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Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Thomas J. Gardella, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. E-mail: gardella{at}helix.mgh.harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For the PTH-1 receptor, the agonist peptides PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) bind with nanomolar affinity and stimulate robust increases (>20-fold) in intracellular cAMP. Previous studies using the PTH-1 receptor have shown that there are two distinct regions of functionality within these ligands; residues 15–34 contribute the majority of the binding energy (8, 9, 10), and residues 1–14 impart most of the stimulatory capacity to the molecule (11, 12). The regions of the PTH-1 receptor that are responsible for these effects have been broadly defined using receptor chimeras (13).
A separate line of work has investigated the molecular basis for the ligand-selectivity of the PTH-2 receptor. With this receptor, ligand residues 5 (Ile in PTH and His in PTHrP) (14) and 23 (Trp in PTH and Phe in PTHrP) were identified as important determinants for signaling and binding selectivity, respectively (15). Subsequent mutational studies have revealed several sites in the PTH-2 receptor that vary from the PTH-1 receptor sequence and play a role in determining ligand selectivity (16, 17, 18). Particularly relevant to the present study is the prior demonstration that the PTHrP analog [Ile5,Trp23,Tyr36]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)NH2 is a potent agonist for both receptor subtypes, exhibiting binding and signaling properties equal to those of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (15).
Several laboratories are currently employing photochemical cross-linking methods to map the topology of the ligand-receptor interface between PTH/PTHrP ligands and the PTH-1 receptor (19, 20, 21). Recently, Behar et al. (22) reported the first mapping of a photochemical cross-link between a PTH ligand and the PTH-2 receptor, and they concluded from this study that photolabile residues at positions 1 and 13 in the ligand contact identical sites in the PTH-1 receptor and PTH-2 receptor.
To expand the current body of data on the ligand-receptor interaction mechanisms for both the PTH-1 receptor and PTH-2 receptor, we prepared a series of analogs of [I5,W23,Y36]-hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)NH2 and characterized their interactions with each receptor subtype. Given the importance of the N-terminal region of the ligand in receptor activation, we focused on this region and introduced either: 1) stepwise deletions of residues 1–4; or 2) single substitutions of the photoreactive amino acid para-benzoyl-L-phenylalanine (Bpa) (23) at positions one through six. The functional and biochemical properties of these peptides reveal ligand-dependent and receptor-dependent profiles of agonism, antagonism, and photochemical cross-linking reactivity, which suggest that the PTH-1 receptor and PTH-2 receptor accommodate large, hydrophobic sidechain substitutions in the N-terminal portion of the ligand in dramatically different ways.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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} used in
this study were prepared on a model 430A peptide synthesizer (PE Applied Biosystems, Norwalk, CT) using
N-(9-fluorenyl)methoxycarbonyl (Fmoc)-protecting group
chemistry. After global deprotection, each of these peptides was
purified by HPLC, lyophilized, reconstituted in 10 mM
acetic acid, and stored at -80 C. The purity, identity, and stock
concentration of each compound were secured by analytical HPLC, mass
spectrometry, and amino acid analysis. Radiolabeling was performed
using 125I-Na (2,200 Ci/mmol, DuPont NEN) and chloramine-T; the resultant
[125I-Tyr]-ligand was purified by HPLC.
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Binding assays
Binding reactions were performed with stably transfected LLC-PK1
cells in 24-well plates. Cells were rinsed with 0.5 ml binding buffer
(50 mM Tris-HCl, 100 mM NaCl, 5 mM
KCl, 2 mM CaCl2, 5% heat-inactivated horse
serum, 0.5% FBS, adjusted to pH 7.7 with HCl), and treated
successively with 100 µl binding buffer, 100 µl binding buffer
containing various amounts of unlabeled competitor ligand, and 100 µl
binding buffer containing ca. 100,000 cpm of 125I-tracer
(ca. 26 fmol; final vol = 300 µl). Incubations were at room
temperature for 2 h, except for homologous binding experiments,
which were performed at 4 C for 6 h to minimize effects of
G-protein coupling and receptor internalization. Cells were then placed
on ice, the binding medium was removed, and the monolayer was rinsed
three times with 0.5 ml cold binding buffer. The cells were
subsequently lysed with 0.5 ml 5N NaOH and counted for
radioactivity. The nonspecific binding for each experiment was
determined by competition with a 1-µM dose of unlabeled
rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36). The
maximum specific binding (B0) was the total
radioactivity bound in the absence of unlabeled PTH ligand, corrected
for nonspecific binding. Binding IC50 values were
determined using nonlinear regression (see below).
