From the Endocrine Unit, Department of Medicine and Children's
Service, Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts 02114
Low resolution mutational studies have indicated
that the amino-terminal extracellular domain of the rat
parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor
(rP1R) interacts with the carboxyl-terminal portion of PTH-(1-34) or
PTHrP-(1-36). To further define ligand-receptor interactions, we
prepared a fully functional photoreactive analog of PTHrP,
[Ile5,Bpa23,Tyr36]PTHrP-(1-36)-amide
([Bpa23]PTHrP, where Bpa is
p-benzoyl-L-phenylalanine). Upon photolysis, radioiodinated [Bpa23]PTHrP covalently and specifically
bound to the rP1R. CNBr cleavage of the broad
80-kDa complex yielded
a radiolabeled
9-kDa non-glycosylated protein band that could
potentially be assigned to rP1R residues 23-63, Tyr23
being the presumed amino-terminus of the receptor. This assignment was
confirmed using a mutant rP1R (rP1R-M63I) that yielded, upon photoligand binding and CNBr digestion, a broad protein band of
46
kDa, which was reduced to a sharp band of
20 kDa upon
deglycosylation. CNBr digestion of complexes formed with two additional
rP1R double mutants (rP1R-M63I/L40M and rP1R-M63I/L41M) yielded
non-glycosylated protein bands that were
6 kDa in size, indicating
that [Bpa23]PTHrP cross-links to amino acids 23-40 of
the rP1R. This segment overlaps a receptor region previously identified
by deletion mapping to be important for ligand binding. Alanine
scanning of this region revealed two residues, Thr33 and
Gln37, as being functionally involved in ligand binding.
Thus, the convergence of photoaffinity cross-linking and mutational
data demonstrates that the extreme amino-terminus of the rP1R
participates in ligand binding.
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INTRODUCTION |
The PTH1/PTHrP receptor
(P1R) mediates the biological actions of PTH and PTHrP (1, 2); both
peptides bind to this common receptor with similar affinities and
stimulate the formation of cAMP and inositol phosphates with similar
efficacies (3-5). In contrast, the recently isolated PTH2 receptor
(P2R) (6) is activated fully by PTH and only poorly by PTHrP
(6-9).
Current information suggests that PTH and PTHrP interact with the P1R
through multiple sites and that these are dispersed throughout the
extracellular surface of the receptor and some portions of the
transmembrane helices (1). Studies with chimeras formed between P1Rs
from different species or different receptor subtypes (P1R or P2R)
indicate that there are interactions between the amino-terminal
extracellular domain of the receptor and region 15-34 of the ligand
and between the core region of the receptor and the amino-terminal
portion of the ligand (9-12). Furthermore, observations from studies
on other members of this peptide hormone receptor family (13, 14), and
particularly with chimeras between the P1R and the calcitonin receptor
(15), suggest that this general orientation of ligand-receptor
interaction may apply to all members of this family of G
protein-coupled receptors.
In addition to mutagenesis approaches, affinity cross-linking methods
can provide valuable information on the location of ligand-receptor
interactions sites in peptide hormone receptors (16, 17). For the P1R,
Zhou et al. (18) recently showed that a PTH-(1-34) analog
containing a photoderivatized lysine 13 cross-linked to a 17-amino acid
segment of the amino-terminal extracellular receptor domain that mapped
close to the junction with the first membrane-spanning helix. In
related experiments, this group showed that another PTH-(1-34) analog,
which contains p-benzoyl-L-phenylalanine (Bpa)
(19) at position 1, cross-linked to a region of the P1R containing
transmembrane helix 6 and extracellular loop 3 (20). The results of
these physicochemical analyses are in agreement with previous
mutational studies that functionally identified similar regions of the
P1R as candidate ligand-interaction sites (12, 21-23). In addition to
these two putative ligand contact regions, mutational studies also
identified segments within the large (
190 amino acids)
amino-terminal domain of the receptor that appear to interact with
region 15-34 of the ligand (10, 24).
