From the Unit on Cell Biology, Laboratory of
Genetics, National Institute of Mental Health,
Bethesda, Maryland 20892-4092 and the § Endocrine
Unit, Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts 02114
Received for publication, October 16, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Ligand binding to the PTH1
receptor is described by a "two-site" model, in which the
C-terminal portion of the ligand interacts with the N-terminal domain
of the receptor (N interaction), and the N-terminal region of the
ligand binds the juxtamembrane domain of the receptor (J interaction).
Previous studies have not considered the dynamic nature of receptor
conformation in ligand binding and receptor activation. In this study
the ligand binding mechanism was compared for the G-protein-coupled
(RG) and uncoupled (R) PTH1 receptor conformations. The two-site
model was confirmed by demonstration of spatially distinct binding
sites for PTH(3-34) and PTH(1-14): PTH(1-14), which binds
predominantly to the J domain, only partially inhibited binding of
125I-PTH(3-34); and PTH(3-34), shown to bind
predominantly to the N domain, only partially inhibited
PTH(1-14)-stimulated cAMP accumulation. To assess the effect of R-G
coupling, ligand binding to R was measured by displacement of
125I-PTH(3-34) with 30 µM guanosine
5'-3-O-(thio)triphosphate (GTP The parathyroid hormone 1 (PTH1)1 receptor is a
cell-surface signal transducer for PTH and PTH-related protein (PTHrP).
PTH plays a central role in calcium homeostasis; the hormone acts on
target cells in bone (osteoblasts) and kidney (renal tubule cells) to
increase blood calcium levels (1). PTHrP is an autocrine factor,
believed to be involved in the maintenance of numerous tissues, and an
important developmental regulator, controlling breast, pancreas, skin,
and bone development (2, 3). PTH and PTHrP are involved in the etiology
and treatment of disease. PTH, when administered intermittently, acts
as a bone anabolic agent potentially useful for the treatment of
osteoporosis (4). PTHrP is overproduced by certain tumors, leading to
hypercalcemia through activation of the PTH1 receptor (5). The
intracellular signaling pathways activated by PTH and PTHrP via the
PTH1 receptor include stimulation of adenylyl cyclase, increases of
intracellular calcium, and activation of phospholipase C and
phospholipase D (6-10).
Owing to its important physiological, pathophysiological, and
therapeutic roles, the molecular mechanisms of PTH1 receptor function
have been studied extensively (11). The receptor belongs to the type II
family of G-protein-coupled receptors (GPCRs), which respond to peptide
ligands of intermediate size such as secretin, glucagon, calcitonin,
corticotropin-releasing hormone, and vasoactive intestinal polypeptide.
The receptor can be divided into two functional domains; the large
extracellular N-terminal domain (N domain) has been proposed to provide
most of the binding energy for receptor-ligand interaction (12, 13),
and the remaining juxtamembrane region of the receptor (J domain) is a
determinant of receptor activation and second messenger generation
(12-15). Likewise the ligand (PTH or PTHrP) can be divided into two
binding regions; the 15-34 portion is a determinant of receptor
binding affinity (12, 16, 17), and the 1-14 portion is a determinant of receptor activation for stimulation of cAMP production (12, 18-21).
(The cAMP-stimulating activity and high affinity binding of PTH and
PTHrP are retained within an N-terminal fragment of 34 residues (22).)
These observations suggested a "two-site" mode of receptor-ligand
interaction (Fig. 1), in which the C-terminal portion of the ligand
interacts with the N domain of the receptor (N interaction), and the
N-terminal ligand region binds to the J domain of the receptor (J
interaction) (11-13, 19). This model has also been demonstrated for
other type II GPCRs (23-26). For the PTH1 receptor, this low
resolution molecular model is supported by a large number of receptor
manipulation and photochemical cross-linking studies, which have also
suggested points of contact and/or proximity between specific amino
acid side chains of the ligand and the receptor (14, 15, 27, 28,
30-32). These observations have been combined with ligand structure
data (33, 34) and computer models of the receptor to provide atomic
resolution structural models of certain regions of receptor-ligand
interaction (28, 33, 35, 36).
Receptor-ligand interaction models for the PTH1 receptor have not taken
into account the dynamic nature of receptor conformation. Conformational change is central to the ability of a GPCR to transduce the extracellular signal of ligand binding across the plasma membrane to the intracellular signal of G-protein activation (37, 38). As a
result, evaluating the effect of this receptor conformational change is
essential for understanding ligand binding and signal transduction
mechanisms (37, 38). These mechanisms have been examined extensively
for GPCRs of the type I family, such as the Reagents and Peptides--
The following peptides were purchased
from Bachem (Torrance, CA) or Peninsula Laboratories (Belmont, CA):
[Nle8,18,Tyr34]bPTH(1-34)-amide,
[Nle8,18,Tyr34]bPTH(3-34)-amide,
[D-Trp12,Tyr34]bPTH(7-34)-amide,
[Nle8,18,Tyr34]bPTH(7-34)-amide, and
[Tyr36]PTHrP(1-36)-amide. hPTH(1-34) and bTIP39 were
obtained from AnaSpec Inc. (San Jose, CA). bTIP(7-39) was obtained
from Biomolecules Midwest (Waterloo, IL).
[MAP22-31,Tyr36]PTHrP(1-36)- amide,
[Ile5,MAP22-31,Tyr36]PTHrP(1-36)-amide,
[Ala3,10,12,Arg11] rPTH(1-14)-amide, and
[Ala1,3,10,12,Arg11,19,Tyr34]hPTH(1-34)
were synthesized by the M. G. H. Biopolymer Synthesis Facility,
Boston. MAP is a model amphipathic Preparation of Radioligands--
The radioligands
125I-[Nle8,18, Tyr34]bPTH(3-34)
and
125I-[MAP22-31,Tyr36]PTHrP(1-36)
were prepared using chloramine T as catalyst, and the
mono-iodinated peptide (2000 Ci/mmol) was purified by high pressure
liquid chromatography, as described previously (41).
Cell Culture, Transfection, and Isolation of Cell
Membranes--
HEK293 cells stably expressing the human PTH1 receptor
were grown in G418-containing media as described previously (42). For
assays of cAMP accumulation cells were transferred to
polyornithine-coated 96-well tissue culture plates 1 day prior to assay
(25,000 cells/well). For preparation of cell membranes cells were grown
in 15-cm tissue culture plates. For transfection COS-7 cells were grown
to confluence, as described previously (41), in 15-cm tissue culture
plates. Cells were transfected using the DEAE-dextran method as
described previously (41) with 25 µg of a plasmid (pCDNA.1)
encoding the human PTH1 receptor (6), in the absence or presence of
varying amounts of a plasmid encoding a G-protein Measurement of cAMP Accumulation--
Previously described
methods were used to measure cAMP accumulation in HEK293 cells
expressing the PTH1 receptor (42) and in COS-7 cells expressing the
PTH1 or PTH1 Measurement of Radioligand Binding to the PTH1
Receptor--
A rapid filtration method was used to measure the
binding of
125I-[Nle8,18, Tyr34]bPTH(3-34)
(125I-PTH(3-34)) and
125I-[MAP22-31,Tyr34]PTHrP(1-36)
(125I-[MAP]PTHrP(1-36)) to membranes prepared from
HEK293 cells or COS-7 cells expressing the PTH1 receptor (44). To
each well of a polypropylene v-bottomed 96-well plate (Nunc,
Naperville, IL), we sequentially added 25 µl of buffer (20 mM HEPES, 100 mM NaCl, 1 mM EDTA, 3 mM MgSO4, pH 7.5, supplemented with 0.3%
nonfat dried milk powder, 100 µM
(4-(2-aminoethyl))benzenesulfonyl fluoride, and 1 µg/ml bacitracin),
25 µl of radioligand, 25 µl of unlabeled ligand, and 50 µl
containing 3-10 µg of membranes for HEK293 cell membranes or 10-20
µg for COS-7 cell membranes. The mixture was incubated for 3 h
at 21 °C prior to separation of bound and free radioligand by
transfer to a Multiscreen filtration plate (MAHVN45, Millipore,
Bedford, MA) on a vacuum manifold. The filters were washed three times
with 150 µl of buffer without supplements. Filters were pre-treated
for 20 min on ice with buffer supplemented with 0.3% nonfat dried
milk. Filters were removed, and radioactivity was determined in a
Wallac 1470 Wizard gamma counter.
