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Endocrine Unit (K.B.J., M.R.J., R.C.G., T.J.G., H.J.), Department of Medicine and Pediatric Endocrine Unit (R.C.G.), MassGeneral Hospital for Children (R.C.G., H.J.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Dr. Harald Jüppner, Endocrine Unit, Wellman 5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: jueppner{at}helix.mgh.harvard.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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In contrast to the firmly established, homeostatic and developmental roles of the PTH1R, the biological role of the PTH2R remains unknown (4). Unlike the widely expressed PTH1R, the PTH2R is found in only a few tissues, including the hypothalamus. Although initial functional characterization of the human PTH2R had shown that it is activated by PTH, but not by PTHrP (5, 6), subsequent radioreceptor studies revealed that PTHrP binds, although poorly, to the human PTH2R (7, 8, 9). The IC50 of PTHrP-(1–36) at the PTH2R was increased 7-fold when Phe23 was replaced by Trp, which is found at position 23 in all PTH species. However, despite improved apparent binding affinity, this Trp23-modified ana-log continued to lack agonist activity at the PTH2R, which implied that the amino-terminus of PTHrP is incompatible with this receptor (7). When His5 was modified to the PTH-specific residue of isoleucine, the resulting PTHrP-(1–36) analog activated the PTH2R with full or nearly full potency (7, 8). Conversely, replacement of Ile5 in PTH-(1–34) with histidine led to an analog with severely impaired capacity to stimulate cAMP accumulation at the PTH2R, implying that position 5 in either ligand is of critical importance for determining receptor signaling selectivity at this receptor (7). Subsequent investigations with [Trp23]PTHrP-(1–36)amide, [Ile5, Trp23]PTHrP-(1–36)amide, and reciprocal PTH1R/PTH2R chimeras led to the identification of regions and individual residues in the PTH2R that play an essential role in determining agonist selectivity of this receptor, particularly regarding residue 5 of the ligand (10). Independently, Turner et al. (11) and Clark et al. (9) used receptor chimeras and mutagenesis studies to explore ligand selectivity of the PTH2R. In each of these studies, residues in receptor regions comprising transmembrane helixes and extracellular loops were found to be involved in determining agonist selectivity for PTH and PTHrP. The availability of two related, but structurally distinct, ligands and of two PTHR subtypes that responded differentially to these ligands thus led to new insights into the molecular determinants of ligand recognition and ligand-dependent activation of the PTH2R.
In contrast to the human PTH2R, which is fully activated by PTH, but
not by PTHrP, recent data indicated that the rat PTH2R is not
responsive to either PTH or PTHrP (12). These findings
suggested that the primary ligand for the PTH2R is not PTH or PTHrP,
and indeed partially purified extracts from bovine hypothalamus were
shown to contain a peptide that stimulated the human and rat PTH2R, but
not the PTH1R (13). Subsequent studies led to the
isolation of TIP39, a 39-amino acid peptide [herein referred to as
TIP-(1–39)] that efficiently activates the PTH2R homologs from
several different species, including zebrafish, but not the PTH1R
(4, 14). The limited amino acid sequence identity shared
by TIP-(1–39), PTH-(1–34), and PTHrP-(1–36) is apparent in the
carboxyl-terminal region, which contains several conserved
resi-dues that have been shown to be functionally important in both
latter peptides (Fig. 1
). By interacting
predominantly with the amino-terminal, extracellular domain of the
PTH1R, the carboxyl-terminal region of PTH-(1–34) and PTHrP-(1–36)
plays a principal role in determining high affinity receptor binding,
and this interaction is thought to position the amino-terminal domain
of either ligand within the region of the receptor that is required for
activation (1, 15, 16, 17, 18, 19). From the apparent structural
homology within the carboxylterminal region of all three peptides,
it appeared plausible that TIP-(1–39) would be able to bind to the
PTH1R without activating it. To test this hypothesis, we synthesized
TIP-(1–39), several truncation mutants of this peptide, as well as
several PTHrP/TIP chimeras and assessed their capacity to functionally
interact with the PTH1R. The results reveal simi-larities and
differences in the receptor interaction properties of TIP-(1–39) and
PTH or PTHrP.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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]:
TIP-(1–39), TIP-(3–39), TIP-(9–39), TIP-(19–39), TIP-(23–39),
[Nle8,21,Tyr34]rat
PTH-(1–34)amide (rPTH), [Tyr34]human
PTH-(1–34)amide [PTH-(1–34)],
[Tyr36]PTHrP-(1–36)amide [PTHrP-(1–36)],
PTHrP-(1–20)amide [PTHrP-(1–20)], PTHrP-(1–6)/TIP-(9–39),
[Ile5]PTHrP-(1–6)/TIP(9–39),
[Ile7]TIP-(1–39), PTHrP-(1–20)/TIP-(23–39),
and
[Leu11,D-Trp12,Trp23,Tyr36]PTHrP-(7–36)amide
[PTHrP-(7–36)].