We have now performed cross-linking studies with a PTHrP analog that
contains photoreactive Bpa at position 23 in place of the native
phenylalanine, a residue recently shown to be involved in the ligand
binding specificity of the PTH2 receptor (7, 11). This new
photoreactive ligand,
[Ile5,Bpa23,Tyr36]PTHrP-(1-36)-amide,
cross-linked to a short segment between residue 40 and the
amino-terminus, which is predicted to be Tyr-23. We also confirmed the
importance of this amino-terminal receptor region by mutational methods
and have identified two amino acid residues that contribute to ligand
binding affinity.
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EXPERIMENTAL PROCEDURES |
Materials--
[Nle8,21,Tyr34]rPTH-(1-34)-amide
(rNlePTH),
[Nle8,18,Tyr34]bPTH-(1-34)-amide
(bNlePTH),
[Ile5,Bpa23,Tyr36]hPTHrP-(1-36)-amide
([Bpa23]PTHrP), [Tyr36]PTHrP-(1-36)-amide
(PTHrP), and
[Leu11,D-Trp12]PTHrP-(7-34)-amide
(PTHrP-(7-34)) were synthesized by the Protein and Peptide Core
Facility at Massachusetts General Hospital (Boston, MA) by solid-phase
method on Perkin-Elmer Model 430A and 431A synthesizers. The Fastmoc
version of Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry
was utilized, and peptides were purified by reversed-phase chromatography.
Na125I (specific activity of 2000 Ci/mmol) and
radioiodinated anti-mouse IgG Fab (Nex162) were purchased from NEN Life
Science Products. Dulbecco's modified Eagle's medium, Ham's F-12
medium, trypsin/EDTA, penicillin G/streptomycin, and horse serum were from Life Technologies, Inc.. Fetal bovine serum,
3-isobutyl-1-methylxanthine, bovine serum albumin, Tricine, and
Me2SO were from Sigma. Trifluoroacetic acid was from
Pierce, and CNBr was from Serva Fine Chemicals/Boehringer Ingelheim
(Heidelberg, Germany). 14C-Methylated protein molecular
mass markers for SDS-PAGE were purchased from Amersham Pharmacia
Biotech, and peptide N-glycosidase F was from New England
Biolabs Inc. (Beverly, MA). DEAE-dextran was from Pharmacia (Uppsala,
Sweden), and X-Omat AR films for autoradiography were from Eastman
Kodak Co. The monoclonal antibody 12CA5 was purchased from Berkeley
Antibodies (Berkeley, CA).
Mutagenesis of the Rat PTH/PTHrP Receptors--
Mutations were
introduced into single-strand plasmid DNA encoding the wild-type rat
PTH/PTHrP receptor (rP1R) (4) or, for cassette replacements and alanine
point mutations, into the P1R with a hemagglutinin (HA) epitope in the
receptor's E2 domain (rP1R-HA) (24) by oligonucleotide-directed
site-specific mutagenesis as described (11, 24, 25). The
oligonucleotide primers were synthesized on an Applied Biosystems Model
380A DNA synthesizer. Positive mutants were verified by nucleotide
sequence analysis of plasmid DNA.
Cell Culture and DNA Transfection--
COS-7 cells were
cultured in Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere containing 95% air and
5% CO2. Cells were seeded in 24-well plates (200,000 cells/well) for radioreceptor and cAMP assays and for preliminary
cross-linking experiments; all other cross-linking experiments were
performed in 150-mm dishes (6 × 106 cells). Once the
cell monolayer reached 90-100% confluency, cells were transfected by
the DEAE-dextran method as described (22) using 200 ng of plasmid
DNA/well or 2 µg/150-mm dish. The medium was replaced daily, and 3 days after transfection, cells were used either for radioligand binding
and cAMP accumulation assays or for cross-linking experiments. ROS
17/2.8 cells were maintained as described (26) in Ham's F-12 medium
supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and
100 µg/ml streptomycin.