The amount of radioligand added varied from 70 to 220 pM
for 125I-PTH(3-34) and from 90 to 220 pM for
125I-[MAP]PTHrP(1-36). For HEK293 cell membranes the
total amount of PTH1 receptor present in the assay varied from 20 to 66 pM, whereas for COS-7 membranes the level varied from 90 to
180 pM. Total binding was less than 15% of the number of
counts added in all cases. Nonspecific binding was measured by
inclusion of a large excess of the unlabeled analogue of the
radioligand (300 nM for PTH(3-34) and 1.00 µM for [MAP]PTHrP(1-36)). When GTP Data Analysis--
Radioligand binding data were analyzed by
nonlinear regression using Prism 2.01 (GraphPad Software Inc. San
Diego, CA). Radioligand saturation of the PTH1 receptor was analyzed
using a single affinity state saturation equation. Displacement of
radioligand binding to the PTH1 receptor was analyzed using a single
affinity state binding model (Equation 1) or a two affinity state model
(Equation 2),
Data for inhibition of 125I-PTH(3-34) binding to the PTH1
receptor by PTH(1-14) were analyzed using an equation that assumes that both ligands can bind simultaneously to the receptor, according to
Model 1 below:
where R is the receptor; L(3-34) is
125I-PTH(3-34); L(1-14) is PTH(1-14);
K(3-34) is the equilibrium association constant
for 125I-PTH(3-34); K(1-14) is
the equilibrium association constant for PTH(1-14); and
Statistical comparison of multiple means was performed by single factor
analysis of variance. Statistical comparison of two means was performed
using a two-tailed Student's t test. Unless otherwise
stated, data points in figures are presented as the mean ± S.E.
of triplicate measurements, and in some cases the error bars are
enclosed within the symbol.
Characterization of 125I-[MAP]PTHrP(1-36), a Novel
Radioligand Selective for the RG State of the PTH1 Receptor--
To
evaluate the effects of receptor-G-protein coupling on the mechanism of
ligand binding to the PTH1 receptor, assays were developed for
measuring the equilibrium binding affinity of ligands for the uncoupled
receptor (R) and the receptor-G-protein complex (RG). For the R state,
the binding affinity of unlabeled ligands was measured by displacement
of the antagonist radioligand
125I-[Nle8,18,Tyr34]bPTH(3-34).
Binding was measured in the presence of a high concentration (30 µM) of GTP
The R/RG binding selectivity of 125I-[MAP]PTHrP(1-36)
was evaluated by measuring the effect of GTP
The R/RG binding selectivity of 125I-[MAP]PTHrP(1-36)
was also evaluated by overexpression of G-protein. COS-7 cells were
transfected with the PTH1 receptor alone or cotransfected with
G-protein. Cotransfection with wild-type rat G
Saturation binding experiments indicated than
125I-[MAP]PTHrP(1-36) bound with high affinity to the
PTH1 receptor in HEK293PTH1 membranes (1.9 ± 0.8 nM,
Fig. 2B). The sites recognized by this radioligand
(Bmax = 180 ± 50 fmol/mg) represented only
a fraction of the sites recognized by 125I-PTH(3-34)
(820 ± 90 fmol/mg), indicating that only a fraction (22%) of the
total receptor population is coupled to G-protein in HEK293PTH1
membranes (Fig. 2B). Radioligand association experiments demonstrated a slow rate of association of
125I-[MAP]PTHrP(1-36) (t1/2 of 1 h). Dissociation of the radioligand was complex (Fig. 2C).
Three phases were detected in the absence of GTP
These findings indicate that the large majority of sites labeled by
125I-[MAP]PTHrP(1-36) represent the RG complex. However
the radioligand is not completely selective for the RG state. We note
that a small fraction (21%) of the binding is not displaced by GTP Comparison of Ligand Binding Affinity for R and RG States of the
PTH1 Receptor--
The effect of RG coupling on ligand binding to the
PTH1 receptor was determined by measuring the affinity of unlabeled
ligands for the R and RG states of the receptor in HEK293PTH1
membranes. Ligand binding affinity for the uncoupled receptor was
measured by displacement of 125I-PTH(3-34) in the presence
of 30 µM GTP
All agonist ligands tested (Fig. 3A) bound with
significantly higher affinity to RG than R (Fig. 4 and Table I). The
extent of the preference for the RG complex over the R state varied
from 12-fold for PTH(1-34) to 160-fold for PTHrP(1-36). In contrast, antagonist ligands did not appreciably discriminate RG from R (Fig.
4B and Table I); PTH(7-34) did not significantly
discriminate RG from R, and PTH(3-34) bound with a slightly higher
affinity (2-fold) to the uncoupled receptor than the RG complex (Table I). For displacement of 125I-PTH(3-34) binding, all
ligands bound according to a single affinity state model, in agreement
with the two-site model for the PTH1 receptor (Fig.
1, Equation 4). The Ki
values obtained provide a measure of the macro-affinity of the ligand
for the R state of the receptor, a value incorporating both the
affinity of the N interaction and of the J interaction
(1/(KN(1 + KNJ)), see legend to
Fig. 1 for explanation of model parameters).
For displacement of 125I-[MAP]PTHrP(1-36) binding, a
single affinity state model described the binding of PTH(1-34) (Fig.
4A) and [Ile5,MAP]PTHrP(1-36) (Fig.
4E), whereas a two affinity state model provided the best
fit for PTHrP(1-36) (Fig. 4C) and [MAP]PTHrP(1-36) (Fig.
4D). The higher affinity of the two states most likely
represents the affinity for RG, since for [MAP]PTHrP(1-36) the
affinity of this state (2.7 nM) is equivalent to that of
125I-[MAP]PTHrP(1-36) (1.9 nM), the binding
of which is largely GTP The 1-2 Region of PTH(1-34) Is a Determinant of R/RG Selectivity
at the PTH1 Receptor--
By assuming that the two-site model (Fig. 1,
Equation 4) is appropriate for analysis of these ligand binding data
(see below), the considerations above indicate that the macro-affinity
(KN(1 + KNJ)) for agonist ligands
was higher at the RG complex than the R state. We next determined the
extent to which this increase resulted from an increase of ligand
affinity for the N domain (KN) and/or for subsequent
ligand interaction with the J domain (KNJ) of the
receptor. The role of the J interaction was investigated first by
examining the effect of modifications in the N-terminal region of the ligand.
The contribution of the 1-2 region of PTH(1-34) on R/RG binding
selectivity was examined by comparing the binding of PTH(1-34) and
PTH(3-34) to the PTH1 receptor. PTH(1-34) bound with 11-fold higher
affinity to RG (displacement of 125I-[MAP]PTHrP(1-36))
than to R (displacement of 125I-PTH(3-34) in the presence
of GTP Residue 5 of [MAP]PTHrP(1-36) Is a Determinant of R/RG
Selectivity and a Determinant of Binding Affinity at the PTH1
Receptor--
We examined further the role of the N-terminal region of
the ligand on R/RG selectivity by modification of residue 5 in
[MAP]PTHrP(1-36). Residue 5 has previously been implicated in
specifying the binding affinity of ligands for PTH receptors. Residue 5 controls the signaling selectivity of PTHrP for the PTH1 receptor over
the PTH2 receptor. (Substitution of His5 in PTHrP by the
equivalent residue in PTH(Ile) enables PTHrP to activate the PTH2
receptor (50, 51).) The same substitution increases the affinity of
PTHrP for the PTH1 receptor (50, 51). In this study, His5
in [MAP]PTHrP(1-36) was replaced with Ile. The substitution modified the R/RG selectivity; [MAP]PTHrP(1-36) bound with 97-fold higher affinity to RG than R (Fig. 4D and Table I). The selectivity was reduced to 17-fold for [Ile5,MAP]PTHrP(1-36) (Fig.
4E and Table I). Position 5 is therefore a determinant of
R/RG selectivity for [MAP]PTHrP(1-36).
The replacement of His5 by Ile also greatly increased the
affinity of [MAP]PTHrP(1-36) for the uncoupled receptor (by 160-fold (Fig. 4, D and E, and Table I)), a larger effect
than that previously observed for PTHrP (7-fold (50, 51)). The
molecular basis of this affinity-enhancing effect is not known. We
investigated the extent to which this effect was preserved at a
receptor from which most of the N-terminal domain had been removed
(PTH1 Detection of R/RG Selectivity of PTH(1-14) Binding to the
PTH1 Receptor--
The ligand modification studies above indicate that
residues in the N-terminal region of the ligand (residues 1-2 and 5)
are determinants of R/RG selectivity at the PTH1 receptor. These
findings implicate ligand interaction with the J domain of the receptor in the preference of agonist ligands for the RG state. This hypothesis was examined more directly by measuring the R/RG selectivity of a
ligand that interacts predominantly with the J domain,
[Ala3,10,12, Arg11]rPTH(1-14) (PTH(1-14)).