cAMP stimulation
Stimulation of stably transfected LLC-PK1 cells was
performed in 24-well plates. Cells were rinsed with 0.5 ml binding
buffer and treated with 200 µl cAMP assay buffer (DMEM containing 2
mM 3-isobutyl-1-methylxanthine, 1 mg/ml BSA, 35
mM HEPES-NaOH, pH 7.4) and 100 µl of binding buffer
containing varying amounts of peptide analog (final vol = 300
µl). The medium was removed after incubation for 1 h at room
temperature, and the cells were frozen (-80 C), lysed with 0.5 ml 50
mM HCl, and refrozen (-80 C). The cAMP content of the
diluted lysate was determined by RIA. Where possible, cAMP
EC50 values were determined using nonlinear regression (see
below).
Antagonism studies
The cAMP stimulation protocol described above was used for
antagonist studies, with some minor modifications. Cells were rinsed
with 0.5 ml binding buffer and treated successively with 100 µl
binding buffer containing varying doses of the candidate antagonist
peptide, 100 µl cAMP assay buffer, and 100 µl cAMP assay buffer
containing varying doses of rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (final vol = 300 µl).
Cells were incubated for 30 min at room temperature and processed as
above. The
[L11,D-W12,W23]-PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
control antagonist inhibited PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) agonist action with both PTH
receptors (see Fig. 5), and with potency similar to that of the
antagonist [L11, D-W12]-PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
described by Nutt et al. (26) (data not shown). The dose of
an antagonist that inhibited the PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)-mediated cAMP response by
50% (IC50A) was calculated as described below.
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100. Hill coefficients
were derived from homologous binding data by extracting the slope from
the linear portion of the plot of
log[B/(B0-B)]
vs. log[competitor]. Antagonist IC50A values
were calculated from the intercept of the linear portion of a plot of
log[E/(Emax-E)]
vs. log[Antagonist], where E and
Emax are the cAMP responses observed in the presence and
absence of antagonist, respectively. The statistical significance
between two data sets was determined using a one-tailed Student’s
t test, assuming unequal variances for the two sets.
Photochemical cross-linking
Photochemical cross-linking was carried out with
stably transfected LLC-PK1 cell lines, which had been
subcultured in 6-well plates; all manipulations were executed on ice
using chilled reagents. Cells were rinsed with 2.0 ml binding buffer,
treated with 2 ml binding buffer containing ca. 4 x
106 cpm (ca. 1 pmol) of a 125I-peptide, and
incubated at 4 C for 6 h. The medium was removed, and the cells
were rinsed twice with 1 ml binding buffer before being covered with
800 µl binding buffer. Cross-linking was induced by irradiation with
a Black-Ray UV lamp (366 nM, 700 µW/cm2,
source-to-cell = 4.5 cm) for 50 min on ice in a cold room (4 C).
The medium was withdrawn, and the cells were rinsed twice with 2 ml of
an acidic buffer (50 mM glycine, 150 mM NaCl,
adjusted to pH 2.5 with HCl) and twice with 2 ml binding buffer. The
cells were lysed with 250 µl of a Triton buffer (50 mM
Tris-HCl, 10% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, 0.05 mg/ml Bacitracin, 150 mM NaCl, pH 7.8) for
45 min at 0 C before being harvested (50 µl rinse with the Triton
buffer) and centrifuged at 2000 x g for 20 min at 4 C.
The supernatant was collected and stored at -20 C. Analysis of
equivolume aliquots from these cross-linking reactions was accomplished
using 5–20% SDS-PAGE (28) with subsequent autoradiography of the
dried gel at -80 C with an intensifying screen.
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Results |
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). The excision of
residues 1–4 did not have a pronounced deleterious effect on the
ability of these analogs to inhibit the binding of
125I-rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) to either the PTH-1 or PTH-2 receptor
stably transfected in LLC-PK1 cells (Fig. 1, A and B; Table 2). Indeed, the truncated analog
[I5,W23]-PTHrP(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) exhibited a 2.3-fold
higher apparent affinity for the PTH-1 receptor than did the Parent
peptide (P = 0.0006), and
[I5,W23]-PTHrP(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) showed a 1.5-fold
higher apparent affinity for the PTH-2 receptor than did the
full-length control (P = 0.007).
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, C and D; Table 2). With the PTH-1 receptor,
[I5,W23]-PTHrP (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) and
[I5,W23]-PTHrP (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) showed 2.2-fold and
3.5-fold reductions in potency relative to the Parent peptide,
respectively (P < 0.07 for each analog vs.