Cell culture
DMEM, trypsin/EDTA, penicillin G/streptomycin, and horse serum
were obtained from Life Technologies, Inc. (Gaithersburg,
MD). LLC-PK1 expressing the recombinant human
PTH1R (HKrk-B7 cells) and SaOS-2 cells expressing the wild-type PTH1R
endogenously were cultured in DMEM supplemented with 10%
heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin
as previously described (7, 20); both cell lines were
maintained in a humidified atmosphere containing 95% air and 5%
CO2. After seeding, medium was replaced daily
until cells were used for radioligand binding or cAMP accumulation
assays.
RRAs and stimulation of cAMP accumulation
Na125I (SA, 2000 Ci/mmol) was purchased
from NEN Life Science Products (Boston, MA). FBS,
3-isobutyl-1-methylxanthine, and BSA were obtained from
Sigma (St. Louis, MO), and trifluoroacetic acid was
purchased from Pierce Chemical Co. (Rockford, IL).
Radiolabeled rPTH-(1–34) and PTHrP-(1–36) were prepared by the
chloramine-T method, followed by HPLC purification using a 30–50%
acetonitril/0.1% trifluoroacetic acid gradient over 30 min;
RRAs were performed in 24-well plates as previously described (7, 21). In brief, each well (final volume, 500 µl) contained
binding buffer [50 mM Tris-HCl (pH 7.7), 100
mM NaCl, 5 mM KCl, 2 mM
CaCl2, 5% heat-inactivated horse serum, and
0.5% heat-inactivated FBS], and the
125I-labeled PTH or PTHrP analog (100,000 to
200,000 cpm) was incubated in the absence or presence of increasing
concentrations of unlabeled peptides. After 4 h at 16 C, buffer
was completely removed, the cells were rinsed with cold binding buffer
and lysed with 1 M NaOH. The entire lysate was counted for
-irradiation. Specific binding was determined after subtracting
radioactivity bound in the presence of maximal concentrations of
unlabeled competing peptide
(10-6 M).
Agonist-dependent stimulation of cAMP accumulation by HKrk-B7 and
SaOS-2 cells (48-well plates; stimulation at room temperature for 45
min) and subsequent measurement of cAMP by RIA were performed as
previously described (7, 21). Data were analyzed and
graphically illustrated using the Prism software package
(GraphPad Software, Inc., San Diego, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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106 receptors/cell) (22), using
either radiolabeled rPTH-(1–34) or PTHrP-(1–36). Native TIP-(1–39)
bound to the PTH1R, although with considerably lower apparent affinity
than did PTH-(1–34) and PTHrP-(1–36) (Fig. 2, A and B; Table 1). Removal of the first two or the first eight amino
acid residues yielded TIP-(3–39) and TIP-(9–39), which exhibited
improvements in apparent binding affinity of up to 6-fold relative to
TIP-(1–39). In fact, the apparent binding affinity of TIP-(9–39) at
the PTH1R was similar to or better than that of the agonist
PTHrP-(1–36). TIP-(19–39), TIP-(23–39), as well as PTHrP-(1–20) did
not inhibit the binding of either radioligand (data not shown).
However, the chimera PTHrP-(1–20)/TIP-(23–39) exhibited high apparent
binding affinity, with an IC50 of 31 ± 8.2
nM when tested with radiolabeled rPTH-(1–34) and 11
± 4.0 nM when tested with radiolabeled PTHrP-(1–36) (Fig. 2, A and B, and Table 1). Thus, the binding affinity of
PTHrP-(1–20)/TIP-(23–39) was only 3- to 4-fold weaker than that of
PTH-(1–34), yet it was 2- to 4-fold higher than that of PTHrP-(1–36)
and 8- to 19-fold higher than that of TIP-(1–39). These findings
suggested that the carboxyl-terminal region of TIP-(1–39) can interact
with the PTH1R, most likely at sites that overlap those used by
PTH-(1–34) and PTHrP-(1–36).
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2-fold over basal) in cAMP accumulation when added
at high molar concentrations; however, a similar increase in cAMP was
observed for this peptide with untransfected
LLC-PK1 cells (data not shown), implying that the
effect was not dependent on the PTH1R.