This N-terminal fragment activates the PTH1
PTH(1-14) displaced 125I-[MAP]PTHrP(1-36) binding to
HEK293PTH1 with the displacement curve fitted best by a two affinity
state model. The high affinity state (540 nM) represented
26% of the specific radioligand binding displaced (Fig. 5B
and Table I). This high affinity fraction was considerably less than
that for full-length (1-34 or 1-36) agonist ligands (83-100%),
suggesting that PTH(1-14) poorly stabilizes the RG state compared with
the full-length ligands. The affinity of the low affinity state (22 µM) was not significantly different from that for the
uncoupled receptor measured by displacement of
125I-PTH(3-34) binding in the presence of GTP PTH(3-34) Binds Predominantly to the N Domain of the Receptor and
Does Not Appreciably Discriminate R and RG States of the PTH1
Receptor--
A direct method with which to address the R/RG
selectivity of the N interaction would be to measure R/RG selectivity
of a ligand that only interacts with the N domain of the receptor. PTH(3-34) is a candidate ligand; circumstantial evidence suggests that
this ligand may bind predominantly to the N domain. This domain of the
receptor is a determinant of the selectivity of N-terminally truncated
analogues of PTH(1-34) for the human PTH1 receptor over the rat PTH1
receptor (13). Dissociation of 125I-PTH(3-34) from the
PTH1 receptor is mono-exponential, consistent with a single site
interaction (in contrast to the complex dissociation of
125I-PTH(1-34) which suggests more than one site of
receptor-ligand interaction (48)). In this study we determined the
extent to which PTH(3-34) binds to the J domain in a direct and
quantitative fashion, by measuring the effect of blocking this domain
on the binding affinity of the ligand. For this purpose we used
[Ala3,10,12, Arg11]rPTH(1-14) (PTH(1-14),
which binds predominantly if not exclusively to the J domain (see above).
The effect of PTH(1-14) on PTH(3-34) binding to the R state of the
PTH1 receptor was measured by displacement of
125I-PTH(3-34) binding in the presence of 30 µM GTP
The PTH(1-14) versus 125I-PTH(3-34)
displacement data were re-analyzed using a model that assumes the
following: 1) binding of PTH(1-14) to the receptor and to the
125I-PTH(3-34)-occupied receptor, and 2) binding of
125I-PTH(3-34) to the receptor and to the
PTH(1-14)-occupied receptor (Equation 3). The fitted values are given
in the legend to Fig. 5. The affinity of 125I-PTH(3-34)
for PTH(1-14)-occupied receptor was only slightly lower than the
affinity of 125I-PTH(3-34) for the nonoccupied receptor
(3.9 and 3.1 nM, respectively). Occupancy of the J domain
by PTH(1-14) therefore minimally affects the binding affinity of
125I-PTH(3-34) for the PTH1 receptor, strongly suggesting
that almost all the binding energy of this ligand is supplied by the N
interaction (Fig. 1). PTH(3-34) does not appreciably discriminate the
R and RG states of the receptor (Fig.
4B), indicating that the N
interaction is insensitive to receptor-G-protein coupling.
Validation of the Simultaneous Binding Model for PTH(1-14) and
PTH(3-34) Interaction with the PTH1 Receptor--
Simultaneous
binding of PTH(3-34) and PTH(1-14) to the PTH1 receptor is suggested
by the binding of 125I-PTH(3-34) to the receptor saturated
with PTH(1-14) (Fig. 5A). Since occupancy of the receptor by PTH(1-14) only partially reduces the binding of PTH(3-34), this binding model predicts that occupancy of the receptor by PTH(3-34) should only partially reduce the binding
of PTH(1-14). We tested this prediction by measuring the effect of
PTH(3-34) on PTH(1-14)-stimulated cAMP production in HEK293PTH1
cells.
PTH(3-34) inhibited 10 µM PTH(1-14)-stimulated cAMP
accumulation at the PTH1 receptor in a
concentration-dependent fashion. However, the antagonist
only partially inhibited the response to PTH(1-14) (Fig.
6A). In the presence of
saturating PTH(3-34) concentrations, the PTH(1-14) response was
39 ± 6% of the cAMP accumulation in the absence of antagonist.
This finding validates the simultaneous binding model for PTH(1-14)
and PTH(3-34) interaction with the receptor. A variety of other PTH1
receptor antagonists were tested for their effect on PTH(1-14)
signaling, using ligands varying in the number of residues truncated
from the N terminus. All antagonist ligands tested only partially
inhibited PTH(1-14)-stimulated cAMP accumulation (Fig. 6B).
The magnitude of the PTH(1-14) response remaining in the presence of
saturating concentrations of ligand was not significantly different for
the different antagonists (p = 0.56 (single-factor
analysis of variance),
[D-Trp12,Tyr34]bPTH(7-34),
31 ± 6% of the PTH(1-14) response in the absence of antagonist;
bTIP39, 31 ± 1%, bTIP (7-39), 38 ± 5%).
The validity of the partial antagonism of the response of PTH(1-14) by
these ligands was evaluated by testing the effect under conditions in
which the antagonist ligands would be expected to inhibit completely
the agonist-stimulated cAMP accumulation. We examined the antagonist
effect on the stimulation of hPTH(1-34) of cAMP accumulation; the N
interaction contributes the bulk of the binding energy for PTH(1-34)
(demonstrated by the >1000-fold lower potency of the ligand at the
N-terminally truncated PTH1
We examined the mechanism of the PTH(3-34)'s partial antagonism of
PTH(1-14)'s stimulation of cAMP accumulation. The concentration dependence of PTH(1-14) for stimulation of cAMP production was measured in the presence of varying concentrations of PTH(3-34) (Fig.
7A), to determine the extent
to which the antagonist affected the EC50 and/or
Emax of PTH(1-14). PTH(3-34) reduced the
Emax for PTH(1-14) (Fig. 7D). The
antagonist also produced an increase of EC50 for
PTH(1-14), but the magnitude of this increase reached a limiting value
(18-fold), producing a hyperbolic Schild plot (Fig. 7C).
Both the reduction of Emax and hyperbolic Schild
plot can be explained by noncompetitive inhibition of the effect of PTH(1-14) by PTH(3-34), fully consistent with the simultaneous binding model for interaction of these two ligands with the PTH1 receptor. The hyperbolic Schild plot indicates that saturation of the
receptor with PTH(3-34) only partially inhibits PTH(1-14)'s binding
to and activation of the receptor (52), consistent with the ability of
PTH(1-14) to bind the PTH(3-34)-occupied receptor. The reduction of
Emax (Fig. 7D) indicates that binding
of PTH(3-34) reduces the strength of signaling of the
PTH(1-14)-occupied receptor (52). Unfortunately it is not possible to
quantify the effect of PTH(3-34) on the binding affinity of PTH(1-14)
without knowing the magnitude of receptor reserve for the agonist
ligand (52). As anticipated (see above), PTH(3-34) behaved as a
competitive antagonist for inhibition of PTH(1-34)'s stimulation of
cAMP accumulation (Fig. 7B). PTH(3-34) did not affect
Emax (Fig. 7D) and produced a linear
increase of log(dose ratio Assessment of Ligand Conformation at the R and RG States of the
PTH1 Receptor--
The observation of higher affinity binding of
agonist ligands to the RG versus the R state of the receptor
implies a different conformation of the receptor when coupled to
G-protein. Other ligand binding data in this study provide
circumstantial evidence for a model of how the conformation of the
receptor differs between the R and RG states. At the uncoupled
receptor, PTH(1-14) and PTH(3-34) bind almost independently of each
other (Fig. 5A), suggesting that the receptor conformation
is "open" enough to allow access of both ligands to their binding
sites on the receptor. PTH(1-14) more effectively blocks
125I-PTH(3-34) binding in the absence versus
the presence of GTP
We tested this hypothesis more directly by increasing the fraction of
PTH1 receptors in the RG state and by testing the effect of PTH(1-14)
on the binding of 125I-PTH(3-34). COS-7 cells were
transfected with either the PTH1 receptor alone or cotransfected with
the PTH1 receptor and the mutant G-protein
G Previous studies employing modified receptors and ligands have
indicated a two-site mode of ligand recognition by the PTH1 receptor.
The C-terminal portion of the 1-34 fragment of PTH or PTHrP interacts
with the extracellular N-terminal receptor region (N interaction), and
the N-terminal portion of the ligand binds to the juxtamembrane domain
of the receptor (J interaction) (11, 12). In this study we examined the
effect of different receptor conformational states arising from
receptor-G-protein interaction on the molecular mechanism of ligand
binding to the PTH1 receptor. The principle findings of this study are
as follows. 1) Receptor-ligand interaction for the unmodified receptor
is well described by the two-site model. Strong, direct evidence for
this binding mode was provided by the novel observation of allosteric
interactions between the binding of PTH(1-14) and PTH(3-34) to the
receptor together with the inference of simultaneous receptor binding
of these two ligands. 2) Agonist ligands bind with higher affinity to
the RG state than to the uncoupled receptor, whereas antagonist ligands
bind with similar affinity to these states. 3) The J interaction is
stabilized by R-G coupling, whereas the N interaction is not appreciably affected by R-G interaction. 4) A more closed receptor conformation is suggested for the PTH1 receptor when coupled to G-protein. These findings are summarized in the model in Fig. 9.