Parent). The efficacy of [I5,W23]-PTHrP
(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) was indistinguishable from that of the Parent peptide, whereas
[I5,W23]- PTHrP (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) was a partial agonist
with this receptor (Maxobs = 32% Parent,
P = 0.003). Similar effects were observed with the
PTH-2 receptor for deletion of ligand residues 1 and 2 (Fig. 1 D). The
shorter-length peptides, [I5,W23]-PTHrP
(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) and [I5,W23]-PTHrP (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) elicited
maximal cAMP responses with each receptor subtype that were at least
10-fold lower than that produced by
[I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36).
Bpa substitutions
To probe further the importance of the N-terminal region of
[I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) in mediating receptor
activation, we prepared a series of photolabile analogs of
[I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) that were singly
modified with Bpa along the first six residues (Table 1) and examined
their functional and biochemical properties with both PTH receptors in
LLC-PK1 cells. In competition binding assays conducted with
the partial agonist 125I-bPTH(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and the PTH-1
receptor, all of the Bpa-substituted peptides bound more weakly than
did the Parent(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) (P < 0.03 for each analog
IC50 vs. Parent IC50), and more than
20-fold reductions in apparent binding affinity were observed with
Bpa-4 and Bpa-5 (Fig. 2A and Table 3). Smaller effects on binding potencies
were seen with the PTH-2 receptor, as Bpa-1 and Bpa-5 bound with
potencies that were not significantly different (P >
0.25) from that of the Parent ligand, and only Bpa-3 and Bpa-4
exhibited more than 2-fold reductions in apparent affinity, relative to
the control peptide (Fig. 2B and Table 3). When the agonist radioligand
125I-rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) was used as the tracer, the binding
profiles observed for these analogs with either receptor were similar
to those seen in the competition studies performed with
125I-bPTH (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), in that Bpa-4 and Bpa-5 exhibited the
lowest apparent binding affinity for the PTH-1 receptor
(IC50’s > 1000 nM, still representing a
>20-fold shift from the Parent), and Bpa-3 and Bpa-4 exhibited the
lowest apparent binding affinity for the PTH-2 receptor (but were still
only 2-fold weaker than the Parent peptide) (Table 3). The binding
IC50 values obtained with the agonist tracer tended to be
higher than those measured with the partial agonist tracer.
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, C and D). For the PTH-1
receptor, lower binding IC50 values were observed in the
homologous binding experiments, compared with the values observed in
the 125I-PTH(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)-based experiments, for Bpa-1 (2.7-fold,
P = 0.002), Bpa-4 (2.8-fold, P =
0.006), and Bpa-5 (24-fold, P = 0.03). For the PTH-2
receptor, lower IC50 values were again noted for Bpa-1
(2.6-fold, P = 0.07) and Bpa-5 (2.8-fold,
P = 0.06). The improvements in the apparent binding
potencies of Bpa-1 and Bpa-5 with either receptor were even more
pronounced when the homologous competition data were compared with the
corresponding results obtained with 125I-rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Table 3). Finally, we noted that the slopes of the homologous binding curves
obtained with the PTH-1 receptor were nonparallel, and in particular,
Bpa-5 exhibited a shallower slope than did peptides of comparable
affinity (e.g. Bpa-1 and Bpa-2; Fig. 2C). The Hill
coefficient derived for Bpa-5 (0.9 ± 0.2) was significantly
different from that of Bpa-1 (1.7 ± 0.03, P =
0.02) or Bpa-2 (1.5 ± 0.2, P = 0.04), perhaps
indicating a change in binding cooperativity (29). In contrast, the
slopes observed with the PTH-2 receptor were parallel (Fig. 2 D), and
the Hill coefficients ranged from 1.0–1.2.
The Bpa-substituted analogs exhibited striking differences in
their ability to stimulate cAMP generation (Fig. 3 and Table 4). Bpa-1 was a potent and efficacious
agonist with both the PTH-1 receptor (EC50 = 8 ±
1 nM, Maxobs = 100% Parent) and PTH-2
receptor (EC50 = 17 ± 11 nM,
Maxobs = 80% Parent). In contrast, Bpa-3 and Bpa-4
were weak partial agonists with both receptors (EC50
values > 85 nM and Maxobs < 35%
Parent for each receptor). The remaining analogs exhibited receptor
subtype- selective agonist properties. Bpa-5 was a ca. 4-fold more
potent and 5-fold more efficacious agonist, relative to Parent, for the
PTH-1 receptor than for the PTH-2 receptor, despite having a binding
profile that dramatically favored the latter, particularly when assayed
by heterologous competition (see Discussion). In contrast,
Bpa-2 was an agonist/partial agonist with the PTH-2 receptor but was
nearly inactive in the cAMP assays performed with the PTH-1 receptor
(Maxobs = 10% Parent, P <
10-5). Bpa-6 was 2-fold more potent as an agonist with the
PTH-2 receptor than with the PTH-1 receptor (EC50
values = 15 ± 10 and 34 ± 1 nM,
respectively, P = 0.05), and elicited only a partial
response with the latter receptor (Maxobs = 50%
Parent, P < 10-5).
