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Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Thomas J. Gardella, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. E-mail: Gardella{at}helix.MGH.Harvard.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Arg mutation in TM 2
(2), the Thr410Pro mutation in TM6 (4, 5),
and the Ile458Arg 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 Jansen’s 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]- PTHrP1–36NH2 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. 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.
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), where varied amounts of DNA were used]. All DNA was
purified by cesium chloride/ethidium bromide gradient centrifugation.
Assays were conducted 72–96 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).
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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 (5–20% 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.
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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
(1.2–300 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 Student’s t test,
assuming unequal variances for the two sets.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 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).
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)—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).
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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
Gly12D-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).
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), 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).
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, 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 (25–30%
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.
|
, 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
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 HisArg and
HisLys 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 3–14 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
His223Arg 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.
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Acknowledgments |
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Footnotes |
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Received September 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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opioid receptors coupled to GTP-binding
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 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]
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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]
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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]
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),
[Leu11,D-Trp12, Trp23,
Tyr36]-PTHrP(
),
[Nle8,21, Tyr34]-rPTH(
),
[Trp2, Ile5, Trp23,
Tyr36]hPTHrP(
),
[Arg2, Ile5, Trp23,
Tyr36]hPTHrP(
), or
[desNH2-Ala1,
Tyr34]-hPTH(