S) present, and binding to
RG was measured by displacement of 125I-[MAP]PTHrP(1-36)
(where MAP is model amphipathic peptide), a new radioligand that binds
selectively to RG. Agonists bound with higher affinity to RG than R,
whereas antagonists bound similarly to these states. The J interaction
was responsible for enhanced agonist binding to RG: residues 1 and 2 were required for increased PTH(1-34) affinity for RG; residue 5 of
MAP-PTHrP(1-36) was a determinant of R/RG binding selectivity, and
PTH(1-14) bound selectively to RG. The N interaction was insensitive
to R-G coupling; PTH(3-34) binding was GTP
S-insensitive. Finally,
several observations suggest the receptor conformation is more
"closed" at RG than R. At the R state, an open conformation
is suggested by the simultaneous binding of PTH(1-14) and PTH(3-34).
At RG PTH(1-14) better occluded binding of 125I-PTH(3-34)
and agonist ligands bound pseudo-irreversibly, suggesting a more closed
conformation of this receptor state. The results extend the two-site
model to take into account R and RG conformations and suggest a model
for differences of receptor conformation between these states.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic
receptor, leading to the development of the ternary complex model and
its extended variants (37-39). These models describe the reciprocal
effects of G-protein (G) and agonist on their binding to the receptor
(R). For type II GPCRs a great deal is known regarding the orientation
of ligand binding, but very little is known regarding how the two-site
binding mechanism is affected by the conformational changes in the
receptor that result from R-G interaction. In this study we evaluated
the effect of R-G interaction on the two-site binding mechanism for the
PTH1 receptor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix
(Glu-Leu-Leu-Gln-Lys-Leu-Leu-Glu-Lys-Leu) substituted for residues
22-31 of PTHrP, a modification originally described for RS-66271 (40).
The MAP-containing analogues used in this study differ from the
originally described RS-66271 by extension of the C terminus to residue
36 and by substitution of position 36 with a tyrosine residue to enable
radioiodination. The letters "b," "r," and "h" designate
the peptide sequence as bovine, rat, or human, respectively. The
peptides were dissolved in 10 mM acetic acid, with the
peptide concentration calculated using the peptide content and weight
provided by the supplier. Aliquots were stored at
80 °C and used
once. 125I-cAMP was obtained from PerkinElmer Life Sciences
and Na125I (2000 Ci/mmol) was from ICN Biomedicals (Costa
Mesa, CA). Cell culture supplies were obtained from Life Technologies,
Inc., except for Dulbecco's modified Eagle's medium which was from
Mediatech (Herndon, VA).
-subunit. (In the
cotransfection of receptor and G-protein the DNA was mixed before
addition of DEAE-dextran.) The G-proteins used were wild-type
G
s in pCDNA.I/Amp and a mutant G-protein,
G
s(
3/
5), in which five
residues in rat G
s were substituted for residues in the
corresponding positions of rat G
i2
(N271K/K274D/R280K/T284D/I285T), also in pCDNA.I/Amp (43). The
mutations decrease the ability of the G-protein to activate adenylyl
cyclase but increase the affinity of receptor for G-protein (43). For
the experiment in Fig. 3B, COS-7 cells were transfected in
24-well plates with 200 µg of pCDNA.1 plasmid DNA encoding the
human PTH1 receptor or a human PTH1 receptor from which residues
24-181 had been removed (PTH1
NT (21)). Previously described methods
were used to isolate cell membranes from HEK293 cells and COS-7 cells
(44, 45). Membrane protein was quantified using the copper
bicinchoninic acid method (Pierce) with bovine serum albumin as the standard.
NT receptors (21).
S was added to
the assay, it was dispensed prior to the radioligands. In
125I-[MAP]PTHrP(1-36) dissociation experiments,
radioligand and membranes were equilibrated for 3 h at 21 °C
prior to the addition of a large excess (1.00 µM) of
unlabeled [MAP]PTHrP(1-36) (with or without GTP
S) to initiate the
dissociation phase of the experiment. In this experiment nonspecific
binding was measured by including 1.00 µM
[MAP]PTHrP(1-36) in the equilibration phase of the assay.
where Y is the total counts/min bound in the presence
of the competing ligand; NSB is nonspecific binding; SB is specific binding in the absence of the competing ligand, and X is the
logarithm of the unlabeled ligand concentration.
(Eq. 1)
(Eq. 2)
where IC50 1 and IC50 2 are the
IC50 values for displacement of radioligand binding to
affinity states 1 and 2, respectively, and F1 is the fraction of
specific radioligand binding displaced from affinity state 1. The best
fit was evaluated by comparing the goodness of fit for Equations 1 and
2 using a partial F test. The IC50 values were
corrected for the radioligand concentration (to obtain a measure of
Ki) using the method of Cheng and Prusoff
(46).
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Model 1
is the
cooperativity factor defining the effect of L(1-14)
occupancy on the receptor binding affinity of L(3-34) and
reciprocally the effect of L(3-34) occupancy on the
receptor binding affinity of L(1-14). Binding of
125I-PTH(3-34) in the presence of PTH(1-14) is described
by Equation 3,
(Eq. 3)
where Y is the total 125I-PTH(3-34)
bound (cpm) in the presence of PTH(1-14); NSB is nonspecific binding;
SB is specific 125I-PTH(3-34) binding in the absence of
PTH(1-14); [L(3-34)] is the concentration of
125I-PTH(3-34); and X is the concentration of
PTH(1-14). A value of
< 1 denotes negative cooperativity, in
which binding of PTH(1-14) blocks binding of
125I-PTH(3-34). If the value of
is not greatly less
than unity, it is possible that the level of inhibition of
125I-PTH(3-34) binding produced by PTH(1-14) may not
reach 100%. (Two other possibilities not observed in this study
include "neutral" cooperativity, in which binding of the unlabeled
ligand does not affect the level of binding of the radioligand, and
positive cooperativity, in which the unlabeled ligand increases the
level of radioligand binding.) This model is mathematically identical
to that proposed for allosteric regulation of radioligand binding to
muscarinic acetylcholine receptors by gallamine and other modulators
and is described in detail in Ref. 52.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S, which binds to the G-protein and
uncouples it from the receptor (47). (Binding of
125I-PTH(3-34) is insensitive to GTP
S (48).) For the RG
state a radioligand was required that bound selectively to this
receptor conformation. We previously showed that the PTHrP analogue
RS-66271 (40) displays a considerable selectivity for the RG state over the R state (44, 49) so we prepared a radiolabeled analogue of this
peptide,
125I-[MAP22-31,Tyr36]PTHrP(1-36).
Membranes prepared from HEK293 cells stably expressing the PTH1
receptor (HEK293PTH1 membranes) exhibited specific high affinity
125I-[MAP]PTHrP(1-36) binding with a total
binding:nonspecific binding ratio of 5:1. No specific binding was
detected in membranes prepared from nontransfected HEK293 cells or
cells transfected with the human PTH2 receptor. Specific binding was
also detected in membranes isolated from COS-7 cells expressing the
human or rat PTH1 receptor and from ROS 17/2.8 cells which endogenously
express the rat PTH1 receptor (data not shown).
S on its binding. At
saturating concentrations GTP
S inhibited 78 ± 2% of
125I-[MAP]PTHrP(1-36) binding to the PTH1 receptor
(measured as the fitted value of the lower plateau of the concentration
dependence curve, Fig. 2A). This new radioligand therefore
binds selectively to the RG
complex.2 It is more
selective than previously described radiolabeled agonists for the PTH1
receptor because binding of
125I-[Nle8,21,Tyr34]rPTH(1-34)
and 125I-[Tyr36]PTHrP(1-36) is reduced
only 32 and 51%, respectively, by GTP
S (48).
s resulted
in a slight increase of 125I-[MAP]PTHrP(1-36) binding,
but the total receptor level (labeled with 125I-PTH(3-34))
was considerably reduced (Fig. 2D). This reduction of
receptor expression probably resulted from cAMP-dependent
down-regulation of the receptor; overexpression of G
s
resulted in an increase of the basal cAMP level in COS-7 cells (from
0.84 ± 0.04 to 2.28 ± 0.01 pmol/well). In an attempt to
minimize this reduction of receptor expression, we used a mutant
G-protein (G
s(
3/
5) (43)) that is impaired in adenylyl cyclase activation. This G-protein mutant
also displays higher receptor binding affinity, which stabilizes high
affinity binding of agonist ligands. Overexpression of
G
s(
3/
5) did not increase
the basal cAMP level in COS-7 cells (0.68 ± 0.04 pmol/well).