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). In most cases, a single band that
migrated with the mobility expected for the glycosylated PTH-1 or PTH-2
receptor (
90,000 Da) was apparent by SDS-PAGE. This photoaffinity
labeling was specific for the PTH receptors, as indicated by: 1) the
ability of a 1-µM dose of noniodinated
[I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) to abolish each of the
cross-links (data not shown); and 2) the difference in the mobilities
of the bands obtained from the reactions performed with the PTH-1
receptor (593 amino acids) and its lower molecular weight PTH-2
receptor counterpart (550 amino acids). With the PTH-1 receptor, Bpa-1,
Bpa-2, Bpa-3, and Bpa-5 each yielded strong band intensities; with the
PTH-2 receptor, Bpa-1, Bpa-4, Bpa-5, and Bpa-6 yielded the strongest
signals. Thus, as with the binding and cAMP signaling assays,
receptor-selective effects were observed in the photochemical
cross-linking of some of these analogs.
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). This assay revealed that Bpa-2 and
Bpa-4 were antagonists, but, in contrast to the truncated PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
analog, these full-length peptides exhibited signs of receptor-subtype
selectivity. A similar analysis of
[I5,W23]-PTHrP(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), -(4–36), and -(5–36)
revealed that [I5,W23]-PTHrP(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) more
efficiently antagonized the actions of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) on both receptor
subtypes than did the other two N-terminally truncated analogs (data
not shown).
When tested against a full-range dose-response curve of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) with
the PTH-1 receptor, a 1-µM dose of either Bpa-2 or
[I5,W23]-PTHrP(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) increased the
EC50 of the PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) response by 8- to 10-fold (Fig. 6A). Inhibition of the PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
response by the PTH-1 receptor partial agonist Bpa-4 was not detected
with the PTH-1 receptor (Fig. 6A). The dose of peptide
(IC50A) that achieved 50% inhibition of the response
elicited by 3.3 nM PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) on the PTH-1 receptor was
0.32 ± 0.07 µM for Bpa-2 and 0.27 ± 0.08
µM for [I5,W23]-PTHrP(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
(Fig. 6C); these two peptides were both 10-fold more potent antagonists
than [L11, D-W12,
W23]-PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) (IC50A = 4.6 ±
1.0 µM, P = 0.02 vs. Bpa-2 or
(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), Fig. 6C). The maximum inhibition observed for Bpa-2 (65%, at
1 µM) differed significantly (P <
0.0001) from that attained by
[I5,W23]-(5–36) (90% at 3 µM)
(Fig. 6C). With the PTH-2 receptor, a 1-µM dose of either
Bpa-4 or [I5,W23]-PTHrP(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) altered the
response to only low doses of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (e.g. <10
nM) and did not alter the EC50 of the
PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) dose-response curve (Fig. 6 B). Although Bpa-2 was a weak or
partial agonist with the PTH-2 receptor, it did not antagonize the
actions of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) with this receptor (Fig. 6B). Bpa-4,
[I5,W23]-PTHrP(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), and [L11,
D-W12, W23]-PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) each inhibited the
response induced by 3.3 nM PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) on the PTH-2 receptor
with similar potencies (IC50A values = ca. 3
µM, P > 0.25, Fig. 6D).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To further explore the role of the N-terminal residues of [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) in receptor activation, we adopted a photochemical cross-linking approach. Although a number of different photolabile amino acids are known (34), we chose to employ para-benzoyl-L-phenylalanine (Bpa) for our study, because the precursor, (S)-Bpa, is readily available and is compatible with the Fmoc amino acid protection scheme for peptide synthesis. In addition, Bpa-substituted peptides frequently exhibit high photochemical cross-linking efficiencies (23, 34). Several groups have used the Bpa modification in cross-linking studies of the PTH receptors (19, 22, 35) and one other Class II receptor (36). In particular, Bissello and co-workers have recently reported on PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) analogs that contain Bpa at positions 1–6 and have shown that [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]-bPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)NH2 cross-links to a site in the third extracellular loop of the PTH-1 and PTH-2 receptors (21, 22).