Radioligand-Receptor Binding Assays and cAMP
Accumulation--
Radiolabeled rNlePTH, PTHrP, and
[Bpa23]PTHrP were prepared by chloramine-T iodination,
followed by high pressure liquid chromatography purification using
a 30-50% acetonitrile in 0.1% trifluoroacetic acid gradient over 30 min. Radioligand-receptor binding assays were performed in 24-well
plates as described (11). In brief, each well (final volume of 500 µl) contained binding buffer (50 mM Tris-HCl (pH 7.7),
100 mM NaCl, 5 mM KCl, 2 mM CaCl,
5% heat-inactivated horse serum, and 0.5% heat-inactivated fetal
bovine serum), 125I-labeled radioligands (100,000-200,000
cpm), and varying concentrations of unlabeled peptide. After 4 h
at 16 °C, the binding mixture was removed, and the cells were rinsed
with cold binding buffer and lysed with 1 M NaOH. The
entire lysate was counted for
-irradiation. Specific binding was
determined after subtracting radioactivity bound in the presence of
maximal concentrations of unlabeled competing peptide
(10
6 M). Agonist-dependent
accumulation of cAMP was determined by radioimmunoassay as described
(11).
Cell-surface Expression of PTH/PTHrP Receptors--
Cell-surface
expression was assessed as described (24) using antibody 12CA5 directed
against the HA epitope in the rP1R-HA receptors and a second
radiolabeled anti-mouse IgG Fab fragment. Relative specific binding of
antibody to each mutant P1R was calculated by subtracting
nonspecifically bound radioactivity (determined in mock-transfected COS
cells; typically 0.1-0.2% of added radioactivity) from the total
bound radioactivity divided by the radioactivity specifically bound to
wild-type rP1R-HA (typically 1-2% of the added radioactivity).
Photoaffinity Labeling of PTH/PTHrP Receptors--
In
preliminary experiments, COS-7 cells that were grown and transfected in
24-well plates were rinsed twice with 1 ml of cold binding buffer, and
the cell monolayer was then incubated for 6 h at 4 °C with
125I-[Bpa23]PTHrP (1 × 106
cpm) diluted in 0.5 ml of binding buffer with or without unlabeled ligand (10
6 M bNlePTH or
[Bpa23]PTHrP). After incubation, cells were rinsed three
times with 1 ml of cold binding buffer before adding 200 µl of
binding buffer and placing the dishes on ice under a UV light source
for 20 min (Blak Ray long-wave lamp; 366 nm, 7000 microwatts/cm2; UV Products, San Gabriel, CA;
source-to-cell distance of
5 cm). After photolysis, cells were
rinsed once with cold phosphate-buffered saline, twice with a cold
acidic buffer (0.05 M glycine and 0.15 M NaCl
(pH 2.5)) to remove noncovalently bound radioligand, and twice with
cold phosphate-buffered saline before solubilization with 0.5 ml of
SDS-PAGE sample buffer (4% (w/v) SDS, 80 mM Tris-HCl (pH
6.8), 20% (v/v) glycerol, 0.2% bromphenol blue, and 100 mM dithiothreitol). The lysate was then passed six times
through a 19-gauge needle to shear genomic DNA.
To prepare larger amounts of the cross-linked ligand-receptor complex,
a similar protocol was followed using COS-7 cells that were grown and
transfected in 150-mm dishes. For each rinsing step, 30 ml of cold
binding buffer were used, and incubation with 125I-[Bpa23]PTHrP (2-4 × 107 cpm) was performed in a final volume of 20 ml of
binding buffer. During UV light exposure, the cell monolayer was
covered with 10 ml of binding buffer, and after photolysis and rinsing,
cells were solubilized with 4 ml of SDS-PAGE sample buffer.
SDS-PAGE Analysis of the Ligand-Receptor Complex--
After
heating to 70 °C, samples (and appropriate size markers) were either
subjected to analytical SDS-PAGE analysis (5-20% acrylamide, 0.75-mm
spacers) according to the method of Laemmli (27) or loaded onto a
16.5% (w/v) Tricine gel (0.75-mm spacers) according to the method of
Schägger and von Jagow (28), with subsequent autoradiography of
the dried gels (1-14 days at
80 °C with intensifying
screens).
