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(Received for publication, August 8, 1994; and in revised form, October 27,
1994) From the
Parathyroid hormone (PTH) and parathyroid hormone-related
peptide (PTHrP) bind to a common PTH/PTHrP receptor. To explore
structure-function relations in these ligands, we synthesized and
functionally evaluated PTH-PTHrP hybrid peptides in which the
homologous 1-14 portions were exchanged. Hybrid-2,
PTH-(1-14)-PTHrP-(15-34)NH
PTH ( Residues 1-14 of hPTH and hPTHrP display considerable amino
acid sequence homology, sharing identities at eight sites(7) .
Beyond residue 14, the two peptides differ significantly, sharing only
three identities within the 15-34 regions. Despite the importance
of the amino-terminal residues in hormone function and their high
degree of evolutionary conservation, peptide fragments containing only
amino-terminal residues, such as PTH-(1-12) or
PTHrP-(1-20), are devoid of biologic
activity(3, 8) . In contrast, short carboxyl-terminal
fragments such as PTH-(14-34) and PTHrP-(14-34) are able to
bind, albeit weakly, to the PTH/PTHrP
receptor(6, 9, 10) . Because of this ability
to bind to the same receptor site, it has been suggested that the
poorly conserved carboxyl-terminal portions of PTH-(1-34) and
PTHrP-(1-34) adopt similar conformations when interacting with
the receptor(9, 10) . The three-dimensional
structures of PTH and PTHrP have not been determined. Conformational
modeling approaches and structure-activity studies have suggested that
PTH-(1-34) and PTHrP-(1-34) are folded in such a way that
the amino- and carboxyl-terminal portions interact (11, 12, 13) ; however, there is no direct
evidence that such interactions occur or are important for ligand
binding. Models such as these, the similar functional properties
displayed by PTH and PTHrP fragments, and the intriguing pattern of
amino acid sequence homology displayed by the two ligands led us to
investigate whether corresponding regions of PTH and PTHrP could be
freely interchanged. We thus synthesized reciprocal PTH-PTHrP hybrid
peptides and evaluated their ability to bind to the rat PTH/PTHrP
receptor. The functional properties of these hybrids provide functional
evidence in support of the hypothesis that the 1-14 and
15-34 domains of the ligand interact.
Figure 1:
Reciprocal PTH-PTHrP
hybrid peptides bind to ROS 17/2.8 cells with different affinities.
Shown are competition binding curves for hybrid-1,
[Tyr
Figure 2:
The carboxyl-terminal incompatibility
determinants of hybrid-1 map to residues 19-21. The sequences of
PTHrP-PTH hybrid peptides and the two parent peptides are shown. Shadedresidues correspond to hPTHrP and unshadedresidues correspond to hPTH. The binding of each peptide
to AR-C40 cells was evaluated in radioreceptor binding assays as
described under ``Experimental Procedures.'' IC
The 19-21 sequence of human PTH is Glu-Arg-Val, and
the corresponding sequence of PTHrP is Arg-Arg-Arg. We thus inferred
that a glutamic acid residue at position 19 and/or a valine residue at
position 21 was not compatible with the 1-14 sequence of PTHrP.
This hypothesis was tested by replacing Arg
Figure 3:
Modifications at positions 19 and 21
impair receptor binding of PTHrP-(1-34). Shown are competition
binding curves for PTHrP-(1-34) and analogs that have Arg
In contrast to
the severe effect of the position 19 change on receptor binding
affinity, the corresponding substitution of Glu
In rat, human, and chicken PTHrP, position 5 is occupied by
histidine, whereas in the five sequenced mammalian PTH molecules,
position 5 is isoleucine (methionine in chicken PTH). When His
Functional
interactions caused by changes in residues 5 and 19 were also apparent
in the context of PTH-(1-34). Thus, the Glu
Figure 4:
Effect of modifications at positions 19
and 21 on the binding of 15-34 fragments. The divergent residue
at position 19 or 21 of PTHrP-(15-34) (A) or position 19
of PTH-(15-34) (B) was changed to the corresponding
residue of the other ligand. The ability of the resulting peptides to
inhibit the binding of
The binding of PTH-(15-34) to AR-C40 cells is
considerably weaker than the binding of PTHrP-(15-34), as has
been found previously with similar carboxyl-terminal fragments of PTH
and PTHrP and cells expressing the cloned rat PTH
receptor(15, 20) . However, cells expressing the
cloned opossum PTH receptor display an inherently higher affinity for
such carboxyl-terminal fragments of PTH-(1-34) in comparison with
cells expressing the cloned rat PTH receptor(15, 20) .
