Endocrinology Vol. 142, No. 4 1534-1545
Copyright © 2001 by The Endocrine Society
Selective and Nonselective Inverse Agonists for Constitutively Active Type-1 Parathyroid Hormone Receptors: Evidence for Altered Receptor Conformations1
Percy H. Carter,
Brian D. Petroni,
Robert C. Gensure,
Ernestina Schipani,
John T. Potts Jr. 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
 |
Abstract
|
|---|
The spontaneous signaling activity of some G protein-coupled receptors
and the capacity of certain ligands (inverse agonists) to inhibit such
constitutive activity are poorly understood phenomena. We investigated
these processes for several analogs of PTH-related peptide (PTHrP) and
the constitutively active human PTH/PTHrP receptors (hP1Rcs)
hP1Rc-H223R and hP1Rc-T410P. The N-terminally truncated antagonist
PTHrP(5-36) functioned as a weak partial/neutral agonist with both
mutant receptors but was converted to an inverse agonist for both
receptors by the combined substitution of Leu11 and
D-Trp12. The N-terminally intact analog
[Bpa2]PTHrP(136)a partial agonist with the wild-type
hP1Rcwas a selective inverse agonist, in that it depressed basal cAMP
signaling by hP1Rc-H223R but enhanced signaling by hP1Rc-T410P. The
ability of [Bpa2]PTHrP(136) to discriminate between
the two receptor mutants suggested that H223R and T410P confer
constitutive receptor activity by inducing distinct conformational
changes. This hypothesis was confirmed by the observations that: 1) the
double mutant receptor hP1Rc-H223R/T410P exhibited basal cAMP levels
that were 2-fold higher than those of either single mutant; and 2)
hP1Rc-H223R and hP1Rc-T410P internalized 125I-PTHrP(536)
to markedly different extents. The overall results thus reveal that two
different types of inverse agonists are possible for PTHrP ligands
(nonselective and selective) and that constitutively active PTH-1
receptors can access different conformational states.
 |
Introduction
|
|---|
THE TYPE-1 PTH receptor (PTH/PTHrP receptor
or P1Rc) mediates the homeostatic calcium-regulating actions of PTH and
the paracrine developmental actions of PTH-related peptide
(1). Three different constitutively active mutant PTH-1
receptors have been identified in patients with Jansens metaphyseal
chondrodysplasia, a rare disease characterized by hypercalcemia and
short stature (2, 3, 4, 5). Each of these activating
mutations occurs near the cytoplasmic terminus of one of the
transmembrane domains: the His223
Arg mutation in TM 2
(2), the Thr410
Pro mutation in TM6 (4, 5),
and the Ile458
Arg mutation in TM7 (3). In COS-7 cell
expression systems, these mutations resulted in 4- to 10-fold increases
in the basal level of cAMP, relative to the basal level of cAMP in
cells expressing the wild-type PTH-1 receptor (2, 3, 4, 5).
Confirmation of the predicted in vivo hyperactivity of these
mutant receptors was provided by a recent study of transgenic mice in
which it was shown that the targeted expression of hP1Rc-H223R in
chondrocytes rescues the lethal phenotype associated with the deletion
of the gene for PTH-related peptide (6).
We have previously reported that certain competitive antagonists for
the PTH receptor act as inverse agonists with these constitutively
active PTH-1 receptors, in that they reduce the elevated basal cAMP
signaling (7). The capacity of a competitive antagonist to
behave as an inverse agonist when studied with a constitutively active
G protein-coupled receptor has been described for a number of other
ligand/receptor systems (for review see Ref. 8). The
molecular mechanisms that underlie inverse agonism and constitutive
receptor activity have been discussed, largely in theoretical terms
(9, 10), but these are still only poorly understood
processes. The continued interest in understanding both the structural
basis for inverse agonism, as well as the mechanisms by which these
ligands modulate receptor signaling activity, is likely to provide
important insights into the fundamental mechanism(s) of ligand
recognition and receptor activation for the G protein-coupled receptors
(11). In our field, potent peptidic inverse agonists for
the constitutively active variants of the PTH-1 receptor could serve as
useful reagents in studying the transgenic mouse models that are now
being developed to dissect the role of the receptor in development
(6). In addition, small molecule mimmetics of these
peptides could provide a therapeutic benefit to patients with Jansens
metaphyseal chondrodysplasia.
Our earlier work revealed that the N-terminally truncated
peptides
[Leu11,D-Trp12]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)NH2
and [D-Trp12]PTH
(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 inverse agonists with both the
hP1Rc-H223R and hP1Rc-T410P constitutively active receptors
(7). These two antagonist/inverse agonist peptides
exhibited 3- to 30-fold higher apparent binding affinities than did the
other (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) antagonist peptides in the study, and each contained Leu
and D-Trp at positions 11 and 12, respectively. These
observations suggested two possibilities regarding the molecular basis
for inverse agonism in PTH/PTHrP ligands: 1) all high affinity
N-terminally truncated antagonists act as inverse agonists for the
constitutively active PTH-1 receptors; and 2)
Leu11, D-Trp12,
or their combination serves as a structural determinant of inverse
agonism. We addressed these possibilities in the present study by
using two newly described high-affinity antagonist
peptides[Ile5,Trp23,Tyr36]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)NH2
and
[Bpa2,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
(12)both of which were previously shown to be
approximately 17-fold more potent as competitive antagonists with the
wild-type PTH-1 receptor than was
[Leu11,D-Trp12,Trp23,Tyr36]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)NH2,
and neither of which contained the Leu11 or
D-Trp12) substitutions. As described
herein, our analysis of these peptide analogs and their derivatives
reveal that neither of our initial hypotheses was entirely correct and
that different mechanistic modes of inverse agonism in the PTH/PTHrP
system are possible. Furthermore, the discovery that
[Ile5,Bpa2,Trp23,Tyr36]-
PTHrP136NH2 acted as a selective inverse
agonist for hP1Rc-H223R led us to reinvestigate the mechanisms of
constitutive activation used by hP1Rc-H223R and hP1Rc-hT410P, and, in
so doing, to determine that the H223R and T410P mutations induce
different activated states of the receptor.
 |
Materials and Methods
|
|---|
Peptides
Amino acid sequences of the peptides used are summarized in
Table 1
. All peptides used in this study
contained a free amino terminus (with the exception of
[desNH2-Ala1,Tyr34]hPTH(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)
and a carboxamide at the C-terminus; all PTHrP(x-36) analogs contained
the modifications of Ile5 (unless truncated at
residue 7), Trp23 and Tyr36
(13). In most cases, these shared peptide modifications
are not indicated in the subsequent textual references to the peptide
analogs. The peptide
[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)} was purchased from Bachem (Torrance, CA).
All other peptides were prepared on a PE Applied Biosystems (Norwalk, CT) model 430A peptide synthesizer
using Fmoc main-chain protecting group chemistry, HBTU/HOBt/DIEA (1:1:2
molar ratio) for coupling reactions, and TFA-mediated
cleavage/sidechain-deprotection (MGH Biopolymer Synthesis Facility,
Boston, MA). Each peptide 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 was
secured by analytical HPLC, mass spectrometry, and amino acid analysis,
respectively. Radiolabeling was performed using
125I-Na (2,200 Ci/mmol, NEN Life Science Products) and chloramine-T; the resultant
[125I-Tyr]-ligand was purified by HPLC.
Cell culture and DNA transfection
Stock solutions of EGTA/trypsin and antibiotics were obtained
from Life Technologies, Inc. (Gaithersburg, MD); FBS was
obtained from HyClone Laboratories, Inc. (Logan, UT).
