Endocrinology Vol. 140, No. 11 4972-4981
Copyright © 1999 by The Endocrine Society
Studies of the N-Terminal Region of a Parathyroid Hormone-Related Peptide(1–36) Analog: Receptor Subtype-Selective Agonists, Antagonists, and Photochemical Cross-Linking Agents1
Percy H. Carter,
Harald Jüppner and
Thomas J. Gardella
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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The N-terminal regions of PTH and PTH-related peptide (PTHrP) are
involved in receptor-mediated signaling and subtype selectivity. To
better understand the molecular basis for these processes, we first
prepared a series of
[I5,W23,Y36]-PTHrP(1–36)NH2
analogs having stepwise deletions of residues 1–4 and characterized
them with the human (h)PTH-1 and hPTH-2 receptor subtypes stably
transfected in LLC-PK1 cells. Deletions beyond residue 2
caused progressive and severe losses in cAMP-signaling efficacy without
dramatically diminishing receptor-binding affinity; consistent with
this, [I5,W23]-PTHrP(5–36) was a potent
antagonist for both PTH receptor subtypes. We then prepared and
characterized photolabile analogs of
[I5,W23,Y36]-PTHrP(1–36)NH2
that were singly modified with
para-benzoyl-L-phenylalanine (Bpa)
along the first six residues. These full-length analogs exhibited
receptor subtype-selective agonism, antagonism, and photochemical
cross-linking profiles. In particular, the [Bpa2]- and
[Bpa4]-substituted analogs selectively antagonized and
preferentially cross-linked to the PTH-1 receptor and PTH-2 receptor,
respectively. These results demonstrate that the 1–5 region of
[I5,W23]-PTHrP(1–36) is critical for
activating the PTH-1 and PTH-2 receptors and suggest that the
individual residues in this region play distinct roles in modulating
the activation states of the two receptors. The cross-linking of both
agonist and antagonist ligands to these PTH receptors lays the
groundwork for identifying critical signaling determinants in the
ligand binding pocket of the receptor.
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Introduction
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THE PTH/PTH-RELATED peptide receptor (PTH-1
receptor) is a member of the class II subfamily of G
protein-coupled seven-transmembrane domain receptors that includes the
receptors for secretin, calcitonin, and glucagon (1, 2, 3, 4). The PTH-1
receptor is well expressed in the kidney, bone, and growth-plate
cartilage and, together with PTH and PTH-related peptide (PTHrP),
serves important endocrine and paracrine functions that are essential
to the maintenance of blood mineral ion homeostasis and skeletal
development (5). Another member of the PTH/secretin/calcitonin family
of receptors was recently identified, the PTH-2 receptor, the
physiological role of which is not known (6). Unlike the PTH-1
receptor, the PTH-2 receptor responds to PTH but not to PTHrP (7).
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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Peptides
[Nle8,18,Tyr34]-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)NH2
{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)} and
[Leu11,D-Trp12]-hPTHrP(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
were purchased from Bachem California (Torrance,
CA).
[Nle8,21,Tyr34]-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)NH2
{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)}, [Leu11,D-Trp12,
Trp23]-hPTHrP(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 {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)},
[Ile5,Trp23,Tyr36]-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
{[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) or Parent}, and
all of the amino-terminally truncated and Bpa-substituted analogs of
[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
{[I5,W23]-PTHrP(X-36) and Bpa-1 through
Bpa-6; cf. Table 1
} 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.
Cell culture
Stably transfected derivatives of the porcine kidney cell
line LLC-PK1 were used for all experiments. The HKRKB7
LLC-PK1 cell line expresses the hPTH-1 receptor at
approximately 1 x 106 receptors/cell (24), and the
hPR2–20 LLC-PK1 cell line expresses the hPTH-2 receptor at
approximately 0.8 x 106 receptors/cell (provided by
H. Takasu and F. R. Bringhurst, Endocrine Unit, Massachusetts
General Hospital). Cells were cultured in T-75 flasks (75
mm2) in DMEM supplemented with 10% heat-inactivated FBS,
100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified
atmosphere containing 95% air and 5% CO2. Cells were
subcultured in 24-well plates and, when confluent, were treated with
fresh media and shifted to 33 C for 12–24 h before the assay (16, 25).
Under these conditions, cell densities at the time of assay were
2.2 ± 0.1 and 2.3 ± 0.2 million cells per single well of a
24-well plate for the HKRKB7 and hPR2–20 cell lines, respectively
(mean ± SE, recorded in triplicate at the time of
assay on four separate occasions).
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.