The functional consequences of introducing the large, hydrophobic benzophenone sidechain of Bpa into the N-terminal region of [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) were strongly position dependent. Thus, Bpa-1 was a potent and fully efficacious agonist for both receptor subtypes, whereas Bpa-3 and Bpa-4 exhibited reduced binding and signaling capabilities with both receptors. Substitution of Bpa at positions 2, 5, and 6 resulted in peptides that showed a dissociation of receptor-binding affinity and signaling potency; these effects were dependent on the PTH receptor subtype examined.
Previous studies with the PTH-1 receptor have shown that certain natural substitutions in the N-terminal region of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) impair cAMP signaling more than receptor-binding affinity (37, 38), thus indicating the potential for full-length PTH receptor antagonists. We found that Bpa-2 was a potent antagonist with the PTH-1 receptor, and that Bpa-4 was an antagonist with the PTH-2 receptor. The ability of Bpa-2 to inhibit PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) activity on the PTH-1 receptor is reminiscent of the antagonist action of [Arg2]-PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), although the efficacy of the latter antagonist depends on the species of PTH-1 receptor used (39), whereas the effectiveness of Bpa-2 does not (data not shown). Although we have previously reported that [W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) (histidine at position 5) is a receptor-subtype selective antagonist for the PTH-2 receptor (15), the ability of an analog with a position 4 modification (e.g. Bpa-4) to antagonize the PTH-2 receptor has not, to our knowledge, been described heretofore. In contrast to Bpa-2 and Bpa-4, the N-terminally truncated antagonists [L11,D-W12,W23]-PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) and [I5,W23]-PTHrP(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) did not effectively discriminate between the two PTH receptors.
Each of the N-terminal Bpa-substituted PTHrP analogs studied here efficiently cross-linked to at least one of the PTH receptor subtypes. Different patterns of cross-linking intensities were observed with the PTH-1 receptor and PTH-2 receptor for this series of analogs. These patterns of subtype-selective cross-linking likely reflect variations in the local receptor environments of each different Bpa benzophenone sidechain at the time of UV irradiation. Importantly, the photolabile Bpa-2 and Bpa-4 analogs strongly cross-linked to the receptors for which they functioned as antagonists. This finding suggests the possibility that contact between the benzophenone sidechain and the receptor is responsible for the observed antagonism, either because of a direct steric inhibition of receptor conformational change or the inducement of indirect allosteric constraints.
For both the PTH-1 receptor and PTH-2 receptor, Bpa-1 and Bpa-5 bound with higher apparent affinity when analyzed by homologous competition methods than when assayed by heterologous competition with 125I-PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or 125I-PTH(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). These differences could indicate that the two Bpa-substituted analogs use a subset(s) of the receptor sites or states used by PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34). Consistent with this hypothesis, Scatchard transformation of the homologous binding data revealed that, for either receptor subtype, the number of binding sites estimated for Bpa-1 and Bpa-5 were similar to each other but 3-fold lower (P < 0.05) than those calculated for PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or the other Bpa analogs (data not shown). Nevertheless, the results indicate that caution be used when interpreting the data derived from the mapping of cross-linking sites for Bpa1- and Bpa5-[I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), and highlight the value of homologous competition experiments for analyzing the binding of photolabile peptide analogs.
The objective of the present study was to elucidate further the chemical basis for the modulation of ligand-induced receptor activation through the use of new N-terminally truncated and Bpa-substituted PTHrP analogs. Taken together, our data show that: 1) residues 2–4 of [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) play critical roles in activating both the PTH-1 receptor and PTH-2 receptor; 2) the insertion of residues containing large, hydrophobic sidechains into the N-terminal region of this peptide generates position-dependent receptor-specific effects on binding and signaling; and 3) both N-terminally truncated and full-length PTHrP analogs can serve as potent antagonists for these receptors. Because receptor/ligand complexes derived from both photolabile agonists (Bpa-1/PTH-1 receptor, Bpa-6/PTH-2 receptor) and antagonists (Bpa-2/PTH-1 receptor, Bpa-4/PTH-2 receptor) have been obtained, these analogs could help in probing the differences between the active and inactive forms of the PTH receptors. Future studies will focus on mapping these receptor/ligand contacts and thus should complement the ongoing efforts to analyze different receptor states using receptor mutagenesis methods (16, 17, 39).
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Acknowledgments |
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Footnotes |
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Received March 8, 1999.
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) to varying doses of a [I5,
W23]-PTHrP(