COS-7 cells were cultured at 37 C in DMEM supplemented with FBS (10%),
penicillin G (20 U/ml), streptomycin sulfate (20 µg/ml), and
Amphotericin B (0.05 µg/ml) in a humidified atmosphere
containing 5% CO2. The wild-type and mutant
PTH-1 receptor complementary DNAs (cDNAs) were contained in the pcDNA1
vector (Invitrogen, Carlsbad, CA) (5, 14).
The plasmid encoding hP1Rc-H223R/T410P was constructed by replacing a
SacI-SacI restriction DNA fragment of
hP1Rc-T410P, which extended from the 5' polylinker region to a site
corresponding to the middle of extracellular loop 2, with the
corresponding fragment of hP1Rc-H223R. The COS-7 cells were transfected
in 24-well plates when the cells were of 8595% confluency using
DEAE-dextran (15). For each well, 200 ng of plasmid DNA
was used [except for the expression normalization studies (see Fig. 6
), where varied amounts of DNA were used]. All DNA was
purified by cesium chloride/ethidium bromide gradient centrifugation.
Assays were conducted 7296 h after transfection, at which point
approximately 20% of the cells expressed surface wild-type PTH
receptors at a density of about 5 x 106 per
cell (15).

View larger version (30K):
[in this window]
[in a new window]
|
Figure 6. Effects of combining the H223R and T410P mutations
on PTH-1 receptor function. A, COS-7 cells were transiently transfected
using plasmid DNA; 200 ng/well) encoding either hP1Rc-WT, hP1Rc-H223R,
hP1Rc-T410P, or hP1Rc-H223R/T410P, and then subsequently treated with
either buffer alone (solid columns) or buffer containing
1 µM [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 (hatched
columns). The data (mean ± SEM) are from
three independent experiments, each performed in duplicate or
triplicate. B and C, COS-7 cells were transfected with hP1Rc-WT DNA at
1.6 ng or 400 ng of DNA per well or with hP1Rc-H223R/T410P DNA at 400
ng/well. In panel B, the cells were treated as in A. In panel C, the
level of cell-surface expression of the receptor was measured using a
primary antibody directed against the receptors extracellular domain
and an iodinated secondary antibody. The data of panels B and C were
extracted from a larger experiment (performed three times in duplicate)
in which the amount of DNA used in the transfection for each receptor
varied from 0.2400 ng per well in 2-fold increments. When the
resulting surface expression levels (and cAMP responses) were measured,
the expression levels of hP1Rc-WT and hP1Rc-H223R/T410P were most
closely matched (P = 0.5) using 1.6 and 400 ng of
DNA encoding the respective receptors. The data (mean ±
SEM) are from the three experiments performed in duplicate.
D, COS-7 were cells transfected with DNA encoding hP1Rc-H223R/T410P
(200 ng/well) and subsequently treated with varying doses of
[Ile5, Trp23,
Tyr36]hPTHrP(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 ( ),
[Leu11,D-Trp12, Trp23,
Tyr36]-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 )NH2 ( ),
[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
(), [Bpa2, 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 ( ),
[Trp2, 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 ( ),
[Arg2, 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 ( ), or
[desNH2-Ala1,
Tyr34]-hPTH(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 (X), and then
assayed for cAMP accumulation. In panels A, B, and D, the differences
between the basal and 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 )-modulated cAMP levels for
hP1Rc-H223R/T410P were statistically different (P =
0.04, 0.00001 and 0.0002, respectively). The data (mean ±
SEM) were derived from three to six independent
experiments, each performed in duplicate; in each experiment, the cAMP
values were normalized to the cAMP level detected in the absence of
added ligand (100%).
|
|
cAMP accumulation assays
Assays of transfected COS-7 cells were performed in 24-well
plates. Cells were rinsed with 0.5 ml of 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 with
100 µl of binding buffer containing varying amounts of peptide analog
and 200 µl of cAMP assay buffer (DMEM containing 2 mM
3-isobutyl-1-methylxanthine, 1 mg/ml BSA, 35 mM HEPES-NaOH,
pH 7.4). The medium (total volume = 300 µl) was removed after a
30 min incubation at room temperature. The cells were then 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. Nonlinear
regression was used to calculate the EC50 values
for cAMP accumulation and to curve-fit the data (see below).
Competitive antagonism studies
The cAMP accumulation protocol described above was used for
studies of antagonist peptides with some minor modifications. Cells
were rinsed with 0.5 ml binding buffer and treated successively with
100 µl of binding buffer containing an antagonist peptide, 100 µl
of cAMP assay buffer, and 100 µl of cAMP assay buffer containing the
agonist 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)
([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)
or inverse agonist
{[Leu11,D-Trp12]-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)
or
[Leu11,D-Trp12]-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)}
for a final volume of 300 µl. The cells were then incubated for 30
min at room temperature and processed as above for quantification of
intracellular cAMP levels.
Competition binding assays
Binding reactions were performed in 24-well plates. Cells were
rinsed with 0.5 ml of binding buffer and then treated successively with
100 µl binding buffer, 100 µl of binding buffer containing various
amounts of unlabeled competitor ligand, and 100 µl of binding buffer
containing approximately 100,000 cpm of
125I-tracer (ca. 26 fmol; final volume = 300
µl). Incubations were 4 h at 15 C, except for experiments
designed for Scatchard analysis, which were incubated 6 h at 4 C.
Cells were then placed on ice, the binding medium was removed, and the
monolayer was rinsed three times with 0.5 ml of cold binding buffer.
The cells were subsequently lysed with 0.5 ml 5 N NaOH and
counted for radioactivity. The nonspecific binding for each experiment
was determined by competition with a 1 µM dose of
unlabeled
[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.
The maximum specific binding (B0) was the
total radioactivity bound in the absence of unlabeled ligand, corrected
for nonspecific binding. Nonlinear regression was used to calculate
binding IC50 values (see below).
Antibody binding analyses
Indirect antibody binding studies were performed to quantify
levels of receptor surface expression in intact transfected COS-7
cells. The primary antibody was an affinity purified rabbit polyclonal
antibody (anti-H2) that recognizes an epitope in the amino-terminal
extracellular domain of the human PTH-1 receptor, and the secondary
antibody was 125I-labeled goat antirabbit Ig. The
binding and washing steps were performed as described
(5).
Photochemical cross-linking
Photochemical cross-linking was carried out with transiently
transfected COS-7 cells as described (12). All
manipulations were executed on ice using chilled reagents. COS-7 cells
in 6-well plates were rinsed with 2.0 ml binding buffer, treated with 1
ml of binding buffer containing ca. 4 x 106
cpm (ca. 1 pmol) of
125I-[Bpa2]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 incubated for 4 h at 13 C. The cells were then chilled on ice,
the medium was removed, and the cells were rinsed twice with 1 ml of
binding buffer. Next, the cells were covered with 800 µl of binding
buffer and exposed to irradiation with a Black-Ray UV lamp (366
nM, 7 mW/cm2, source-to-cell =
4.5 cm) for 15 min on ice in a cold room (4 C). The medium was
withdrawn and the cells were rinsed twice with 2 ml of ice-cold
acid-saline buffer and twice with 2 ml of ice-cold 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
30 min at 0 C before being harvested (50 µl rinse with the Triton
buffer) and centrifuged at 2,000 x g for 20 min at 4
C. The supernatant was collected and equal-volume aliquots were
analyzed using SDS-PAGE (520% acrylamide gradient) (16)
followed by autoradiography of the dried gel at -80 C with an
intensifying screen. For the experiment shown (see Fig. 5
), the
relative cross-linking efficiency for each receptor was determined by
excising the major band (
80 kDa) from the dried gel, counting the
radioactivity, dividing the resultant CPMs by the total radioactivity
that specifically bound to that receptor (determined in a parallel
experiment in which the cells were washed with neutral buffer instead
of acid buffer), and normalizing the resulting value to the
corresponding value obtained for hP1Rc-WT.