Purification of [Bpa23]PTHrP·P1R
Complexes--
To isolate larger amounts of radiolabeled
ligand-receptor complexes from cells cultured in 150-mm dishes, we used
preparative SDS-polyacrylamide gels (5-20% acrylamide, 3-mm spacers);
the complexes were identified by autoradiography of the wet gels
(exposure time of 4-12 h at room temperature) and electroeluted from
an excised gel slice using a Model 422 electroeluter (Bio-Rad). The isolated radiolabeled ligand-receptor complex was stored at
20 °C in elution buffer (25 mM Tris, 192 mM glycine,
and 0.02% SDS) before chemical/enzymatic treatment (see below).
CNBr Cleavage--
CNBr was dissolved in 100%
trifluoroacetic acid and then added to the partially purified
radiolabeled ligand-receptor complex to give a final concentration of
100 mM CNBr in 70% trifluoroacetic acid. After an
overnight, light-protected incubation, the digest was reduced in volume
under a stream of nitrogen and then repeatedly lyophilized to remove
trifluoroacetic acid and CNBr. Once the apparent molecular mass of each
CNBr-derived ligand-receptor fragment had been established,
trifluoroacetic acid and CNBr were eliminated more efficiently by
ultrafiltration using a Microcon 3 concentrator (Amicon, Inc., Beverly,
MA) and repeated dilution of the retentate with H2O.
Peptide N-Glycosidase F Digestion--
The CNBr-cleaved and
concentrated radioligand-receptor complex was treated with peptide
N-glycosidase F (2500 units) for 1 h at
37 °C in 30 µl of 50 mM sodium phosphate (pH 7.5), 0.5% SDS, 1%
-mercaptoethanol, and 1% Nonidet P-40 according to
the protocol provided by the supplier.
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RESULTS |
Functional Characterization of
[Bpa23]PTHrP--
[Bpa23]PTHrP was
tested in competition binding studies performed with COS-7 cells
expressing the native rP1R and was found to have an apparent binding
affinity that is indistinguishable from that of bNlePTH and of other
analogs of PTH and PTHrP (7, 10). The Bpa-containing PTHrP analog was
also fully functional in cAMP accumulation assays and exhibited a
potency that was indistinguishable from that of bNlePTH (data not
shown).
Photoaffinity Labeling of Rat PTH/PTHrP Receptors--
After
binding and photoactivation, the covalent complex formed between
radioiodinated [Bpa23]PTHrP and the rP1R was visualized
by analytical SDS-PAGE and subsequent autoradiography. The complex
migrated as a single broad band corresponding to a glycosylated
protein with a molecular mass of
80 kDa (Fig.
1, lane 1). This
size of the ligand-receptor complex is comparable to that previously
seen with other photoreactive PTH or PTHrP analogs using either cells
expressing endogenous PTH/PTHrP receptors (29-32) or HEK-293 cells
expressing the cloned P1R (18). Coincubation of transfected COS-7 cells
with 125I-[Bpa23]PTHrP and unlabeled
bPTH-(1-34) (10
6 M) or unlabeled
[Bpa23]PTHrP (10
6 M) completely
eliminated the formation of the radiolabeled ligand-receptor complex (data not shown).

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Fig. 1.
Analysis of the CNBr-digested
[Bpa23]PTHrP·P1R complex using COS-7 cells expressing
the wild-type rat PTH/PTHrP receptor and the M63I receptor mutant.
As described under "Experimental Procedures," the partially
purified complex of ligand and wild-type P1R (lanes
1 and 2) or the M63I receptor mutant
(lanes 3 and 4) was incubated in 70%
trifluoroacetic acid in the absence (lanes 1 and
3) or presence (lanes 2 and
4) of CNBr (100 mM). After repeated
lyophilization, samples were analyzed by SDS-PAGE with subsequent
autoradiography (overnight at 80 °C). The positions of different
size markers are indicated in kilodaltons.