We were therefore able to assess the binding of PTH-(15-34) to
AOK-B50 cells, which are LLC-PK1 cell derivatives that stably express
the cloned opossum PTH receptor (80,000 receptors/cell(15) ).
When the Glu This study of PTH-PTHrP hybrid peptides evolved from our
interest in understanding how two distinct ligands bind to a common
receptor and as an approach to revealing possible long range
intramolecular interactions within these ligands. The ability of
hybrid-2, PTH-(1-14)-PTHrP-(15-34), to bind to the
PTH/PTHrP receptor with high affinity indicates that, despite the high
level of amino acid divergence, the 15-34 domain of PTHrP can
substitute for the 15-34 domain of PTH, and thus the 1-14
portion of PTH is compatible with the 15-34 region of either
ligand. The results with hybrid-2 alone would suggest, therefore, that
the 1-14 portions of PTH and PTHrP are freely interchangeable.
However, the very weak binding properties exhibited by the reciprocal
peptide, hybrid-1, clearly demonstrate that the 1-14 region of
PTHrP is incompatible with the 15-34 region of PTH, thus the
conserved amino-terminal domains are not functionally equivalent. With additional hybrid peptides and derivative analogs, we
determined that the amino acid divergences at positions 5, 19, and 21
were responsible for the binding defect of hybrid-1. Most importantly,
the particular combination of histidine at position 5 and glutamate at
position 19, whether in the context of PTH, PTHrP, or hybrid backbones,
consistently resulted in peptides with poor binding affinity (IC Our observations are
consistent with those of Caulfield and co-workers (9) who
found that two reciprocal PTH-PTHrP hybrid molecules, which were based
on the 7-34 fragments of PTH and PTHrP and recombined at the
18/19 position, bound to PTH receptors with affinities that were nearly
equal to those of the parental peptides,
[Nle The molecular basis by which the
residues at positions 5, 19, and 21 contribute to receptor binding is
currently unclear. Our functional studies cannot directly assess ligand
conformation; however, the results lead to the speculation that the
1-14 and 15-34 regions of the ligand interact and that
residues 5, 19, and 21, directly or indirectly, play an important role
in this interaction. It is difficult to reconcile the phenotypes of the
hybrid peptides and the interactive effects of mutations in two
distinct domains of the ligand without invoking such long range
interactions. Furthermore, the finding that mutations at positions 19
and 21 had no effect on the binding of the 15-34 fragment of
PTHrP, whereas the same mutations dramatically reduced the affinity of
PTHrP-(1-34), supports this interpretation, because such results
indicate that the mutations do not have pronounced local effects. The
local effects of mutations at position 5 could not be similarly
assessed, because short amino-terminal fragments of PTH and PTHrP are
not active in competitive binding assays. However, it seems unlikely
that the 100-fold reduction in binding affinity caused by the His The speculation that our results reflect tertiary interactions
between the amino- and carboxyl-terminal portions of the ligand are
consistent with previous conformational models of PTH-(1-34) and
PTHrP-(1-34) (11, 12, 13, 22) . Recently,
McFarlane et al.(23) analyzed fragments of
PTHrP-(1-34) by Fourier transform infrared spectroscopy and found
that amino- or carboxyl-terminal deletions destabilized secondary
structure in the distal portion of the peptide; thus, mutations in one
domain can influence residues in a distal domain. However, neither this
Fourier transform infrared study nor a number of NMR analyses performed
on PTHrP (24) and PTH (25, 26, 27, 28) provides direct
evidence for any long range tertiary interaction. Most of these studies
did, however, find evidence for a flexible hinge segment and/or a
Volume 270,
Number 12,
Issue of March 24, 1995 pp. 6584-6588
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
