuscript.
 |
FOOTNOTES |
This investigation was supported in part by research grants from the
National Institute of Diabetes, Digestive and Kidney Diseases (DK-46655
and DK-10206 to A. H. Tashjian, Jr. and DK-47940 to M. Rosenblatt).
Present addresses: S. Fukayama, Department of Medicine, Brockton/West
Roxbury VA Medical Center and Harvard Medical School, West Roxbury, MA
02132; M. Royo, Department of Organic Chemistry, University of
Barcelona, Mazti Franques 1-11 E-08028, Barcelona, Spain.
Address for reprint requests: A. H. Tashjian, Jr., Dept. of Molecular
and Cellular Toxicology, Harvard School of Public Health, 665 Huntington Ave., Boston, MA 02115.
Received 4 September 1997; accepted in final form 10 November
1997.
 |
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