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To identify the region of the P1R that interacted with the
Bpa23 moiety, we isolated larger amounts of the
radiolabeled ligand-receptor complex using preparative SDS-PAGE,
cleaved the partially purified complex with CNBr, and separated the
cleavage products on analytical gels. After CNBr cleavage, most of the
radioactivity migrated on SDS-PAGE as a single sharp protein band
corresponding to a size of <14 kDa (Fig. 1, lane
2). A minor fraction migrated as a diffuse band at the
46-kDa size marker and probably corresponded to a partially cleaved
glycosylated ligand-receptor complex. Tricine/SDS-PAGE analysis was
used to achieve higher resolution in the low molecular mass range, and
this suggested a molecular size of
9 kDa for the principal
radiolabeled CNBr-generated fragment (see also Fig. 5, lane
1). Since [Bpa23]PTHrP has a molecular size of
4.286 kDa, the receptor fragment contributing to the complex was
estimated to have a molecular size of
5 kDa. The same results were
obtained when analyzing the complex formed between radiolabeled
[Bpa23]PTHrP and the endogenous PTH/PTHrP receptor of ROS
17/2.8 cells (data not shown).
The above results suggested that Bpa at position 23 of PTHrP interacts
with an
5-kDa non-glycosylated CNBr-generated portion of the
receptor. Inspection of the amino acid sequence of the rP1R showed that
several fragments delimited by methionine residues are within this
molecular size range (Fig. 2). Because of
the predicted overall architecture of ligand-receptor interaction (1,
11, 15), we considered the hypothesis that Bpa23 interacts
with the receptor segment defined by Met63 and the
amino-terminus, which is predicted to be Tyr23 by a
recently developed algorithm (33). A mutant rP1R was generated, rP1R-M63I, in which methionine at position 63 was replaced by isoleucine (Fig. 3B); this
mutant receptor had functional (data not shown) and cross-linking (Fig.
1, lane 3) properties that were indistinguishable
from the wild-type rP1R. CNBr cleavage of the covalently labeled
rP1R-M63I mutant yielded to a broad radioactive band comigrating with
the 46-kDa marker (Fig. 1, lane 4, and Fig.
4, lane 1); this
cleavage product was reduced to a sharp protein band of <20 kDa upon
further digestion with peptide N-glycosidase F (Fig. 4,
lane 2). The increase in size from
9 to
46
kDa of the CNBr-derived ligand-receptor complex that occurred as a
result of the M63I mutation and the subsequent size reduction of this
larger receptor fragment upon deglycosylation indicated that, in the
mutant receptor, Bpa23 interacts with a receptor segment
that extends from the amino-terminus to Met174. These
results confirmed that cross-linking of [Bpa23]PTHrP to
the wild-type P1R occurred between Met63 and the
receptor's amino-terminal residue.

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Fig. 2.
Schematic model of the rat P1R showing the
seven putative transmembrane domains and the locations of all 14 methionine residues in the mature protein. The amino-terminus
is at the top. Potential N-linked glycosylation sites ( ),
the putative signal peptide, and the predicted first residue
(Tyr23) of the mature receptor are shown. The sites for
leucine (L) to methionine (M) and for methionine
(M) to isoleucine (I) substitutions and the
location of methionine residues ( ) are indicated (see also Fig.
3).
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Fig. 3.
Schematic drawing of the amino-terminus of
the wild-type rat PTH/PTHrP receptor and two receptor mutants. The
predicted signal sequence cleavage site (arrow), the sites
for N-linked glycosylation ( ), and the locations of
methionine residues ( ) and the introduced isoleucine ( ) are
indicated. The calculated molecular sizes of CNBr fragments are
indicated in daltons. A, wild-type P1R; B,
P1R-M63I; C, P1R- M63I/L40M.
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Fig. 4.