14 and 1534 Domains of the
Ligand (*)
, bound to LLC-PK1
cells expressing the cloned rat PTH/PTHrP receptor with high affinity
(IC
7 nM). In contrast, hybrid-1,
PTHrP(1-14)-PTH-(15-34)NH, bound with much
weaker affinity (IC 8,700 nM). Thus, the
1-14 region of PTHrP is incompatible with the 15-34 region
of PTH. The carboxyl-terminal incompatibility site was identified as
residues 19-21 (Glu-Arg-Val in PTH and Arg-Arg-Arg in PTHrP);
extending the amino-terminal PTHrP sequence to residue 21 but not to 18
cured the hybrid's binding defect. The amino-terminal
incompatibility site was identified as position 5 (Ile in PTH and His
in PTHrP), because Ile
-hybrid-1 bound with high affinity
(IC 20 nM). The importance of these
identified residues in the native ligands was established by evaluating
the effects of substitutions at these sites in a series of PTH and
PTHrP analog peptides. Overall, the results are consistent with the
hypothesis that, in both PTH and PTHrP, the 1-14 and 15-34
domains interact when binding to the receptor and that residues 5, 19,
and 21 contribute either directly or indirectly to this interaction.
)and PTHrP bind with near equal affinity to
receptors on the surface of bone and kidney cells. PTH functions
throughout life as the key regulator of serum mineral ion levels,
whereas PTHrP, originally discovered as the causative agent of
hypercalcemia of malignancy, has important developmental roles
(reviewed in (1) ). Synthetic fragments of PTH and PTHrP
containing residues 1-34 display full biological activity in most
assay systems (2, 3) . Studies with truncated variants
of these peptides have shown that the amino-terminal residues are
essential for activating the cAMP response pathway and also contribute
modestly to the overall binding energy(4, 5) . The
majority of the receptor binding energy is provided by residues in the
carboxyl-terminal portion of the 1-34 peptide(6) .
Peptide Synthesis
All peptides were based on the
native human PTH or human PTHrP sequences except for residue 34, which
was always Tyr-amide. Peptides were prepared by the solid-phase
procedure (14) using an Applied Biosystems model 430 peptide
synthesizer and purified by gel filtration and sequential HPLC. Amino
acid composition, sequence analysis, and pulse-desorption mass
spectroscopy verified the authenticity of each peptide.Cell Culture
Cell lines were maintained in a
humidified atmosphere containing 95% air, 5% CO in 250-ml
culture flasks (75 cm) in media containing 20 units/ml
penicillin G, 20 µg/ml streptomycin sulfate, and 0.05 µg/ml
amphotericin B. ROS 17/2.8 cells were cultured in Ham's F-12
medium supplemented with 5% fetal bovine serum (Hyclone Laboratories,
Logan, UT). The AR-C40 and AOK-B50 cell lines are derivatives of the
LLC-PK1 cell line and stably express the rat and opossum PTH/PTHrP
receptor cDNAs, respectively(15) . These cells were cultured in
Dulbecco's modified Eagle's medium supplemented with 7%
fetal bovine serum. For receptor binding assays, cells were subcultured
in 24-well plates and maintained as confluent monolayers for 3-5
days (ROS 17/2.8) or 1-2 days (AR-C40 and AOK-B50) prior to
assay.Competitive Radioreceptor Binding
Binding
reactions (300 µl) contained binding buffer (50 mM Tris-HCl, pH 7.7, 100 mM NaCl, 5 mM KCl, 2
mM CaCl, 5% heat-inactivated horse serum, 0.5% fetal bovine
serum), an unlabeled PTH or PTHrP analog diluted in binding buffer, and
radiolabeled
[Nle
,Tyr
]bPTH-(1-34)NH (2.2 
10
mCi/mmol, 100,000 cpm/well) diluted
in binding buffer. The tracer was prepared by chloramine-T iodination
using 
I-sodium (DuPont NEN) and was purified by
HPLC(16) . After 4 h at 16 °C, the binding mixture was
removed, and the monolayer was rinsed 3 times with binding buffer,
lysed with 5 M NaOH, and counted. Nonspecific binding and
maximal binding were determined from wells containing 1 µM unlabeled [Tyr]hPTH-(1-34)NH and no unlabeled PTH, respectively. IC values were
obtained from the midpoints of competition binding curves.