Coexpression with the PTH1 receptor resulted in a large increase of
125I-[MAP]PTHrP(1-36) binding with only a slight
reduction of the level of receptor expression, as measured by the level
of 125I-PTH(3-34) binding (Fig. 2D). This
additional 125I-[MAP]PTHrP(1-36) binding represents the
agonist high affinity RG state. Expression of
G
s(
3/
5) increases the
fraction of [MAP]PTHrP(1-36) binding in the high affinity state, and
this component is sensitive to GTP
S (Fig. 8A).
S as follows: a very
rapid phase (t1/2 < 10 s, representing 18 ± 2% of specific binding), a more slowly dissociating phase (t1/2 = 47 ± 1 min, 50 ± 1% of
binding), and a pseudo-irreversible phase (32 ± 2% of binding).
30 µM GTP
S removed most of the pseudo-irreversible
component (now representing 4 ± 1% of specific binding), reduced
the fraction of binding and t1/2 of the slower phase
(30 ± 0%, 29 ± 1 min), and increased the amount of binding
dissociating at the very rapid rate (to 66 ± 1%). These data
suggest that GTP
S converts the slow and pseudo-irreversible phases
to the rapidly dissociating phase.
S
and that a similar fraction (18%) dissociates very rapidly. These
components likely represent a low affinity state, since a low affinity
state was detected in homologous-displacement experiments (Fig.
4D). (This low affinity state was not detected in the
radioligand saturation experiments because the maximum concentration of
radioligand that could be used was limited (to about 10 nM), due to the decrease of the specific
binding:nonspecific binding ratio with increasing radioligand
concentration.) This low affinity state might represent the uncoupled
receptor or a GTP
S-insensitive form of the RG complex. In support of
the former, the affinity of the low affinity component in
125I-[MAP]PTHrP(1-36) displacement assays was not
significantly different from that for the R state, measured by
displacement of 125I-PTH(3-34) binding in the presence of
GTP
S (Table I). Association of the
radioligands with the low affinity R state might not be directly
detectable by measurement of equilibrium binding. This low affinity
state may arise instead by spontaneous dissociation of G-protein from
the RG complex.
Ligand binding to the human PTH1 receptor, measured by inhibition of
125I-[MAP]PTHrP(1-36) binding, and by inhibition of
125I-PTH(3-34) binding in the presence of GTPS
S provides a
measurement of ligand binding affinity for the uncoupled receptor.
Statistical significance of the difference between
pKi(GTP
S) and
pKi(high) was tested by Student's
t test. NA, not applicable.
S. (This concentration of GTP
S was
near-saturating for removal of the RG state. It reduced specific
binding of 125I-[MAP]PTHrP(1-36) binding by 77%, close
to the lower plateau value of the displacement curve in Fig.
2A of 78%.) Ligand binding to the RG state was measured by
displacement of 125I-[MAP]PTHrP(1-36) in the absence of
exogenous guanine nucleotides. It is important to note that this assay
provides only an approximate measurement of ligand affinity for RG,
since a fraction of specific 125I-[MAP]PTHrP(1-36)
binding is insensitive to guanine nucleotides (Fig. 2A), and
a fraction of binding is pseudo-irreversible (Fig. 2C).
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Fig. 1.
Two-site model of receptor-ligand interaction
for the PTH1 receptor. The 1-34 fragment of PTH and PTHrP
interacts with the PTH1 receptor according to a two-site model (11,
12). The C-terminal portion of the ligand interacts with the large
extracellular N-terminal domain (N domain) of the receptor, and the
N-terminal region of the ligand interacts with the juxtamembrane domain
(J domain) comprising the transmembrane -helices and interconnecting
loops. It is assumed that ligand (L) first interacts with the N domain
(defined by the equilibrium association constant KN)
and then subsequently interacts with the J domain (defined by the
isomerization constant KNJ). This assumption of a
sequential mechanism is based upon the much lower binding affinity of
ligands for the J domain compared with the N domain. PTH(1-34)
displays >1000-fold lower potency at a receptor from which the
N-terminal domain has been removed (21). As a result, the equilibrium
fraction of receptors with ligand bound only to the J domain
(RLJ) is very small compared with the fraction with ligand
bound to the N domain or bound to N and J domains (RLN and
RLNJ, respectively). For this reason RLJ has been
excluded from the model. The equation describing equilibrium binding of
L to R is shown in Equation 4,
where KL = KN(1 + KNJ) and [Rtot] is the total
concentration of receptors. The equation is mathematically identical to
a single affinity state binding isotherm, in which
KL is the macro-affinity constant for
receptor-ligand interaction. The Ki value
measured in the experiments in Fig. 4 provide a measure of
1/KL. For the 1-34 ligand fragment, the
contribution of the micro-affinity constants KN and
KNJ to the value of KL cannot be
determined by measurement of equilibrium binding of L to R. To assess
ligand binding to the N domain and J domain, ligands were used that
interact almost exclusively with each of these domains (PTH(3-34) and
PTH(1-14), respectively, see text for details).
(Eq. 4)
S-sensitive (Fig.
2A). Therefore
Ki(high) (Table I) for displacement of
125I-[MAP]PTHrP(1-36) binding provides an approximate
measurement of the macro-affinity of the ligand
(1/(KN(1 + KNJ))) for the RG
state. (Ki(high) is the affinity from
the single state fit for PTH(1-34) and
[Ile5,MAP]PTHrP(1-36), and the high affinity state for
PTHrP(1-36) and [MAP]PTHrP(1-36).)
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Fig. 2.
Characterization of
125I-[MAP]PTHrP(1-36) binding to the human PTH1
receptor. A, effect of GTP S on equilibrium
125I-[MAP]PTHrP(1-36) binding to the PTH1 receptor in
HEK293 cell membranes. The displacement curve is a two affinity state
fit, which provided a significant improvement compared with a single
affinity state model (p < 0.0001). Nonspecific binding
was defined using 1 µM unlabeled [MAP]PTHrP(1-36).
Note that a fraction of specific binding (19%) is not displaced by
GTP
S. Total binding was 2988 cpm; nonspecific binding was 417 cpm;
and the total 125I-[MAP]PTHrP(1-36) added was 122,000 cpm (0.22 nM). The fitted lower plateau value of the
displacement curve was 896 cpm. Data are from a representative
experiment that was performed three times with similar results.
B, comparison of 125I-[MAP]PTHrP(1-36) and
125I-PTH(3-34) saturation of the PTH1 receptor in HEK293
cell membranes. Varying concentrations of radioligand were incubated
with membranes, and the data were analyzed as described under
"Experimental Procedures." Pooled data are presented as the
Scatchard transformation for comparative purposes. C,
dissociation of 125I-[MAP]PTHrP(1-36) from the PTH1
receptor in HEK293 cell membranes. Radioligand and membranes were
incubated together for 3 h prior to addition of 300 nM
unlabeled [MAP]PTHrP(1-36) (with or without GTP
S) to initiate the
dissociation phase of the assay. Data were fitted best by a
bi-exponential decay function, which significantly improved the
goodness of fit compared with a mono-exponential function
(p = 0.017 and p < 0.0001 for the
absence and presence of GTP
S, respectively). Nonspecific binding
was defined using 1 µM unlabeled [MAP]PTHrP(1-36).
Total binding and nonspecific binding were 3275 and 380 cpm,
respectively, in the absence of GTP
S and 3194 and 374 cpm in the
presence of the nucleotide. In the absence of GTP
S the fitted value
of the lower plateau (1361 cpm) was greater than nonspecific binding
(380 cpm), indicating a pseudo-irreversible phase of
125I-[MAP]PTHrP(1-36) binding. The experiment was
performed three times with similar results. D, binding of
125I-[MAP]PTHrP(1-36) and 125I-PTH(3-34) to
membranes prepared from COS-7 cells expressing the PTH1 receptor alone
or coexpressed with G-protein
-subunits. COS-7 cells in 15-cm tissue
culture plates were transfected with 25 µg of plasmid DNA encoding
the PTH1 receptor, with or without 100 µg of plasmid encoding
wild-type rat G
s or the mutant G-protein
G
s(
3/
5) (43), as described
under "Experimental Procedures." Radioligand binding to 10 µg of
membrane protein was measured for each condition. The experiment was
preformed twice with similar results, using membranes from different
transfections in the two experiments.
S) (Fig. 4A and Table I). In contrast, PTH(3-34)
bound with a similar affinity to the RG and R states of the PTH1
receptor (Fig. 4B and Table I). Therefore PTH(3-34) does
not appreciably discriminate R and RG states, and the 1-2 region of
PTH(1-34) is a determinant of ligand selectivity for the RG state.