Analysis of the CNBr-digested
[Bpa23]PTHrP·P1R-M63I complex using COS-7 cells
expressing the M63I receptor mutant. As described under
"Experimental Procedures," the partially purified, CNBr-cleaved
radioligand-receptor complex (lane 1) was
digested with peptide N-glycosidase F (lane
2). Samples were analyzed by Tricine/SDS-PAGE with
subsequent autoradiography (14 days at 80 °C). The positions of
different size markers are indicated in kilodaltons.
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To further define the site of cross-linking, we introduced
methionine substitutions at either Leu40 or
Leu41 in the rP1R-M63I mutant to yield the double mutants
rP1R-M63I/L40M and rP1R-M63I/L41M, respectively (Fig. 3C).
Like rP1R M63I, these two mutants had biological properties that were
indistinguishable from those of the wild-type rP1R (data not shown).
Leu40 and Leu41 were chosen because their
substitution with methionine is a conservative replacement and because
CNBr cleavage at these positions would yield ligand-receptor conjugates
whose size upon SDS-PAGE analysis would easily distinguish between the
two possible sites of interaction with [Bpa23]PTHrP.
Thus, cross-linking to a site amino-terminal of Met40 or
Met41 would yield non-glycosylated, low molecular mass
complexes corresponding to receptor residues 23-40 and 23-41,
respectively, whereas cross-linking to a site carboxyl-terminal of
either mutation would yield glycosylated, high molecular mass complexes
corresponding to residues 41-174 and 42-174, respectively. As shown
in Fig. 5 (lane 3),
CNBr cleavage of the affinity-labeledM63I/L40M mutant yielded a small
radiolabeled, non-glycosylated complex of
6 kDa, as did cleavage of
the M63I/L41M mutant (data not shown). This indicated that the covalent
interaction between Bpa23 and the rP1R occurred between the
receptor's amino-terminus and Leu40.

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Fig. 5.
Analysis of the CNBr-digested
[Bpa23]PTHrP·P1R complex using COS-7 cells expressing
the wild-type rat PTH/PTHrP receptor and two different receptor
mutants. As described under "Experimental Procedures," the
partially purified ligand-receptor complexes were incubated in 70%
trifluoroacetic acid in the presence of CNBr. After repeated
lyophilization, samples were analyzed by Tricine/SDS-PAGE with
subsequent autoradiography (5 days at 80 °C). Lane
1, wild-type P1R; lane 2, P1R-M63I;
lane 3, P1R-M63I/L40M. The positions of different
size markers (in kilodaltons) and of
125I-[Bpa23]PTHrP (arrowhead) are
indicated.
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Effects of Point Mutations in the Amino-terminal
Extracellular Domain of the PTH/PTHrP Receptor on Ligand
Binding--
The amino-terminal receptor fragment identified by the
above physicochemical approach overlaps a P1R region previously shown by functional studies to be important for ligand binding (24). Two
mutant receptors with deletions of residues 26-60 (the E1 region) or
31-47 (E1a) were shown to have only moderately reduced receptor
expression levels in COS-7 cells (22 ± 1 and 36 ± 3% of
the wild type, respectively) and little or no capacity to bind radiolabeled PTH (24). To further examine the functional importance of
residues in this amino-terminal E1a region, we first made four cassette
mutant receptors, termed E1a-1 through E1a-4, in which four or five
adjacent residues were replaced by either alanine or valine (Fig.
6, A and B). Each
mutant receptor was adequately expressed on the surface of COS-7 cells
(>35% of the wild-type) (Fig. 6C). The two mutants in
which residues 31-35 and 36-39 were altered displayed diminished
125I-rNlePTH binding capacity, whereas the two mutants with
substitutions of residues 40-43 and 44-47, respectively, maintained
high levels of PTH binding (Fig. 6D).

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Fig. 6.
Cassette mutagenesis of the E1a region of the
rat PTH/PTHrP receptor. The location of the E1a region, previously
identified by deletion analysis as a ligand-binding site (24), is shown
in A. The four cassette substitutions used to further divide
the E1a region are shown in B. The effects of each cassette
mutation on surface expression as assessed by antibody binding to the
HA epitope in the E2 domain of each receptor and
125I-[Nle8,21,Tyr34]rPTH-(1-34)-NH2
binding are shown in C and D, respectively.