View larger version (53K):
[in this window]
[in a new window]
|
Figure 5. Cross-linking of [Bpa2]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 wild-type and constitutively active PTH-1 receptors in COS-7 cells.
The analog 125I-[Bpa2, 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 was
bound to COS-7 cells transiently transfected with either hP1Rc-WT,
hP1Rc-H223R, or hP1Rc-T410P and, after washing the cells with neutral
binding buffer to remove unbound ligand, was exposed to UV irradiation.
The cells were then washed again with acid-saline buffer, lysed with
SDS loading buffer, and equal-volume aliquots of the resulting lysates
were analyzed by SDS-PAGE/autoradiography. The relative cross-linking
efficiencies calculated in this experiment for each receptor mutant,
compared with hP1Rc-WT (1.0), were 3.4 (H223R) and 1.6 (T410P). The
results were replicated in two other independent experiments.
|
|
Internalization assays
Cells were treated as above for competition binding assays, but
incubations were at room temperature. At each time point, the binding
process was terminated by the removal of the binding medium and the
cell monolayer was washed three times with either 0.5 ml of binding
buffer (pH 7.7), to determine total cell-associated cpm, or 0.5 ml of
acid-saline buffer (50 mM glycine, 150 mM NaCl,
adjusted to pH 2.5 with HCl), to determine the internalized cpm. The
cells were then lysed with 0.5 ml 5 N NaOH and counted for
radioactivity. Nonspecific binding (subtracted) was determined at each
time point and for each washing condition using COS-7 cells transfected
with pcDNA1.
Other data calculation
Calculations were performed using Microsoft Corp.
Excel. Nonlinear regression analyses of binding and cAMP dose-response
data were performed using the four-parameter equation:
yP = Min + [(Max - Min)/(1 +
(IC50/x)slope)]. The Excel
Solver function was used for parameter optimization, as described
previously (12, 17). Scatchard transformations of
homologous competition binding data performed with iodinated 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)
(26 fmol/well) and varying amounts of the same noniodinated ligand
(1.2300 pmol/well) were used to calculate cell surface expression
levels and apparent dissociation constants
(kDapps). The calculations assumed a transfection
efficiency of 20% a cell density of 500,000 cells per well (verified
in several representative transfections), and a single class of
ligand-binding sites. The statistical significance between two data
sets was determined using a one-tailed Students t test,
assuming unequal variances for the two sets.
 |
Results
|
|---|
Based on the hypotheses described above, we first compared the
abilities of two newly described N-terminally truncated PTHrP
antagonist analogs to function as inverse agonists in COS-7 cells
transiently transfected with the constitutively active mutant PTH
receptors hP1Rc-H223R and hP1Rc-T410P. As previously found for
[Leu11,D-Trp12]-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),
[Leu11,D-Trp12]-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)NH2
depressed intracellular cAMP levels in a dose-dependent fashion in
cells expressing either hP1Rc-H223R or hP1Rc-T410P (Fig. 1
, B and C). At the highest peptide
concentration tested, the maximum reductions achieved for the H223R and
T410P receptors were 42% and 51% of the corresponding basal cAMP
levels, respectively, and these reductions from basal were significant
(P < 0.0001). In contrast,
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)NH2 did not act as an inverse agonist
with either hP1Rc-H223R or hP1Rc-T410P, but instead, induced a weak
partial agonist response with hP1Rc-H223R (27% of the maximum
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)-induced response for this receptor) and was nearly inert
with hP1Rc-T410P (Fig. 1
, B and C). The inability of 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) to
behave as an inverse agonist with the two constitutively active
receptors was not due to weak binding affinity, as the
IC50 values observed for this analog in
competition binding analyses performed with either mutant receptor were
3- to 4-fold lower than the corresponding IC50
values observed for
[Leu11,D-Trp12]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)
(hP1Rc-H223R: 7.2 ± 1.8 nM vs.
28 ± 9 nM, P = 0.02; hP1Rc-T410P: 6.0
±1.5 nM vs. 22 ± 9
nM, P = 0.05, Table 2
).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 1. cAMP-signaling responses 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 ) and
N-terminally truncated PTHrP(x-36) analogs in COS-7 cells expressing
wild-type and constitutively active PTH-1 receptors. Shown are the
effects of the analog ligands, [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, (),
[Ile5, Trp23,
Tyr36]hPTHrP(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 ( ), or
[Leu11,D-Trp12, Trp23,
Tyr36]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 ( ) on the
cAMP-signaling properties of hP1Rc-WT (A), hP1Rc-H223R (B), or
hP1Rc-T410P (C) in transiently transfected COS-7 cells. The cells were
treated with varying doses of each peptide for 30 min at room
temperature, and the resulting intracellular cAMP levels were
quantified by RIA. The dotted lines in B and C indicate
the basal cAMP levels. The data (mean ± SEM) are from
three independent experiments, each performed in duplicate.
|
|
The experiments described above eliminated our first possibility
(vide supra), in that not all high-affinity N-terminally
truncated PTHrP fragments exhibited inverse agonism. To address the
second possibility, namely that inverse agonism arises from the
specific substitutions at positions 11 and/or 12, we synthesized three
peptides[Leu11]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),
[D-Trp12]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
[Leu11,D-Trp12]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)
(Table 1
)and tested their effects on cAMP-signaling by the
wild-type and constitutively active mutant receptors. In cells
expressing hP1Rc-WT, none of the three 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) analogs produced a
detectable change in basal cAMP levels (data not shown). In cells
expressing the mutant receptors, the singly substituted analogs,
[Leu11]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
[D-Trp12]-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),
behaved similarly to the unmodified 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) parent peptide and
induced weak increases or no change in cAMP production levels (Fig. 2
, A and B). However, the doubly
substituted analog
[Leu11,D-Trp12]-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)
depressed cAMP levels with both hP1Rc-H223R and hP1Rc-T410P (Fig. 2
, A
and B). Dose response analyses (Fig. 2
, C and D) indicated that with
both hP1Rc-H223R and hP1Rc-T410P the potency of
[Leu11,D-Trp12]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)
as an inverse agonist was not significantly different from that seen
with
[Leu11,D-Trp12]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),
as the EC50s obtained for the respective peptides
with hP1Rc-H223R were 59 ± 20 nM vs. (34 ± 12
nM, P = 0.2) and with hP1Rc-T410P they were
63 ± 12 nM vs. 65 ± 21
nM (P = 0.5).

View larger version (31K):
[in this window]
[in a new window]
|
Figure 2. Effects of position 11 and 12 modifications on the
inverse agonism of 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 ). A and B, COS-7 cells were transiently
transfected with either hP1Rc-H223R (A) or hP1Rc-T410P (B) and treated
with buffer alone (basal), buffer containing [Ile5,
Trp23,
Tyr36]hPTHrP(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, or buffer
containing an analog of that same peptide with the substitutions
of Lys11 Leu, Gly12 D-Trp, or
both. The peptide concentrations were 3 µM and the
treatment was for 30 min at room temperature. C and D, COS-7 cells were
transiently transfected with hP1Rc-H223R (C) or hP1Rc-T410P (D), and
treated with varying doses of [Ile5,
Leu11,D-Trp12, Trp23,
Tyr36]hPTHrP(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 ( ) or
[Leu11,D-Trp12, Trp23,
Tyr36]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 () for 30 min at
room temperature. The dashed lines indicate the basal
cAMP levels. Shown are data (mean ± SEM) from three
(A, B) or four (C, D) independent experiments, each performed in
duplicate.
|
|
Although the introduction of both the Leu11 and
D-Trp12 substitutions was sufficient
to confer inverse agonism to 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), it had a surprisingly
deleterious effect on receptor-binding affinity. Thus, with each
receptor, the IC50 observed for the disubstituted
peptide was approximately 15-fold higher than that observed for
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) (P < 0.002). This reduction in apparent
binding affinity could be largely attributed to the
Gly12
D-Trp
substitution, which by itself reduced affinity approximately 45-fold,
relative to the binding affinity that 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) exhibited for each
receptor (P < 0.05) (Table 2
). This result contrasts
with the 5- to 13-fold improvement in binding affinity that the
Gly12
D-Trp substitution produced in
PTH(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(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) (18, 19). Consistent with the
high apparent binding affinity of unmodified 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), relative to
that of
[Leu11,D-Trp12]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
[Leu11,D-Trp12]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),
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) could inhibit the inverse agonist actions of the latter
two peptides (Fig. 3
, A and B, and data
not shown). We tested whether or not the inverse agonism inherent to
[Leu11,D-Trp12]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)
would compensate for its weaker binding affinity when assayed for
competitive 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) at the wild-type PTH-1 receptor.