NT, residues 24-181 removed (21)). [MAP]PTHrP(1-36) failed
to stimulate cAMP accumulation in COS-7 cells transfected with the
PTH1
NT receptor (Fig. 3B).
Substitution of His5 with Ile restored the ability of the
ligand to stimulate cAMP accumulation via the PTH1
NT receptor (Fig.
3B). This finding indicates that the enhancing effect of the
substitution is preserved at a receptor from which the N domain has
been largely removed, suggesting direct and/or indirect effects of
residue 5 on ligand interaction with the J domain of the receptor.
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Fig. 3.
Effect of various ligands on cAMP
accumulation at the wild-type PTH1 receptor and an N-terminally
truncated PTH1 receptor. cAMP accumulation was measured as
described under "Experimental Procedures." The following ligands
were tested: [Nle8,18,Tyr34]bPTH(1-34),
[Nle8,18,Tyr34]bPTH(3-34),
[Tyr36]PTHrP(1-36),
[MAP22-31,Tyr36]- PTHrP(1-36),
[Ile5,MAP22-31,Tyr36]PTHrP(1-36),
[Ala3,10,12,Arg11]- rPTH(1-14), and
[Ala1,3,10,12,Arg11,19,Tyr34]hPTH(1-34).
The curves are fit to a four-parameter logistic equation, except
for the linear regression fit for the PTH(3-34) data. Data points are
the mean ± range of duplicate measurements. A, cAMP
accumulation in HEK293 cells stably expressing the PTH1 receptor. Data
were normalized by calculating the response as a fraction of the
maximal response to hPTH(1-34). Basal cAMP was 0.2 ± 0.10 pmol/well, and the maximal response to PTH(1-34) was 11.2 ± 1.0 pmol/well (96-well plate). The experiment was performed three times
with similar results. B, cAMP accumulation in COS-7 cells
expressing an N-terminally truncated PTH1 receptor (PTH1 NT, residues
24-181 deleted (21)). Data were normalized as the percentage of the
maximal response to
[Ala3,10,12,Arg11,19,Tyr34]hPTH(1-34),
the most efficacious agonist identified for the truncated receptor
(M. Shimizu and T. J. Gardella, unpublished observations.)
The basal cAMP accumulation was 2.4 ± 0.2 pmol/well, and the
maximal response to
[Ala3,10,12,Arg11,19,Tyr34]hPTH(1-34)
was 159 ± 6 pmol/well (24-well plate). The experiment was
performed 3 times with similar results. The [MAP]PTHrP(1-36) and
[Ile5,MAP]PTHrP(1-36) responses at the wild-type PTH1
receptor in COS-7 cells were similar to the responses at the PTH1
receptor in HEK293 cells (data not shown).
NT and wild-type
receptors with equivalent potency (21). The ligand is a full agonist
for stimulation of cAMP accumulation in HEK293 cells expressing the
human PTH1 receptor (Fig. 3A).
S (Table
I). We confirmed that the high affinity state of PTH(1-14) binding
represented the RG state by examining the effect of overexpression of
the G
s(
3/
5) mutant
G-protein in COS-7 cells. A high affinity state for PTH(1-14) was not
detected in membranes prepared from COS-7 cells expressing the PTH1
receptor alone (Fig. 5C). (The lack of detectable high
affinity binding in COS-7 cells is probably a result of the very high
level of receptor expression in these cells, 1-2 million
receptors/cell compared with 100,000 receptors/cell for the PTH1
receptor stably expressed in HEK293 cells.) Coexpression of the PTH1
receptor with G
s(
3/
5)
produced a high affinity state of binding for PTH(1-14)
(Ki(high) = 300 nM,
representing 40 ± 9% of 125I-[MAP]PTHrP(1-36)
binding displaced, Fig. 5C), demonstrating that the high
affinity state is the RG complex. The detection of high affinity
binding to the RG state for PTH(1-14) supports the hypothesis that
ligand interaction with the J domain of the receptor controls R/RG
selectivity of agonist binding to the PTH1 receptor.
S. PTH(1-14) only partially inhibited binding
of the radioligand (Fig. 5A); analysis of the displacement
data with a single affinity state model (Equation 1) indicated that
PTH(1-14), at saturating concentrations, inhibited only 18 ± 5%
of specific 125I-PTH(3-34) binding to the R state. This
suggests that 125I-PTH(3-34) and PTH(1-14) can bind
simultaneously to the receptor, since 125I-PTH(3-34) can
still bind when the receptor is saturated with PTH(1-14). This in turn
suggests that the binding sites on the receptor for
125I-PTH(3-34) and PTH(1-14) are, at least to an extent,
spatially distinct, a finding that is fully consistent with the
two-site model which assumes different binding sites on the receptor
for N- and C-terminal portions of the ligand (12).
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Fig. 4.
Inhibition of 125I-PTH(3-34) and
125I-[MAP]PTHrP(1-36) binding to the PTH1 receptor by
unlabeled agonist and antagonist ligands. Binding of ligands to
the PTH1 receptor in HEK293 cell membranes was measured in competition
against 125I-PTH(3-34) in the presence of 30 µM GTP S (closed symbols, R state), and
against 125I-[MAP]PTHrP(1-36) in the absence of GTP
S
(open symbols, RG state). A,
[Nle8,18,Tyr34]bPTH(1-34); B,
[Nle8,18,Tyr34]bPTH(3-34); C,
[Tyr36]PTHrP(1-36); D,
[MAP22-31,Tyr36]PTHrP(1-36); and
E,
[Ile5,MAP22-31, Tyr36]PTHrP(1-36)).
Data were fit to single and two affinity state models (Equations 1 and
2, respectively), and the best fit was determined using a partial
F test. For PTH(1-34), PTH(3-34), and
[Ile5,MAP]PTHrP(1-36), a single affinity state model
provided the best fit for inhibition of both radioligands, the
two-state fit providing no improvement (p > 0.05).
Inhibition of 125I-[MAP]PTHrP(1-36) binding by
PTHrP(1-36) and [MAP]PTHrP(1-36) was better described by a
two-state fit (p < 0.05), whereas a single state model
adequately described inhibition of 125I-PTH(3-34) binding
by these two ligands (p > 0.05). Data were normalized
as the percent of specific binding in the absence of unlabeled ligand.
With one exception, the value of nonspecific binding used for
normalizing the data was the fitted lower plateau of the displacement
curve, which was in good agreement with the measured value of
nonspecific binding (determined in the presence of 1.00 µM [MAP]PTHrP(1-36) or 300 nM
[Nle8,18,Tyr34]bPTH(3-34)). The exception
was inhibition of 125I-PTH(3-34) binding by
[MAP]PTHrP(1-36), for which the measured value of nonspecific
binding was used for normalizing the data and to define the lower
plateau of the displacement curve in the curve-fitting analysis. Data
are from representative experiments that were performed three times
with similar results.
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Fig. 5.
Inhibition of 125I-PTH(3-34) and
125I-[MAP]PTHrP(1-36) binding to the PTH1 receptor by
PTH(1-14). A, displacement of
125I-PTH(3-34) binding to the PTH1 receptor in HEK293 cell
membranes by [Ala3,10,12,Arg11]rPTH(1-14) in
the absence ( ) and presence (
) of GTP
S. Note that the
PTH(1-14) analogue only partially inhibits binding of
125I-PTH(3-34). The curves are the best fits to an
allosteric binding model (Equation 3) that allows for simultaneous
binding to the receptor of both 125I-PTH(3-34) and
PTH(1-14). The best fit parameters (mean ± S.E. from analysis of
3 or 4 independent experiments) were as follows: 30 µM
GTP
S, K (1-14) = 96,000 ± 26,000 M
1 (10.4 µM),
= 0.81 ± 0.06; no GTP
S,
K(1-14) = 71,000 ± 33,000 M
1 (14 µM),
= 0.63 ± 0.03. The
values are
significantly different (p = 0.025).
K(3-34) was held constant in the analysis at
the value determined from the 125I-PTH(3-34) saturation
experiments (3.2 × 109
M
1, Fig. 2B). The
experiment was performed three times for the presence of GTP
S and
four times for the absence of the nucleotide, with similar results.
B, displacement of 125I-[MAP]PTHrP(1-36)
binding to the PTH1 receptor in HEK293 cell membranes by PTH(1-14).
The curve is a two affinity state fit that provided a better fit than a
single affinity state model (p = 0.0045). In this
experiment the high affinity state (IC50 of 250 nM) represents 13% of the total displacement of
125I-[MAP]PTHrP(1-36) binding. Data are from a single
representative experiment that was performed three times with similar
results. C, PTH(1-14) displacement of
125I-[MAP]PTHrP(1-36) binding to membranes from COS-7
cells expressing the PTH1 receptor alone or coexpressed with the mutant
G-protein G
s(
3/
5) (43).