In contrast to the findings with full-length TIP-(1–39) and its
truncated analogs, the peptide chimera PTHrP-(1–20)/TIP-(23–39) was a
full and potent agonist for the PTH1R and stimulated cAMP accumulation
in HKrk-B7 cells with an EC50 of 1.40 ± 0.3
nM (Fig. 3a;
Table 2). This potency was comparable to the
EC50 values observed for PTH-(1–34) and
PTHrP-(1–36). When tested with SaOS-2 cells, an osteoblast-like cell
line expressing lower levels of the PTH1R (30,000 receptors/cell)
(24), the PTHrP-(1–20)/TIP-(23–39) chimera induced cAMP
accumulation with a potency (EC50, 0.38 ±
0.12 nM) similar to that obtained with PTH-(1–34) or
PTHrP-(1–36) (EC50, 0.30 ± 0.12 and
0.25 ± 0.15 nM, respectively; Fig. 3B and Table 2).
To begin exploring which site(s) within the amino-terminus of
TIP-(1–39) prevent(s) signal transduction at the PTH1R, three
additional peptides were synthesized; the chimeras
PTHrP-(1–6)/TIP-(9–39) and
[Ile5]PTHrP-(1–6)/TIP-(9–39), as well as
[Ile7]TIP-(1–39), the latter having
Asp7 replaced by the corresponding isoleucine of
PTH (see Fig. 1). None of these peptide stimulated cAMP formation in
HKrk-B7 cells (data not shown).
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), in the absence or presence of
increasing concentrations of TIP-(1–39), TIP-(9–39), or PTHrP-(7–36)
(Fig. 4, A–F). TIP-(9–39) inhibited
agonist-stimulated cAMP accumulation with an efficiency similar to that
of PTHrP-(7–36) (22). In HKrk-B7 cells, the
IC50 values were approximately 300 nM
for TIP-(9–39) compared with about 100 nM for
PTHrP-(7–36) (Fig. 4, A–C); TIP-(1–39) also functioned as an
antagonist and showed a half-maximal inhibition of agonist-induced cAMP
accumulation at approximately 1000 nM. Similar results were
observed in SaOS-2 cells (Fig. 4, D–F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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In contrast to PTH-(1–34) and PTHrP-(1–36), TIP-(1–39) failed to
stimulate cAMP accumulation in HKrk-B7 and SaOS-2 cells, confirming
earlier studies with this peptide that had been performed in
transfected COS-7 and HEK293 cells expressing the PTH1R (4, 23). However, TIP-(1–39) bound to the PTH1R, albeit with low
affinity. To explore the structural features in TIP-(1–39) that
determine its interaction with the PTH1R, we tested several TIP-(1–39)
deletion mutants for receptor binding affinity and the capacity to
induce cAMP accumulation. As amino acid sequence alignment of
PTH-(1–34), PTHrP-(1–36), and TIP-(1–39) revealed that the latter
peptide has an amino-terminus extended by two amino acid residues
(4), it seemed plausible that this extension could account
for the reduced binding affinity at the PTH1R as well as the lack of
agonist activity. In fact, in comparison to TIP-(1–39), TIP-(3–39)
exhibited a 2- to 3-fold improvement in IC50 when
tested with either radiolabeled rPTH-(1–34) or PTHrP-(1–36). However,
despite the improved apparent binding affinity, this truncated analog
failed to stimulate cAMP accumulation in HKrk-B7 cells, consistent with
previous findings in transfected COS-7 and HEK293 cells (4, 23). Thus, the first two residues of TIP-(1–39) are clearly not
the structural elements that prevent PTH1R activation. Deletion of an
additional six residues from the amino-terminus increased binding
affinity further, as the resulting TIP-(9–39) had, compared with
TIP-(1–39), a 5- to 6-fold improvement in IC50.
However, despite its high binding affinity, which was similar to that
of the agonist PTHrP-(1–36), TIP-(9–39) failed to stimulate cAMP
accumulation. Similarly, Hoare et al. found that TIP-(7–39)
efficiently inhibited radioligand binding to the PTH1R, but showed no
agonist activity (23). The current data thus suggest that
TIP-(1–39) and related analogs might be potent antagonists at the
PTH1R. We therefore directly tested TIP-(1–39) and TIP-(9–39) for
their antagonist activity on the PTH1R. TIP-(9–39) was able to inhibit
the actions of PTH-(1–34), PTHrP-(1–36), and
PTHrP-(1–20)/TIP(23–39) with a potency similar to that of
PTHrP-(7–36) (22). Taken together, our findings suggest
that the carboxyl-terminal regions of three different peptides share
sufficient structural homology to allow efficient binding to the same
or similar sites in the PTH1R. Consistent with this conclusion, a
recent nuclear magnetic resonance imaging study of TIP-(1–39) revealed
a secondary structure profile similar to that of PTH-(1–34),
i.e. two 
-helixes connected by a flexible linker region
of yet undefined structure (26).