As shown in Fig. 3C
, this was not the case, as
[Leu11,D-Trp12]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)
was approximately 4-fold less effective in inhibiting
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)-induced cAMP signaling at the wild-type receptor than was
the unmodified 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) analog (IC50s =
590 ± 170 nM vs. 145 ± 30
nM, respectively, P = 0.02), and
a lower maximum inhibition was observed for the modified peptide than
for the parent peptide (47 ± 7% vs. 79 ± 3%,
P = 0.001).

View larger version (16K):
[in this window]
[in a new window]
|
Figure 3. Antagonism of inverse agonists and classical
agonists by 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 ) analogs. A and B, Shown is the capacity of the
near-neutral antagonist analog, [Ile5, Trp23,
Tyr36]hPTHrP(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, to block the inverse
agonist action of [Leu11,D-Trp12
Trp23, Tyr36]-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 )NH2 in
COS-7 cells expressing hP1Rc-H223R (A) or hP1Rc-T410P (B). The cells
were treated with varying doses of the 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 ) inverse agonist
analog alone () or with a constant dose (1 µM) of the
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 ) analog ( ). Note the small partial agonist effect that
the 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 ) analog has on these two mutant receptors. C, Shown are
the capacities of [Ile5, Trp23,
Tyr36]hPTHrP(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 ( ) or
[Ile5, Leu11,D-Trp12,
Trp23, Tyr36]hPTHrP(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
( ) to inhibit the agonist response induced by [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 (3 nM) in
COS-7 cells transiently transfected with the wild-type hP1Rc. All data
(mean ± SEM) were derived from three independent
experiments, each performed in duplicate.
|
|
In parallel with the studies described above on truncated peptides, we
also examined the antagonist [Bpa2]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)
(12) (Table 1
), as well as several new structurally
related positition-2 modified 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, for inverse agonism
on the constitutively active mutant PTH-1 receptors (Fig. 4
). These experiments allowed us to test
whether or not the Leu11 and
D-Trp12 modifications, along with the
N-terminal truncation, were strictly required for inverse agonism.
The effect of these position-2-substituted 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
on cAMP accumulation in COS-7 cells expressing the wild-type human
PTH-1 receptor is shown in Fig. 4A
, and their receptor-binding
properties are presented in Table 3
. Each
of the peptides displayed a diminished maximum signaling response with
hP1Rc-WT, in comparison to 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 the rank order of
potency/efficacy was: 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) >
[Arg2]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) >
[Trp2]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) >
[Bpa2]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) >
[D-Bpa2]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)
[Bpa2]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) (Fig. 4A
). The
IC50 values obtained for these peptides in
competition binding analyses were nearly identical with those observed
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) (Table 3
), and each of the peptides was capable of
antagonizing the agonist 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 the hP1Rc-WT (data
not shown).

View larger version (23K):
[in this window]
[in a new window]
|
Figure 4. cAMP signaling properties of 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
modified at position 2. COS-7 cells were transiently transfected with
either hP1Rc-WT (A), hP1Rc-H223R (B) or hP1Rc-T410P (C) and then
treated with varying doses of [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 (),
[Arg2, 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 ( );
[Trp2, 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 ( ),
[Bpa2, Ile5, Trp23,
Tyr36]hPTHrP(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 ( ),
[D-Bpa2 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 ( ), and
[Bpa2, 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 ( ). The
dashed lines indicate the basal cAMP levels observed for
each receptor. Shown are data (mean ± SEM) from three
independent experiments, each performed in duplicate. The analog
[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 (not shown) exhibited
cAMP response profiles with each receptor that were comparable with
those exhibited by [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.
|
|
When assayed with the constitutively active hP1Rc-T410P, each of
the position-2 modified analogs induced a weak partial agonist
response. However, when assayed with hP1Rc-H223R, one of the peptides,
[Bpa2]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), exhibited inverse agonist
behavior (Fig. 4
, B and C). An EC50 value for
this response was not derived, due to the absence of a clear maximum.
The response threshold occurred at a concentration of 10 nM
and a 32% reduction in the basal cAMP levels occurred at the highest
peptide concentration tested (1 µM, Fig. 4B
); this
reduction was significant (P = 0.002). There was strong
structural specificity for this effect, as
[D-Bpa2]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 was comparable to [Bpa2]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
regards to its cAMP-signaling and receptor-binding properties with both
hP1Rc-WT and hP1Rc-T410P, was not an inverse agonist with hP1Rc-H223R.
In addition, the effect was dependent on an intact amino-terminus, as
[Bpa2]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 not an inverse agonist
(Fig. 4B
). The selectivity exhibited by
[Bpa2]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) was not altered by the
hP1Rc-I458R constitutively active receptor, for which only
[Leu11,D-Trp12]-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
[Leu11,D-Trp12]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)
were inverse agonists (data not shown). In addition to showing that
N-terminal truncation is not strictly required for inverse agonism, the
current results with [Bpa2]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)
demonstrate that it is possible to discriminate between constitutively
active PTH-1 receptors on the basis of inverse agonism.
The above results suggested that the benzophenone sidechain moiety of
[Bpa2]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) was the specific structural
feature of the ligand that mediated inverse agonism. Accordingly, we
tested whether or not the photochemical cross-linking potential of the
benzophenone group could be used to explore further its interaction
with the receptors. Following binding to transfected COS-7 cells and UV
irradiation, the radioiodinated Bpa2 analog specifically cross-linked
to the wild-type PTH-1 receptor and each mutant receptor, as
SDS-PAGE/autoradioradiographic analyses revealed a single conjugate of
approximately 80 kDa for each receptor (Fig. 5
). This band was not observed in
untransfected COS-7 cells (data not shown). As can be seen in Fig. 5
, the intensity of the band obtained with hP1Rc-H223R was greater than
that obtained with hP1Rc-WT or hP1Rc-T410P (observed in three
independent experiments). The cross-linking efficiency calculated for
hP1Rc-H223R was approximately 3.5-fold greater than that of hP1Rc-WT,
while the cross-linking efficiency
calculated for hP1Rc-T410P was similar to that of the wild-type
receptor. Thus [Bpa2]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) cross-linked
with greater intensity to the receptor for which it was an inverse
agonist (hP1Rc-H223R) than it did to the receptors for which it was a
partial agonist (hP1Rc-WT and T410P) (Fig. 5
). Previously, we observed
that [Bpa2]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) cross-linked less
efficiently to the wild-type PTH-2 receptor, for which it is a full
agonist, than it did to the hP1Rc-WT (12). Thus, the data
suggest that there is a negative correlation between the cross-linking
efficiency of [Bpa2]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 its
ability to stimulate receptor-mediated increases in cAMP
accumulation.