COS-7 cells in 15-cm tissue culture plates were transfected with 25 µg of plasmid DNA encoding the PTH1 receptor, with or without 100 µg of plasmid encoding
G
s(
3/
5), as described
under "Experimental Procedures." The displacement data were fit
best (p = 0.25) by a single affinity state curve for
the PTH1 receptor expressed alone (Equation 1), whereas a two affinity
state fit (Equation 2) was required to fit the data for the receptor
coexpressed with G-protein (p = 0.0005). The experiment
was performed three times with similar results.
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Fig. 6.
Inhibition of PTH(1-14)- and
PTH(1-34)-stimulated cAMP accumulation in HEK293 cells expressing the
PTH1 receptor by antagonist ligands. Antagonism of cAMP
accumulation was measured as described under "Experimental
Procedures" for inhibition of the effect of 10 µM
[Ala3,10,12,Arg11]rPTH(1-14) (A)
and 2 nM hPTH(1-34) (B). The following
antagonists were tested:
[Nle8,18,Tyr34]bPTH(3-34),
[D-Trp12,Tyr34]bPTH(7-34),
bTIP(7-39), and bTIP39. Both the agonist and antagonist were added
simultaneously, following a 30-min preincubation with assay buffer
containing a phosphodiesterase inhibitor (Ro 20-1724). The assay was
terminated after a 30-min incubation with the ligands. The data were
analyzed using a four-parameter logistic equation to obtain estimates
of IC50, pseudo-Hill slope, total cAMP produced in the
presence of the agonist but in the absence of antagonist (max), and
cAMP produced in the presence of the agonist and a saturating
concentration of the antagonist (min). For presentation purposes the
data have been normalized as follows: % response = ((y basal)/(max
basal)) × 100, where
y is the cAMP produced at a given concentration of
antagonist and basal is the amount of cAMP produced in the absence of
any ligand. Note that the ligands only partially antagonize the
PTH(1-14) response (min is greater than basal). Data points are the
mean ± range of duplicate measurements. Data are from
representative experiments that were performed three times with similar
results.
NT receptor compared with the wild-type
receptor (21)). As a result ligands that are proposed to interact
predominantly with the N domain, such as PTH(3-34), should effectively
and completely antagonize the response to PTH(1-34). The data are in
good agreement with this prediction-PTH(3-34), and the other
antagonist ligands completely inhibited the response to 2 nM PTH(1-34) at the PTH1 receptor (Fig. 6B;
PTH(3-34), 0.8 ± 7% of the PTH(1-34) response remaining; [D-Trp12,Tyr34]bPTH(7-34),
3 ± 1%; bTIP39, 3 ± 1%; bTIP(7-39), 1 ± 1%).
1) with a Schild slope of 1.14 ± 0.17 (Fig. 7C).
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Fig. 7.
Antagonism of PTH(1-14)- and
PTH(1-34)-stimulated cAMP accumulation by PTH(3-34) at the PTH1
receptor expressed in HEK293 cells. Both the agonist and
antagonist were added simultaneously, following a 30-min preincubation
with assay buffer containing a phosphodiesterase inhibitor (Ro
20-1724). The concentration dependence of agonist-stimulated cAMP
accumulation was measured in the presence of varying concentrations of
[Nle8,18,Tyr34]bPTH(3-34), for
[Ala3,10,12,Arg11]rPTH(1-14) (A)
and hPTH(1-34) (B). PTH(3-34) alone had no detectable
effect on cAMP accumulation, consistent with the data in Fig. 3. The
curves are the best fits to a four-parameter logistic equation. Data
points are the mean ± S.E. of duplicate measurements. Data are
from representative experiments that were performed three times with
similar results. C, Schild plot of PTH(3-34)'s antagonism
of PTH(1-14)- and PTH(1-34)-stimulated cAMP accumulation. The dose
ratio was calculated by dividing the EC50 in the presence
of antagonist by the EC50 in the absence of the antagonist.
Note the hyperbolic relationship between PTH(3-34) concentration and
the shift of PTH(1-14) EC50, but the linear relationship
for the antagonism of the PTH(1-34) effect. D, effect of
PTH(3-34) on the Emax for PTH(1-14) and
PTH(1-34). For C and D data points are the
mean ± S.E. of measurements from three or four experiments or
mean ± range of measurements from two experiments.
S; the negative cooperativity between the
binding of 125I-PTH(3-34) and PTH(1-14) is significantly
greater in the absence of GTP
S (Fig. 5A). Although this
difference is small, only a small fraction of the receptor population
is coupled to G-protein (22%), suggesting a considerable reduction of
PTH(3-34) binding affinity resulting from PTH(1-14) occupancy at the
RG state. This suggests that the conformation of the receptor is more
"closed," preventing simultaneous access of both ligands to their
binding sites on the receptor. Further circumstantial evidence for a
more closed conformation at the RG state is provided by the observation of pseudo-irreversible binding of agonist radioligands to the RG state
(Fig. 2C (48)), suggesting that the ligand is trapped within
the RG complex.
s(
3/
5). Coexpression with
G-protein substantially increased the fraction of PTH1 receptors in the
RG state. In membranes prepared from COS-7 cells expressing the PTH1
receptor alone, a high affinity state for [MAP]PTHrP(1-36) could not
be detected in a 125I-PTH(3-34) displacement assay (Fig.
8A). In membranes containing the receptor and G
s(
3/5), the high
affinity state (IC50 = 270 pM) represented
40 ± 4% of the 125I-PTH(3-34) binding displaced by
[MAP]PTHrP(1-36) (Fig. 8A). The additional high affinity
binding produced by expression of the mutant G-protein was not entirely
sensitive to GTP
S; the nucleotide did not completely remove the high
affinity state (Fig. 8A). (The mechanism underlying this
effect, which has also been observed for the
2-adrenergic receptor (43), is not presently
understood.) PTH(1-14) did not appreciably inhibit binding of
125I-PTH(3-34) to the PTH1 receptor expressed alone;
significant inhibition was observed at the highest concentration tested
(100 µM), but we were unable to reliably fit the data to
a single affinity state binding model (Equation 1) or the simultaneous
binding model (Equation 3). In contrast, PTH(1-14) inhibited
125I-PTH(3-34) binding to membranes containing the PTH1
receptor and G
s(
3/
5), with
a maximal inhibition of 48% (Fig. 8B) and with an affinity
(190 nM) similar to that for the RG state measured in
competition against 125I-[MAP]PTHrP(1-36) (300 nM, Fig. 5C). Further evidence that this high
affinity state represented the RG state was its removal by GTP
S
(Fig. 8B). These data suggest that PTH(1-14) almost or
completely inhibits the binding of 125I-PTH(3-34) to the
40% of the receptor population in the RG state. This finding is in
good agreement with the hypothesis of a more closed conformation of the
RG state that prevents simultaneous binding of PTH(1-14) and
PTH(3-34).
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Fig. 8.
Effect of coexpression of the PTH1 receptor
and
G s(
3/
5)
on displacement of 125I-PTH(3-34) binding to the PTH1
receptor by [MAP]PTHrP(1-36) and PTH(1-14). The mutant
G-protein G
s(
3/
5) (43) was
coexpressed in COS-7 cells with the PTH1 receptor, and cell membranes
were prepared as described under "Experimental Procedures."
A, inhibition of 125I-PTH(3-34) binding to the
PTH1 receptor by
[MAP22-31,Tyr36]PTHrP(1-36), expressed
alone or coexpressed with
G
s(
3/
5), in the absence
and presence of 30 µM GTP
S. No high affinity
[MAP]PTHrP(1-36) binding was detected for the PTH1 receptor
expressed alone, either in the absence of the presence of GTP
S, the
displacement curve adequately described by a single affinity state
model (Equation 4, p = 0.44 and 0.19, respectively, for
comparison with a two affinity state fit, Equation 2). A high affinity
state of binding was detected for the PTH1 receptor coexpressed with
G
s(
3/
5), both in the
absence and presence of GTP
S, the two affinity state fit providing
an improvement (p = 0.016 and 0.013, respectively). The
experiment was performed three times for one transfection and once for
a second transfection with similar results. B, inhibition of
125I-PTH(3-34) binding to the PTH1 receptor by
[Ala3,10,12,Arg11]rPTH(1-14), for the
receptor expressed alone or coexpressed with
G
s(
3/
5). A single affinity
state binding curve describes the displacement for the PTH1 receptor
coexpressed with the G-protein in the absence of GTP
S (Equation 1).
We were unable to reliably fit data from the other experimental
conditions to this equation. The experiment was performed twice for one
transfection and once for a second transfection with similar
results.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (17K):
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Fig. 9.
Model for modulation of ligand binding to the
PTH1 receptor by G-protein. A, the C-terminal portion
of the ligand interacts with the N domain of the receptor.