Data calculation
All calculations were performed using Excel (Microsoft Corp., Redmond, WA). Nonlinear regression analysis of
binding and cAMP stimulation data were performed using four parameters,
defined as the minimum (Min), maximum (Max), midpoint
(IC50), and slope of the response curve. The
predicted response (yp) for a given dose
(x) of peptide was calculated using the following equation:
yp = Min + [(Max - Min)/(1 +
(IC50/x)slope)]. The initial
parameter values were estimated from the primary data, and the Excel
Solver function was then used to vary the four parameters to minimize
the differences between the predicted and actual responses
(least-squares method) (27). For cAMP EC50 calculations,
the Max was not constrained to the maximum response observed
(Maxobs), but was constrained to 
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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N-terminal deletions
To compare the importance of the N-terminal region of an agonist
peptide in mediating signal transduction with the PTH-1 receptor and
PTH-2 receptor, we synthesized 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), which encompassed
stepwise deletions from the N-terminus (Table 1
). 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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Figure 1. Binding and cAMP-stimulation properties of
[I5, W23]-PTHrP(X-36) analogs. A and B,
Competition binding of the [I5,
W23]-PTHrP(X-36) analogs (peptide lengths are indicated in
the symbol key) to the PTH-1 receptor (A) and the PTH-2
receptor (B) stably expressed in LLC-PK1 cells was
performed in the presence 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 ), as described
in Materials and Methods. Shown are data (mean ±
SE) combined from three individual experiments, each of
which was performed in duplicate on a separate day. C and D,
Stimulation of intracellular cAMP accumulation in LLC-PK1
cells expressing the PTH-1 receptor (C) and PTH-2 receptor (D) was
performed as described in Materials and Methods. Shown
are data (mean ± SE) combined from three individual
experiments, each of which was performed in duplicate on a separate
day. For some data points, the error bars are smaller
than the symbols.
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In contrast to its effect on binding, stepwise removal of residues 1–4
progressively diminished the cAMP stimulation potency and efficacy of
the [I5,W23]-PTHrP(X-36) analogs with both
receptors (Fig. 1, 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.
We also conducted binding experiments under more homologous conditions,
wherein each radiolabeled analog was bound in the presence of varying
doses of the same noniodinated peptide (Fig. 2, 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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Figure 3. cAMP stimulation with
Bpa-substituted-[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 )
analogs. Each panel shows the response of LLC-PK1 cells
stably expressing either the PTH-1 receptor (•) or PTH-2 receptor
( ) to varying doses of a [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 ) analog containing the Bpa substitution
indicated above the graph. Assays were performed as
described in Materials and Methods. Data shown
(mean ± SE) are combined from three individual
experiments, which were performed in duplicate on separate days. The
ordinate values are expressed as a percent of the maximum cAMP response
observed in parallel cells treated with [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 ) (100 nM); for the three
combined experiments, these maximum cAMP values were 129 ± 22
pmol/well for the PTH-1 receptor and 294 ± 39 pmol/well for the
PTH-2 receptor. The corresponding basal cAMP values (not subtracted)
were 1.5 ± 0.8 and 2.1 ± 1.0 pmol/well.
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Photochemical cross-linking
The ability of each of the radioiodinated photolabile
analogs to cross-link to the two PTH receptor subtypes was analyzed in
stably transfected LLC-PK1 cells (Fig. 4). 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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Figure 4. Photochemical cross-linking of Bpa analogs to the
PTH-1 and PTH-2 receptors. Shown is the cross-linking of the six
radioiodinated Bpa-substituted [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 ) analogs to LLC-PK1 cells
stably expressing either the PTH-1 or PTH-2 receptor. After radioligand
binding, cells were collected and separated by SDS-PAGE (5–20%
gradient), under reduced conditions, and visualized by autoradiography,
as described in Materials and Methods. The position of
the Bpa-substitution is indicated at the lane origins. The differential
mobilities of the PTH-1 receptor and PTH-2 receptor complexes seen in
the 97K region of the gel are consistent with the lower MW of the PTH-2
receptor (550 amino acids vs. 593 amino acids for the
PTH-1 receptor). The autoradiograph shows a single experiment and is
representative of two other experiments performed in similar fashion.
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Antagonism 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)
The strong dissociation of apparent binding affinity and signaling
potency observed for several of the Bpa-substituted
[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) analogs suggested that
these compounds might act as competitive antagonists. This hypothesis
was confirmed upon testing four of these analogs and a 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 peptide (each at 1 µM) for the ability to inhibit
the stimulation of the PTH-1 receptor or PTH-2 receptor by a
half-maximal stimulatory dose (3.3 nM) 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) (Fig. 5). 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,
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Abstract
Introduction