The receptor selectivity exhibited by
[Bpa2]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 regards to inverse
agonism and photochemical cross-linking was consistent with the notion
that the H223R and T410P mutations result in constitutive activity by
inducing distinct conformational changes in the receptor
(5). As an independent test of this hypothesis, we
constructed the double mutant receptor hP1Rc-H223R/T410P. When this
double mutant receptor was expressed in COS-7 cells, the resulting
basal levels of cAMP production were 2.0- to 2.6-fold higher than those
observed for either single mutant receptor, and 26-fold higher that
seen for hP1Rc-WT (Fig. 6A
); each of
these differences was significant (P < 0.0001). The
differences in the basal signaling levels of the double and single
mutant receptors were not due to changes in receptor expression, as the
three mutant receptors were expressed at similar levels (
2530%
wild-type hP1Rc, cf. Table 4
). The apparent hyperactivity of
hP1Rc-H223R/T410P relative to the two single mutant receptors indicates
that the H223R and T410P mutations have approximately additive
effects on basal signaling, and therefore supports the hypothesis
that the two mutations alter receptor function through distinct
conformational changes.
As shown in Fig. 6A
, treatment of the double mutant receptor with 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) resulted in a small, yet significant (P =
0.04), reduction in the intracellular cAMP level, relative to the level
observed with the untreated double mutant receptor. To assess whether
or not the basal or 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)-modulated signaling activities of
hP1Rc-H223R/T410P were commensurate with that of the agonist-stimulated
hP1Rc-WT, we compared the two receptors at equal levels of surface
expression. This equalization was achieved by adjusting the DNA
concentrations used in the COS-7 cell transfections (1.6 ng and 400 ng
of DNA per well, for hP1Rc-WT and hP1Rc-H223R/T410P, respectively) to
obtain equal amounts of PTH-1 receptor antibody bound to the cell
surface (Fig. 6C
). Under these conditions, the basal and
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)-treated cAMP levels observed for the double mutant were
2.8-fold and 1.9-fold higher, respectively, than the cAMP level
observed for the maximally stimulated hP1Rc-WT (Fig. 6B
); these
differences were significant (P = 0.003 and 0.005,
respectively). These results suggest that the signaling activity of
hP1Rc-H223R/T410P, in either the free or 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)-occupied state, is
higher than that of the agonist-stimulated wild-type receptor.
All of the peptides used so far in the study acted as inverse agonists
with hP1Rc-H223R/T410P (Fig. 6D
). We also noted that the position
2-substituted 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 were more effective inverse agonists
with this receptor than were the truncated peptides (Fig. 6D
). We
therefore screened several position 1-modified PTH analogs for effects
on the double mutant receptor. Among these,
[desNH2-Ala1]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)
was found to elicit the strongest inverse agonist response, resulting
in a maximum 75% decrease in cAMP accumulation, relative to the
untreated receptor, with an EC50 of 9.1 ± 3.8
nM (Fig. 6D
). The
[desNH2-Ala1]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)
analog exhibited an apparent binding affinity for hP1Rc-H223R/T410P (11
± 2 nM, n = 3) that was similar to that observed for
the other ligands (
10 nM, Tables 1
and 2
). With the two
other constitutively active receptors and with hP1Rc-WT,
[desNH2-Ala1]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)
acted as a weak partial agonist for cAMP production (data not shown),
as it did previously in cells expressing native PTH receptors
(20). The order of efficacy for inverse agonism on
hP1Rc-H223R/T410P, therefore, did not correlate with either 1) the
ability of the peptides to stimulate cAMP formation with the wild-type
receptor, 2) the ability of the peptides to bind to the double-mutant
receptor, or 3) the ability of the peptides to act as inverse agonists
with the single mutant receptors.
In recent studies on other G protein-coupled receptors, it was found
that different activating mutations had varied effects on the receptor
internalization process, leading to the conclusion that the mutations
induced different conformational states of the receptor
(21, 22, 23). Because our results so far suggested that H223R
and T410P confer constitutive activity by inducing different
conformational changes, we examined their effects, as well as that of
the combined mutation, on PTH-1 receptor internalization. For the
wild-type PTH-1 receptor, it has been shown that agonists induce
rapid internalization, whereas antagonists induce little or no
internalization (24, 25, 26, 27). In the current studies we used
radioiodinated 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), which elicited varied agonist/inverse
agonist responses with the different receptors, as well as
radioiodinated 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), which functioned as a near-neutral
antagonist for each receptor. Internalization was measured as the
specifically bound radioactivity resistant to washing with an
acid/saline buffer (50 mM glycine/150 mM NaCl,
pH 2.5). 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) radioligand was internalized to approximately
the same extent by the wild-type, as well as each mutant receptor: 75%
of total specifically bound peptide was resistant to acid washing
within 60 min of incubation (Fig. 7
). The
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) radioligand revealed differences in the internalization
patterns of the different receptors. With hP1Rc-WT and hP1Rc-H223R,
less than 25% of the specifically bound peptide was internalized by
the 60 min time-point (14% and 21%, respectively; Fig. 7
, A and B).
In contrast, with hP1Rc-T410P and hP1Rc-H223R/T410P, most of the bound
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) was internalized by 60 min (59% and 63%, respectively,
Fig. 7
, C and D), as was observed with these two mutant receptors and
radiolabeled 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). These results indicate that T410P (as well as
H223R/T410P) results in an activated PTH-1 receptor with
internalization properties that are distinct from those induced by
H223R.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 7. Internalization properties of wild-type and
constitutively active PTH-1 receptors. COS-7 cells transiently
transfected with hP1Rc-WT (A), hP1Rc-H223R (B), hP1Rc-T410P (C), or
hP1Rc-H223R/T410P (D) were incubated at room temperature with the
analogs 125I-[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 () or
125I-[Ile5, Trp23,
Tyr36]hPTHrP(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 ( ) (100,000 cpm per
well for each radioligand). At the indicated times, the medium was
removed and the cells were washed with buffer at a pH of 7.7 (to
determine total bound radioactivity) or pH 2.5. (to determine
internalized radioactivity). The cells were then lysed, and the
radioactivity was counted. The specifically bound acid-resistant
(internalized) radioactivity at each time point is shown as a
percentage of the total specifically bound radioactivity at that time
point. The data (mean ± SEM) are from three
independent experiments, each performed in duplicate.
|
|
 |
Discussion
|
|---|
In this study, we explored the structure-activity relationships
that underlie constitutive activity and inverse agonism in PTH-1
receptors and PTH/PTHrP peptide ligands. Our previous finding that
[Leu11,D-Trp12]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)
and [D-Trp12]PTH(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) were
inverse agonists with both hP1Rc-H223R and hP1Rc-T410P (7)
left us with two possibilities: 1) all high affinity
antagonists also act as inverse agonists for the constitutively active
PTH-1 receptors; or 2) that the
D-Trp12 substitution (with
or without Leu at position-11), was an essential determinant for
inverse agonism. To discriminate between these alternatives, we tested
two series of peptide analogs that were based on two structurally
distinct high affinity P1Rc antagonists, 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
[Bpa2]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) (12), both of
which contained the native glycine at position 12, for the capacity to
act as inverse agonists with hP1Rc-H223R and hP1Rc-T410P. The key
results of these investigations were as follows: 1) despite having the
highest binding affinity (IC50s = 6.011
nM) and inhibitory potency on the wild-type
receptor of any antagonist peptide that we have examined thus far,
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) was not an inverse agonist with either constitutively
active mutant receptor; 2) neither the Leu11 nor
the D-Trp12 substitution
was alone sufficient to confer inverse agonistic property to
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), rather, both substitutions were required and resulted in
inverse agonism at both H223R and T410P; 3)
[Bpa2]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) was an inverse agonist for
hP1Rc-H223R but not for hP1Rc-T410P; and 4) the structural requirements
at position 2 for inverse agonism with hP1Rc-H223R were highly
specific, in that
[D-Bpa2]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),
[Bpa2]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),
[Arg2]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
[Trp2]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 not inverse agonists,
even though they each exhibited a marked dissociation of binding
affinity and cAMP-stimulating potency. These data suggest that, whereas
a peptide simply needs high binding affinity and low signaling efficacy
to be a competitive antagonist, specific structural features are
required to be an inverse agonist with hP1Rc-H223R or hP1Rc-T410P. We
have thus far identified Bpa2 (in an intact
peptide), and the combination of Leu11 and
D-Trp12) (in
amino-terminally truncated peptides) as two structurally distinct
ligand determinants of inverse agonism for constitutively active mutant
PTH-1 receptors. Notably, the effectiveness of these two structural
determinants at inducing inverse agonism was dependent on the length of
the peptide in which they were studied. Thus, the combined
substitutions Leu11 and
D-Trp12 were ineffective in
the context of a full-length peptide, as
[Leu11,D-Trp12]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)
elicited agonist responses that were comparable with 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) with the wild-type, H223R, and T410P receptors (data not
shown). Likewise, Bpa2 was ineffective in the
context of a truncated peptide, as
[Bpa2]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 not an
inverse agonist for H223R (Fig. 4
).