Subsequently, the N-terminal portion of the ligand binds to the J
domain of the receptor (B). C, R-G interaction
increases the affinity of the J interaction, possibly by producing a
closure of the receptor conformation. Reciprocally, interaction of the
ligand with the J domain increases the affinity of receptor for
G-protein, stimulating G-protein activation. Binding of G-protein to
the other states of the receptor (R and RLN) has been omitted
for clarity.
The observation of allosteric interactions between the binding of PTH(1-14) and PTH(3-34) confirms the two-site model for the unmodified PTH1 receptor. The inference of simultaneous binding of PTH(1-14) and PTH(3-34) is consistent with spatial and functional independence of receptor-binding sites that interact with the 1-14 and 15-34 regions of the ligand, as proposed previously (12). The allosteric analysis also provided insight into the mechanism of PTH(3-34) binding. PTH(1-14), used to block interactions of PTH(3-34) with the juxtamembrane domain (21), had very little effect on the receptor binding affinity of 125I-PTH(3-34) (1.25-fold increase of Ki at the G-protein-uncoupled receptor). This finding indicates that PTH(3-34) does not interact appreciably with the juxtamembrane domain, implying that almost all the binding energy for PTH(3-34) is provided by the N interaction.
The effect of R-G-coupling on ligand binding to the receptor was
evaluated by comparing ligand affinity for the uncoupled receptor with
the affinity for the RG complex. The former was measured by
displacement of 125I-PTH(3-34) binding in the presence of
30 µM GTPS. For the latter we developed a new
radioligand that selectively labels the RG state,
125I-[MAP22-31,Tyr36]PTHrP(1-36),
an analogue of the bone anabolic agent RS-66271 (40). The R/RG
selectivity of this radioligand was demonstrated by the 78% reduction
of binding produced by GTP
S, which breaks down the RG complex (47),
and by the increase of binding produced by coexpression of the PTH1
receptor with a mutant G
s G-protein, G
s(
3/
5), that stabilizes
the RG state ((43) Fig. 8). All agonist ligands bound with higher
affinity to the RG complex than to the uncoupled receptor, whereas
antagonist ligands bound with similar affinity to these states,
confirming the findings of previous studies of the PTH1 receptor (48,
53) and in agreement with a large number of studies of type I GPCRs
(38). This finding suggests that agonists enhance G-protein activation
by stabilizing the RG state. For other GPCRs, measurement of ligand
binding to R and RG states for a range of agonists has been used to
test models of ligand-receptor-G-protein interaction. In this study we
obtained estimates of the difference of ligand affinity for R and RG
states
(Ki(GTP
S)/Ki(high)) and the fraction of 125I-[MAP]PTHrP(1-36) binding
displaced with high affinity by the unlabeled ligand
(%(high)). The binding data in this study cannot be
accounted for by the simplest form of the ternary complex model, since
there was no correlation between these two measurements (39). This
discrepancy suggests additional states of the receptor (such as the
active and inactive forms of the receptor proposed in the extended
ternary complex model (38)) and/or additional states of the G-protein
in complex with the receptor. With respect to the latter, the role of
the G-protein
dimer in agonist binding to the PTH1 receptor
remains to be determined. For type I GPCRs the
dimer does not
detectably affect agonist binding alone but is required together with
the
-subunit for the detection of the high affinity agonist binding
ternary complex state (54, 55).
Since the macro-affinity of agonist ligands was increased by R-G interaction, we next investigated the extent to which the micro-affinity constants within the two-site binding mechanism (KN and/or KNJ) were affected by R-G coupling. Modification of the N-terminal region of the ligand altered the RG/R binding selectivity as follows. 1) Removal of the two N-terminal residues from PTH(1-34) resulted in the loss of RG/R selectivity. 2) Replacement of His5 of [MAP]PTHrP(1-36) with Ile reduced the RG/R selectivity from 97- to 17-fold, demonstrating a previously unknown role for residue 5 in the control of RG/R binding selectivity. These findings suggest that the affinity of ligand interaction with the J domain is increased by RG interaction, a hypothesis strongly supported by the detection of selective binding of PTH(1-14) to the RG state. In contrast ligand binding to the N domain is not appreciably sensitive to RG coupling; PTH(3-34) bound with similar affinity to the R and RG states of the receptor. Therefore, agonists enhance R-G interaction through ligand interaction with the J domain (KNJ) and not through ligand binding to the N domain (KN) (Fig. 9).
An increase of ligand affinity for RG compared with R indicates different receptor conformations at the G-protein-coupled and -uncoupled receptor states. At the uncoupled receptor, the allosteric binding data suggest than the conformation is open enough to permit the simultaneous binding of PTH(1-14) and PTH(3-34). For the RG state, three observations are consistent with the hypothesis of a more closed receptor conformation. 1) Agonist binding is pseudo-irreversible, suggesting that the ligand is trapped within the ligand-receptor-G-protein complex (48). 2) PTH(1-14) produces a greater reduction of the binding affinity of PTH(3-34) at the RG state, suggesting that simultaneous binding of one ligand better occludes binding of the second. 3) PTH(3-34) reduces the Emax of PTH(1-14)-stimulated cAMP accumulation. This suggests a reduced ability of the PTH1 receptor to adopt an active conformation that couples to G-proteins when both PTH(3-34) and PTH(1-14) are bound to the receptor. The terms open and closed are used operationally in this model. We offer no structural interpretation of the postulated open and closed states, since such an interpretation is beyond the scope of the present data. The hypothesis, currently based on indirect measurements of receptor conformation (ligand binding data), requires more direct examination.
Whereas this study has focused on the conformation of the receptor, the
conformation of the ligand when bound to the receptor is largely
unknown. For the free peptide, a recent crystal structure of PTH(1-34)
indicates an extended helical conformation (33). NMR studies of PTH and
PTHrP under secondary structure-inducing conditions are consistent with
an -helix in the N- and C-terminal portions linked by a region of
variable structure (34, 56). When bound to the receptor some studies
suggest an extended
-helical ligand conformation (57), whereas other
studies suggest tertiary interactions between the N- and C-terminal
ligand domains (29). In this study, a comparison of the binding of
PTH(3-34) and PTH(7-34) is difficult to reconcile with the extended
conformation hypothesis. PTH(7-34) binds with much lower affinity to
the PTH1 receptor than PTH(3-34). However, the weak effect of
PTH(1-14) binding on the affinity of PTH(3-34) suggests that very
little of the binding energy of PTH(3-34) is provided by receptor
interactions involving the 3-14 portion of the ligand. As a result,
the large loss of affinity resulting from deletion of resides 3-6 is
difficult to explain by the loss of a strong direct interaction of this region of the ligand with the juxtamembrane region of the receptor. The
effect could be accounted for by the loss of intramolecular stabilization interactions within the ligand, which result in the
reduction of affinity of the 15-34 ligand region with the receptor.
In conclusion, for the first time we have extended the two-site model
to take into account different conformations of the PTH1 receptor, the
R and RG states. Agonist ligand interaction with the J domain of the
PTH1 receptor discriminates the RG state from the R state suggesting
that the J interaction increases receptor-G-protein interaction and
enhances subsequent second messenger generation. Ligand binding to the
N domain is insensitive to R-G interaction. Finally, the increase of
agonist affinity for the RG state may result from a "closure" of
the receptor conformation. Given the commonality of the low resolution
binding mechanism for type II GPCRs, these findings may well be
relevant to an understanding of the signal transduction mechanism of
other members of this receptor family.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Catherine Berlot for supplying us
plasmids encoding Gs(
3/
5)
and for helpful discussion of the data.
![]() |
FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Laboratory of Genetics, National Institute of Mental Health, Rm. 3D06, Bldg 36, 36 Convent Dr., Bethesda, MD 20892-4092. Tel.: 301- 402-6976; Fax: 301-435-5465; E-mail: usdin@codon.nih.gov.
Published, JBC Papers in Press, December 6, 2000, DOI 10.1074/jbc.M009395200
2
The RG state identified likely contains
G-protein with an empty nucleotide-binding site because the diphosphate
nucleotides GDP and GDPS inhibited
125I-[MAP]PTHrP(1-36) binding to a similar extent as
GTP
S (data not shown).
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
PTH, parathyroid
hormone;
PTHrP, PTH-related protein;
b, bovine;
h, human;
r, rat;
MAP, model amphipathic peptide;
TIP(7-39), tuberoinfundibular peptide
(7-39);
G, G-protein;
GPCR, G-protein-coupled receptor;
R, G-protein-uncoupled receptor state;
RG, G-protein-coupled receptor
state;
GDPS, guanosine 5'-2-O-(thio)diphosphate;
GTP
S, guanosine 5'-3-O-(thio)triphosphate.
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REFERENCES |
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