Included as controls are COS-7 cells transfected with a mutant P1R in
which the entire E1a region was deleted (del.E1a) and
mock-transfected COS-7 cells. Data are the means ± S.E. of
triplicate values from one of two equivalent experiments.
WT, wild-type.
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To further localize candidate binding residues within region 31-39
(E1a-1 and E1a-2), an alanine-scanning approach was used. Several of
the individual alanine substitutions in this region, which had little
or no effect on cell surface expression (Fig. 7A), resulted in small
reductions in 125I-rNlePTH binding capacity (Fig.
7B). A reduction in PTH binding of >25% occurred with two
substitutions, T33A and Q37A (Fig. 7B). In addition, each of
these two point mutations had a more severe effect on
125I-PTHrP binding than on 125I-rNlePTH binding
(Fig. 7C). In competition binding studies with 125I-rNlePTH as tracer radioligand, the apparent binding
affinity of rNlePTH for wild-type and mutant P1Rs was comparable (Fig. 8A). The apparent binding
affinity of bNlePTH for the T33A and Q37A mutant receptors was 5.0- and
2.3-fold weaker, respectively, than it was for the wild-type receptor
(Fig. 8B). Consistent with the reduced maximal binding of
radiolabeled PTHrP, the apparent binding affinity of PTHrP-(1-36) for
these two mutant receptors was 14- and 48-fold weaker, respectively,
than it was for the wild-type receptor (Fig. 8C). Both
receptor mutations also abolished binding of an amino-terminally
truncated PTHrP analog,
[Leu11,D-Trp12]PTHrP-(7-34)-amide,
indicating that Thr33 and Gln37 affect
interactions with region 7-34 of the ligand rather than with region
1-6 (Fig. 8D and Table
I).

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Fig. 7.
Alanine-scanning mutagenesis of residues
31-39 of the rat PTH/PTHrP receptor. Individual residues in the
receptor regions defined by the E1a-1 and E1a-2 cassette mutations (see
Fig. 6) were replaced by alanine; the resulting mutants were
transfected into COS-7 cells and tested for surface expression
(A), binding of
125I-[Nle8,21,Tyr34]rPTH-(1-34)-NH2
(B), and binding of
125I-[Tyr36]PTHrP-(1-36)-NH2
(C). Data are the means ± S.E. of three experiments,
each performed in triplicate. WT, wild-type.
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Fig. 8.
Effects of alanine substitution of
Thr33 or Gln37 in the PTH/PTHrP receptor on
ligand binding. The rP1R (wild type (WT); ) and the
T33A ( ) and Q37A ( ) mutants were expressed in COS-7 cells and
evaluated for binding PTH or PTHrP analogs. Competition binding
analyses were performed using
125I-[Nle8,21,Tyr34]rPTH-(1-34)-NH2
as a tracer radioligand and
[Nle8,21,Tyr34]rPTH-(1-34)-NH2
(A),
[Nle8,18,Tyr34]bPTH-(1-34)-NH2
(B), [Tyr36]hPTHrP-(1-36)-NH2
(C), or
[Leu11,D-Trp12]hPTHrP-(7-34)-NH2
(D) as an unlabeled competitor ligand. Each graph shows data
(mean ± S.E.) from four or five experiments, each performed in
duplicate.
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Table I
Peptide analog binding to wild-type and mutant PTH/PTHrP receptors
Competition binding studies were performed with
125I-[Nle8,21,
Tyr34]rPTH(1-34)-NH2 and the indicated unlabeled
peptides as described under "Experimental Procedures" and in the
legend to Fig. 8. Values are the means ± S.E.