The capacity of [Bpa2]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
distinguish between the H223R and T410P mutant receptors on the basis
of inverse agonism supports the notion that the two mutations induce
constitutive activity via different mechanisms. This hypothesis was
also suggested by our previous mutagenesis studies, in which we found
that nearly all amino acid substitutions at position 410 in the hP1Rc
resulted in constitutive activity, whereas only the His
Arg and
His
Lys substitutions did so at position 223 (5).
However, neither this earlier work, nor our present data with
[Bpa2]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), answer the question of how
the two mechanisms might differ: for example, from these data alone we
could not determine whether the H223R and T410P mutations induce two
different activated states of the receptor or whether they induce the
same activated state via different molecular rearrangements
(one of which is selectively blocked by the Bpa2
peptide). As a means to address this question, we constructed the
double mutant receptor hP1Rc-H223R/T410P, as this would allow us to
determine whether or not the mutations were additive or redundant, with
respect to their effects on receptor function. We also examined the
internalization properties of the receptors to see if they were
regulated in a similar fashion. We found that the H223R and T410P
mutations exerted additive effects on basal signaling (Fig. 6
), and
that hP1Rc-H223R and hP1Rc-T410P were internalized differently when
occupied by the near-neutral antagonist
125I-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. 7
). Thus, we conclude
that hP1Rc-H223R and hP1Rc-T410P adopt different active conformations,
each of which is able to couple to G protein in an agonist-independent
fashion, but which recruits the proteins involved in receptor
internalization with different efficiencies. It is interesting to note
that both of these active conformations are only partially active, in
that their signaling is further stimulated 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). One
possibility is that these two partially active conformations are both
accessed in normal PTH stimulation of the wild-type receptor, perhaps
as intermediate steps in a pathway leading to the fully active state
(11). The additive effect of combining the H223R and T410P
mutations on signaling is consistent with such a notion. It is
interesting to speculate as to whether or not individual structural
elements 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) also induce distinct conformational changes
(similar to H223R and T410P) and that the sum of these changes is what
is responsible for the full agonist response. By this scenario, a
partial agonist is a compound that stimulates the receptor incompletely
because it lacks all of the structural elements needed to induce all of
the conformational changes; thus the partial agonist stabilizes a
partially active conformational state. This hypothesis is clearly
distinct from a two-state model, which posits that a partial agonist
simply has different affinities for the inactive (R) and fully active
(R1) receptor conformations (11). Overall, our results are
consistent with current theories (11), which suggest that
G protein-coupled receptors have the potential to adopt multiple
activated conformational states, possibly in a sequential manner. Each
of these states may have different affinities for ligands, G proteins,
and the proteins that control receptor internalization.
Three other important questions were raised by our current studies: 1)
What is the molecular basis for the difference in the behavior of the
inverse agonists
[Leu11,D-Trp12]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 [Bpa2]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)? 2) What is the
molecular basis for the difference in the behavior of hP1Rc-H223R and
hP1Rc-T410P? and 3) Why did every ligand tested with the
hP1Rc-H223R/T410P act as an inverse agonist? Although our results do
not yet answer these questions completely, there are enough data in our
study and in the literature to provide some plausible explanations. A
previous cross-linking study (28), as well as a mutational
analysis (29), have shown that the position 2 benzophenone
sidechain group of [Bpa2]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 is
clearly the determinant of inverse agonism in this ligand, interacts
with the wild-type receptor near Met425 situated at the extracellular
boundary of TM6. We have recent data to suggest that the benzophenone
moiety of this ligand cross-links to and interacts with the same region
in the constitutively active mutant hP1Rc-H223R (data not shown). It is
not yet clear where in the wild-type or mutant receptor the sidechains
of Leu11 and
D-Trp12 in the N-terminally truncated
peptides interact. The boundary of the N-terminal extracellular domain
and TM1 is one candidate site, because this was the contact site for a
benzophenone group attached to the position 13 sidechain of a
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) cross-linking analog (30) and was also shown to
contain residues that functionally interact with the 314 region of
PTH (31). In any case, it seems likely that the putative
receptor contact sites for
Leu11/D-Trp12
are removed from the TM6 contact site of Bpa2, as suggested by the
recent computer model of the PTH/PTH receptor complex provided by
Rölz et al. (32). Although it is
attractive to speculate that the functional differences observed
between [Bpa2]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
[Leu11,D-Trp12]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)
arise from differences in receptor contacts used by
Bpa2 and
Leu11/D-Trp12,
it is also worth considering that the two substitutions might induce
different conformations in the peptide backbone (33), and
that this is the basis for their different pharmacological
profiles.
As far as the molecular basis for the differences in functional effects
of H223R and T410P is concerned, it is relevant to consider how these
mutations might have different effects on receptor conformation,
specifically in regards to movements of the seven transmembrane
helices. Evidence has been recently presented that in the WT PTH-1
receptor, a movement of the cytoplasmic terminus of TM3 away from the
neighboring TM6 is required for receptor isomerization to the active
state (34). It is possible that in the basal state of the
wild-type receptor the threonine sidechain at position 410 plays a key
role in preventing this movement. The finding that a wide array of
mutations at this position resulted in constitutive activation
(5) could thus be explained by the inability of the
sidechains of the substituted amino acids to restrain this TM3/TM6
movement. Based on their computer modeling analysis, Rölz
et al. (32) have suggested that the
His223
Arg mutation in TM2 results in a Coulombic interaction with
Glu465 at the base of TM7, and that this interaction promotes receptor
activation. What is remarkable about our current functional data are
that the benzophenone moiety of
[Bpa2]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 interacts with the
extracellular boundary of TM6, does not block the constitutive activity
of hP1Rc-T410P, which is altered in TM6, but rather, does so for the
mutant receptor altered in TM2. This suggests that the putative
interdomain interaction between TM2 and TM7 (32) can be
affected by functional determinants in the ligand that bind near the
extracellular terminus of TM6. Further work is clearly needed to
elucidate the mechanisms involved in these processes.