Previous studies by us and others have led to the conclusion that the interaction of PTH-(1–34) [and PTHrP-(1–36)] with the PTH1R involves two distinct principal receptor components (for review, see Ref. 1). According to this model, which is supported by several different cross-linking studies (18, 19, 27, 28, 29), the carboxyl-terminal region of the ligand interacts predominantly with the amino-terminal, extracellular domain of the PTH1R to provide binding energy, and the amino-terminal portion of the ligand interacts with the receptor’s membrane-spanning helices and the connecting extracellular loops to induce signal transduction. Fragments of TIP-(1–39) that are truncated at the amino-terminus, i.e. TIP-(3–39) and TIP-(9–39), bound to the PTH1R with reasonably high affinity, and at least TIP-(9–39) inhibited the actions of PTH-(1–34), PTHrP-(1–36), and the PTHrP/TIP chimera, as efficiently as PTHrP-(7–36) (22). Taken together with the observation that PTHrP-(1–20)/TIP-(23–39) activated the PTH1R as efficiently as PTH-(1–34) and PTHrP-(1–36), it appears likely that the interaction between the PTH1R and TIP-(1–39) involves residues in the ligand’s carboxyl-terminus and the receptor’s amino-terminal, extracellular domain. This hypothesis is supported by recent observations by Hoare et al. (23), who demonstrated that a PTH1R/PTH2R chimera (containing the amino-terminal, extracellular domain, and the first membrane-spanning helix of the PTH1R fused to the remaining portions of the PTH2R), but not the reciprocal PTH2R/PTH1R chimera, is efficiently activated by TIP-(1–39).
In our studies TIP-(19–39) and TIP-(23–39) showed no detectable binding to the PTH1R, even though this portion of TIP contains several amino acid residues that are functionally important in PTH-(1–34) or PTHrP-(1–36), i.e. Glu21, Arg22, Arg23, Trp25, and Leu26 (1, 19, 30). Previous investigations had indicated that PTH-(15–34)amide binds with very low (micromolar) affinity to the PTH1R (15), and it is therefore not too surprising that TIP analogs comprising only the most carboxyl-terminal portion of the ligand exhibited no detectable binding to this receptor. These results furthermore imply that region 9–18 of TIP-(1–39) contributes to binding affinity. Because TIP-(1–39) showed antagonist activity at the PTH1R, it conceivably could act as an endogenous inhibitor of PTH and/or PTHrP action at the PTH1R if present at sufficiently high concentrations. Conversely, synthetic PTH and PTHrP analogs that bind to the PTH1R could have unwarranted effects in those tissues where the PTH2R is most abundantly found (5, 6).
The amino-terminal domain of TIP-(1–39) is probably positioned at least near the activation pocket of the PTH1R when bound to this receptor, but it remains uncertain what prevents it from inducing activation. The lack of activation is clearly not related to the presence of the two-amino acid extension at the amino-terminus (this study and Ref. 23); however, several other candidate residues in the amino- terminal region of TIP-(1–39) might be involved. Most substitutions in the 1–9 region of PTH have been recently shown to impair PTH1R activation (31), and it may well be that one or more of the divergent residues in the corresponding region of TIP-(1–39), i.e. Asp7, Ala8, Ala9, Phe10, and Arg11, prevent a productive interaction with the PTH1R. It is furthermore likely that one or several of the first eight ligand residues impair PTH1R binding affinity (23). Although the underlying mechanisms are unknown, a recent computer modeling study of the ligand-receptor complex has suggested that some of the amino-terminal residues of TIP-(1–39), such as Asp7, would not fit productively into the agonist-binding pocket of the PTH1R (26). Because Asp7 aligned with PTH residue Ile5, which determines PTH2R agonist selectivity (2, 7, 8), we replaced this residue with Ile. However, no activation of the PTH1R was observed with [Ile7]TIP-(1–39) (data not shown). Taken together with the lack of PTH1R activation by PTHrP-(1–6)/TIP-(9–39) and [Ile5]PTHrP-(1–6)/TIP-(9–39), it thus appears likely that other divergent amino acid residues in the amino-terminal region of TIP-(1–39) are involved in preventing agonist actions at the PTH1R. It should be possible to identify these residues through the development of additional TIP chimeras and analogs; the resulting information is likely to provide new insights into functionally important regions of the PTH1R.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgments |
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Footnotes |
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Received July 20, 2000.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
), TIP-(3–39) (
), TIP-(9–39) (
), PTH-(1–34) (
),
PTHrP-(1–36) (
), or PTHrP-(1–20)/TIP-(23–39) (
). Data are
expressed as a percentage of maximal specific binding and represent the
results (mean ± SE) of at least three independent
experiments.