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DISCUSSION |
Previous studies have suggested that the amino-terminal
extracellular region of the PTH/PTHrP receptor interacts with
carboxyl-terminal region 15-34 of either PTH or PTHrP; a similar
architecture of ligand-receptor interaction may well apply to other
members of this family of G protein-coupled receptors (9-15). In this
study, we confirmed and extended these predictions for the P1R with a PTHrP analog containing photoreactive Bpa at position 23, a residue with apparent functional significance based on its ability to determine
ligand binding specificity in the P2R (7, 11). After CNBr digestion of
[Bpa23]PTHrP·P1R complexes, an
9-kDa radiolabeled
protein was detected upon Tricine/SDS-PAGE analysis. This fragment was
likely to represent 125I-[Bpa23]PTHrP
covalently coupled to a receptor fragment extending from Tyr23, the first residue after the predicted cleavage site
for the signal peptide (33), to Met63, the first methionine
in the mature receptor sequence. We confirmed this assignment and
refined the mapping further by using site-directed mutagenesis to
introduce or remove methionines at strategic sites in the receptor.
First, the rP1R- M63I mutant was generated and shown to be fully
functional. When the ligand-receptor complex formed with this receptor
was cleaved with CNBr, the
9-kDa band was replaced by an
46-kDa
glycosylated band corresponding to the receptor fragment extending from
the amino-terminus to Met174. This receptor segment
contains three of the four potential N-linked glycosylation
sites, and glycosylation is consistent with the broadness of the
46-kDa complex on SDS-PAGE and its reduction to a smaller,
non-glycosylated protein band by peptide N-glycosidase F
treatment. These results confirmed that cross-linking between BPA23 and the rat PTH/PTHrP receptor involved residues that
are located between the amino-terminus and Met63. The M63I
mutation allowed us to exclude other CNBr-derived receptor fragments of
similar size, such as Ala426-Met450. Two
additional, fully functional receptor double mutants, rP1R-M63I/L40M and rP1R-M63I/L41M, were prepared to further refine the cross-linking site. Both mutants contained the M63I mutation to eliminate the natural
CNBr cleavage site at position 63. CNBr cleavage of the complexes
formed between 125I-[Bpa23]PTHrP and either
of these two mutant receptors resulted in low molecular mass
radiolabeled protein conjugates (Fig. 5). This result established that
Bpa23 cross-linked to a side amino-terminal to
Met41 in the rP1R and clearly excluded segment 41-174 as
the site of interaction.
Earlier mutagenesis studies had indicated that deletion of a portion of
the rP1R that included 17 residues (the E1a region) close to the
amino-terminus of the mature receptor abolished binding of radiolabeled
PTH or PTHrP, with only moderate effects on receptor expression (24).
To further map functional binding residues in this region, we
constructed four "cassette" mutants in which four or five adjacent
amino acids were replaced by alanine or valine. Two of these cassette
mutants, E1a-1 and E1a-2, showed normal cell-surface expression, but
little or no binding of radiolabeled PTH or PTHrP. These results
suggested that residues within segments 31-35 and 36-39 contribute to
ligand interaction. The replacement of each of these nine residues with
individual alanine substitutions confirmed this hypothesis. Two
mutants, rP1R T33A and rP1R Q37A, exhibited the weakest capacity to
bind the radioligand. Interestingly, the effects of these mutations on
ligand binding were more pronounced with PTHrP than with PTH; this
pattern might be attributable to the divergence in region 15-34 of
these two ligands, a hypothesis supported by the observation that the
mutations also impaired PTHrP-(7-34) binding.
In summary, our physicochemical observations indicate that
Bpa23 (and presumably Phe23 in the native PTHrP
molecule) interacts with residues at the extreme amino-terminus of the
PTH/PTHrP receptor. Mutational analysis of this receptor region
supported this conclusion and identified two amino acid residues,
Thr33 and Gln37, as possible sites for ligand
interaction. The combined use of the two techniques, photoaffinity
cross-linking and receptor mutagenesis, should enable the definition of
other receptor segments that comprise contact points for PTH and
PTHrP.
We thank Drs. Henry T. Keutmann,
Ashok Khatri, Abdul-Badi Abou-Samra, Henry M. Kronenberg, and John
T. Potts, Jr. for continuous support and helpful discussions and John
H. Davies, Cindy W. Su, and Peter Lyons for technical assistance.