The question as to why all ligands tested were inverse agonists with
hP1Rc-H223R/T410P is an intriguing one. In related studies, we have
observed that the corresponding double mutant mouse P1Rc and human P2Rc
are also hyperactive, relative to the respective single mutant
receptors, and exhibit inverse agonist responses to ligands that are
otherwise agonists (data not shown). Ganguli and colleagues have
recently reported that an analogous double mutant secretin receptor
(H156R/T322P) is hyperactive, relative to the two single mutant
receptors, and that it responds to native secretin in an inverse
fashion (35). Such results seem to be consistent with
recent theoretical models that postulate that the intrinsic efficacy of
a ligand can vary with changing receptor conditions, and that even full
agonists can behave as inverse agonists if the receptor has a very high
spontaneous activation rate (36, 37). But other factors
may also be involved. We considered the possibility that 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)
causes the double mutant receptor to couple to the inhibitory G protein
(Gi); however, we found no evidence for this in
experiments performed with pertussis toxin-treated cells: the reagent
did not alter the inverse agonist response profiles obtained for the
mutant receptor (data not shown). Another possibility invokes altered
rates of receptor internalization/desensitization. For the wild-type
PTH-1 receptor, receptor internalization correlates positively with
second messenger signaling response (24, 25). We initially
postulated, therefore, that because 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) results in a lower cAMP
accumulation level with hP1Rc-H223R/T410P than does 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), the
former peptide would be internalized by that receptor less efficiently
than would be the latter. Our results clearly indicated that this was
not the case, however, as 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) was more completely internalized
by hP1Rc-H223R/T410P than was 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. 7D
). Whether or not
this higher level of internalization seen 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) with the
double mutant receptor is linked to its inverse agonist behavior is
currently unknown. In this regard, however, it is worth noting that
recent studies on lutropin/CG receptors (23) and
-1b
receptors (21) showed that classical agonist ligands,
human CG and epinephrine, respectively, elicited neutral or weak
inverse agonist responses with constitutively active receptor mutants,
and the ligands were more completely internalized by those receptors
than by the wild-type receptors. Further work is still needed to
elucidate the mechanisms of inverse agonism for the constitutively
active PTH-1 receptors and the role that receptor internalization might
play in this process.
In summary, our study with PTHrP analogs and constitutively
active PTH-1 receptors has revealed a number of new structure/function
relationships pertinent to both inverse agonism and constitutive
activity for this peptide hormone/G protein-coupled receptor pair.
Although there is still a high degree of uncertainty regarding the
receptor conformations that are adopted by the wild-type and
constitutively active PTH-1 receptors, our current work indicates that
a variety of activated receptor states are possible and that these may
be subject to different modes of subcellular regulation. Moreover, it
seems clear that structurally distinct peptide ligands can use the
varying conformational features of the constitutively active PTH-1
receptors to induce inverse agonism.
 |
Acknowledgments
|
|---|
We thank Ashok Khatri of the M.G.H. Biopolymer Core Facility for
synthesis of the peptides, and Michael D. Luck for technical assistance
during the early stages of this work.
 |
Footnotes
|
|---|
1 This study was supported by NIH Grant DK-11794. 
Received September 21, 2000.
 |
References
|
|---|
-
Kronenberg H, Abou-Samra A, Bringhurst F, Gardella
TJ, Jüppner H, Segre G 1997 The PTH/PTHrP receptor: one
receptor for two ligands. In: Thakker R (ed) Genetics of Endocrine and
Metabolic Disorders. Chapman & Hall, London, pp 389420
-
Schipani E, Kruse K, Jüppner H 1995 A
constitutively active mutant PTH-PTHrP receptor in Jansen-type
metaphyseal chondrodysplasia. Science 268:98100[Abstract/Free Full Text]
-
Schipani E, Langman C, Hunzelman J, Le Merrer M, Loke
K, Dillon MJ, Silve C, Jüppner H 1999 A novel parathyroid
hormone (PTH)/PTH-related peptide receptor mutation in Jansens
metaphyseal chondrodysplasia. J Clin Endocrinol Metab 84:30523057[Abstract/Free Full Text]
-
Schipani E, Langman C, Parfitt A, Jensen G, Kikuchi S,
Kooh S, Cole W, Jüppner H 1996 Constitutively activated
receptors for parathyroid hormone and parathyroid hormone related
peptide in Jansens metaphyseal chondrodysplasia. N Engl J
Med 335:708714[Abstract/Free Full Text]
-
Schipani E, Jensen G, Pincus J, Nissenson R, Gardella
TJ, Jüppner H 1997 Constitutive activation of the cyclic
adenosine 3',5'-monophosphate signaling pathway by parathyroid hormone
(PTH)/PTH-related peptide receptors mutated at the two loci for
Jansens metaphyseal chondrodysplasia. Mol Endocrinol 11:851858[Abstract/Free Full Text]
-
Schipani E, Lanske B, Hunzelman J, Kovacs CS, Lee K,
Pirro A, Kronenberg HM, Jüppner H 1997 Targeted expression
of constitutively active PTH/PTHrP receptors delays endochondral bone
formation and rescues PTHrP-less mice. Proc Natl Acad Sci USA 94:1368913694[Abstract/Free Full Text]
-
Gardella TJ, Luck M, Jensen G, Schipani E, Potts Jr JT,
Jüppner H 1996 Inverse agonism of amino-terminally truncated
parathyroid hormone (PTH) and PTH-related peptide (PTHrP) analogs
revealed with constitutively active mutant PTH/PTHrP receptors.
Endocrinology 137:39363941[Abstract]
-
de Ligt R, Kourounakis A, IJzerman A 2000 Inverse
agonism at G protein coupled receptors: (patho)physiological relevance
and implications for drug discovery. Br J Pharmacol 130:112[CrossRef][Medline]
-
Samama P, Pei G, Costa T, Cotecchia S, Lefkowitz
RJ 1994 Negative antagonists promote an inactive conformation of
the ß2-adrenergic receptor. Mol Pharmacol 45:390394[Abstract]
-
Costa T, Herz A 1989 Antagonists with negative
intrinsic activity at
opioid receptors coupled to GTP-binding
proteins. Proc Natl Acad Sci USA 86:73217325[Abstract/Free Full Text]
-
Gether U 2000 Uncovering molecular mechanisms
involved in activation of G protein-coupled receptors. Endocr Rev 21:90113[Abstract/Free Full Text]
-
Carter PH, Jüppner H, Gardella TJ 1999 Studies of the N-terminal region of a parathyroid hormone-related
peptide(136) analog: receptor subtype-selective agonists,
antagonists, and photochemical cross-linking agents. Endocrinology 140:49724981[Abstract/Free Full Text]
-
Gardella TJ, Luck M, Jensen G, Usdin T, Jüppner
H 1996 Converting parathyroid hormone-related peptide (PTHrP) into
a potent PTH-2 receptor agonist. J Biol Chem 271:1988819893[Abstract/Free Full Text]
-
Schipani E, Karga H, Karaplis AC, Potts Jr JT,
Kronenberg HM, Segre GV, Abou-Samra AB, Jüppner H 1993 Identical complementary deoxyribonucleic acids encode a human renal and
bone parathyroid hormone (PTH)/PTH-related peptide receptor.
Endocrinology 132:21572165[Abstract/Free Full Text]
-
Bergwitz C, Jusseaume S, Luck M, Jüppner H,
Gardella TJ 1997 Residues in the membrane-spanning and
extracellular loop regions of the PTH-2 receptor determine signaling
selectivity for PTH and PTH-related peptide. J Biol Chem 272:2886128868[Abstract/Free Full Text]
-
Laemmli UK 1970 Cleavage of structural proteins
during the assembly of the head of bacteriophage T4. Nature 227:680685[CrossRef][Medline]
-
Bowen W, Jerman J 1995 Nonlinear regression using
spreadsheets. Trends Pharmacol Sci 16:413417[CrossRef][Medline]
-
Goldman ME, McKee RL, Caulfield MP, Reagan JE, Levy JJ,
Gay CT, DeHaven PA, Rosenblatt M, Chorev M 1988 A new highly
potent parathyroid hormone antagonist:
[D-Trp12,Tyr34]bPTH-(734)NH2. Endocrinology 123:25972599[Abstract/Free Full Text]
-
Chorev M, Goldman ME, McKee RL, Roubini E, Levy J, Gay
T, Reagan JE, Fisher JE, Caporale LH, Golub EE, Caulfield MP, Nutt RF,
Rosenblatt M 1990 Modifications of position 12 in parathyroid
hormone and parathyroid hormone related protein: toward the design of
highly potent antagonists. Biochemistry 29:15801586[CrossRef][Medline]
-
Parsons J, Rafferty B, Gray D, Reit B, Zanelli J,
Keutmann H, Tregear G, Callahan E, Potts Jr JT 1975 Pharmacology
of parathyroid hormone and some of its fragments and analogues In:
Talmage R, Owen M, Parsons J (eds) Proceedings of the Vth Parathyroid
Conference. Excerpta Medica, Amsterdam
-
Mhaouty-Kodja S, Barak LS, Scheer A, Abuin L, Diviani D,
Caron MG, Cotecchia S 1999 Constitutively active alpha-1b
adrenergic receptor mutants display different phosphorylation and
internalization features. Mol Pharmacol 55:339347[Abstract/Free Full Text]
-
Leurs R, Smit MJ, Alewijnse AE, Timmerman H 1998 Agonist-independent regulation of constitutively active
G-protein-coupled receptors. Trends Biochem Sci 23:418422[CrossRef][Medline]
-
Min KS, Liu X, Fabritz J, Jaquette J, Abell AN, Ascoli
M 1998 Mutations that induce constitutive activation and mutations
that impair signal transduction modulate the basal and/or
agonist-stimulated internalization of the lutropin/choriogonadotropin
receptor. J Biol Chem 273:3491134919[Abstract/Free Full Text]
-
Malecz N, Bambino T, Bencsik M, Nissenson RA 1998 Identification of phosphorylation sites in the G protein-coupled
receptor for parathyroid hormone. Receptor phosphorylation is not
required for agonist-induced internalization. Mol Endocrinol 12:18461856[Abstract/Free Full Text]
-
Ferrari S, Behar V, Chorev M, Rosenblatt M, Bisello
A 1999 Endocytosis of ligand-human parathyroid hormone receptor 1
complexes is protein kinase C-dependent and involves arrestin2. J
Biol Chem 274:2996829975[Abstract/Free Full Text]
-
Huang Z, Bambino T, Chen Y, Lameh J, Nissenson RA 1999 Role of signal transduction in internalization of the G
protein-coupled receptor for parathyroid hormone (PTH) and PTH-related
protein. Endocrinology 140:12941300[Abstract/Free Full Text]
-
Tawfeek H, Abou-Samra A 1999 Over-expression of
beta arrestin-2 in LLCPK-1 cells stably expressing a green fluorescent
protein-tagged PTH/PTHrP receptor increases internalization. J
Bone Miner Res Suppl 14 (Abstract SU444), p S542
-
Behar V, Bisello A, Bitan B, Rosenblatt M, Chorev M 1999 Photoaffinity cross-linking identifies differences in the
interactions of an agonist and an antagonist with the parathyroid
hormone/parathyroid hormone-related protein receptor. J Biol Chem 275:917[Abstract/Free Full Text]
-
Carter PH, Gardella TJ 1999 Residues within the
third extracellular loops of the parathyroid hormone type-1 and type-2
receptors determine the receptor-selective antagonist properties of
[Bpa2,Ile5,Trp23]-PTHrP(136). J Bone Miner Res Suppl 14
(Abstract SU447), p S547
-
Adams A, Bisello A, Chorev M, Rosenblatt M, Suva L 1998 Arginine 186 in the extracellular N-terminal region of the human
parathyroid hormone 1 receptor is essential for contact with position
13 of the hormone. Mol Endocrinol 12:16731683[Abstract/Free Full Text]
-
Carter PH, Shimizu M, Luck M, Gardella TJ 1999 The
hydrophobic residues phenylalanine 184 and leucine 187 in the type-1
parathyroid hormone (PTH) receptor functionally interact with the
amino-terminal portion of PTH (134). J Biol Chem 274:3195531960[Abstract/Free Full Text]
-
Rölz C, Pellegrini M, Mierke D 1999 Molecular
characterization of the receptor-ligand complex for parathyroid
hormone. Biochemistry 38:63976405[CrossRef][Medline]
-
Chorev M, Behar V, Yang Q, Rosenblatt M, Mammi S,
Maretto S, Pellegrini M, Peggion E 1995 Conformation of
parathyroid hormone antagonists by CD, NMR, and molecular dynamics
simulations. Biopolymers 36:485495[CrossRef][Medline]
-
Sheikh SP, Vilardarga JP, Baranski TJ, Lichtarge O, Iiri
T, Meng EC, Nissenson RA, Bourne HR 1999 Similar structures and
shared switch mechanisms of the beta2-adrenoceptor and the parathyroid
hormone receptor. Zn(II) bridges between helices III and VI block
activation. J Biol Chem 274:1703317041[Abstract/Free Full Text]
-
Ganguli SC, Park CG, Holtmann MH, Hadac EM, Kenakin TP,
Miller LJ 1998 Protean effects of a natural peptide agonist of the
G protein-coupled secretin receptor demonstrated by receptor
mutagenesis. J Pharmacol Exp Ther 286:593598[Abstract/Free Full Text]
-
Chidiac P, Nouet S, Bouvier M 1996 Agonist-induced
modulation of inverse agonist efficacy at the beta 2-adrenergic
receptor. Mol Pharmacol 50:662669[Abstract]
-
Kenakin T 1997 Protean agonists. Keys to receptor
active states? Ann NY Acad Sci 812:116125
This article has been cited by other articles:

|
 |

|
 |
 
Y.-L. Zhang, J. A. Frangos, and M. Chachisvilis
Mechanical stimulus alters conformation of type 1 parathyroid hormone receptor in bone cells
Am J Physiol Cell Physiol,
June 1, 2009;
296(6):
C1391 - C1399.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
T. J. Gardella
Mimetic Ligands for the PTHR1: Approaches, Developments, and Considerations
IBMS BoneKEy,
February 1, 2009;
6(2):
71 - 85.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
D. M. Perez and S. S. Karnik
Multiple Signaling States of G-Protein-Coupled Receptors
Pharmacol. Rev.,
June 1, 2005;
57(2):
147 - 161.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
N. Shimizu, T. Dean, J. C. Tsang, A. Khatri, J. T Potts Jr, and T. J. Gardella
Novel Parathyroid Hormone (PTH) Antagonists That Bind to the Juxtamembrane Portion of the PTH/PTH-related Protein Receptor
J. Biol. Chem.,
January 21, 2005;
280(3):
1797 - 1807.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
M. R. Papasani, R. C. Gensure, Y.-L. Yan, Y. Gunes, J. H. Postlethwait, B. Ponugoti, M. R. John, H. Juppner, and D. A. Rubin
Identification and Characterization of the Zebrafish and Fugu Genes Encoding Tuberoinfundibular Peptide 39
Endocrinology,
November 1, 2004;
145(11):
5294 - 5304.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
R. C. Gensure, B. Ponugoti, Y. Gunes, M. R. Papasani, B. Lanske, M. Bastepe, D. A. Rubin, and H. Juppner
Identification and Characterization of Two Parathyroid Hormone-Like Molecules in Zebrafish
Endocrinology,
April 1, 2004;
145(4):
1634 - 1639.
[Abstract]
[Full Text]
[PDF]
|
 |
|

|
 |

|
 |
 
R. C. Gensure, P. H. Carter, B. D. Petroni, H. Juppner, and T. J. Gardella
Identification of Determinants of Inverse Agonism in a Constitutively Active Parathyroid Hormone/Parathyroid Hormone-related Peptide Receptor by Photoaffinity Cross-linking and Mutational Analysis
J. Biol. Chem.,
November 9, 2001;
276(46):
42692 - 42699.
[Abstract]
[Full Text]
[PDF]
|
 |
|