PTH-PTHrP Hybrid Peptides
We first examined the
effects of exchanging the homologous 1-14 regions of PTH and
PTHrP on the ability of the ligands to bind to ROS 17/2.8 cells. Fig. 1shows that hybrid-2, PTH-(1-14)-PTHrP-(15-34),
bound to ROS 17/2.8 cells with high apparent affinity (IC 1.4 nM). In contrast, the reciprocal peptide
hybrid-1, PTHrP-(1-14)-PTH-(15-34), was severely defective
in its ability to bind to ROS 17/2.8 cells (IC
2,700 nM). Similar results were obtained with the AR-C40 cells (Table 1); these cells are derivatives of the porcine kidney cell
line LLC-PK1 that stably express the cloned rat PTH receptor (100,000
receptors/cell)(15) .
]hPTHrP-(1-14)-hPTH-(15-34)NH (
) and hybrid-2,
[Tyr]hPTH-(1-14)-hPTHrP-(15-34)NH (
). Radioreceptor binding assays using I-[Nle,Tyr]bPTH-(1-34)NH as a tracer were performed as described under ``Experimental
Procedures.'' Data are the average ± S.E. of three
experiments, each performed in triplicate.
Carboxyl-terminal Incompatibility Sites
The poor
binding affinity of hybrid-1 indicated that the 1-14 region of
PTHrP was not compatible with a site (or sites) in the 15-34
region of PTH. To localize the putative incompatibility sites in the
PTH-(15-34) domain of hybrid-1, we synthesized the additional
hybrid peptides shown in Fig. 2. Like hybrid-1, the two hybrids
that had the amino-terminal PTHrP sequence extending to residue 16 or
18 bound very poorly to AR-C40 cells (IC 8,700
nM). When the amino-terminal PTHrP sequence extended to
position 21 or 24, then receptor binding affinity improved dramatically
(ICs 3 or 24 nM, respectively). These
results strongly suggested that residues 19-21 of PTH were the
carboxyl-terminal determinants responsible for the binding defect of
hybrid-1.
values are the average ± S.E. of three or more
experiments, each performed in triplicate.
or Arg
of PTHrP-(1-34) by Glu or Val, respectively. As predicted
by the hybrid peptides, each of these substitutions resulted in a
severe reduction in receptor binding affinity as compared with the
binding affinity of the control peptide, PTHrP-(1-34) (Fig. 3). The two substitutions together had the greatest impact
on receptor binding affinity, as
[Glu,Val]PTHrP-(1-34) bound
with very weak affinity (IC 5,500 nM);
this affinity was comparable with that of hybrid-1.
and/or Arg changed to the corresponding PTH
residues, Glu and Val, respectively. ,
[Tyr] hPTHrP-(1-34)NH;
,
[Val,Tyr]hPTHrP-(1-34);
, [Glu,Tyr]
hPTHrP-(1-34)NH; 
,
[Glu,Val,Tyr]hPTHrP-(1-34)NH.
Competition binding assays were performed with AR-C40 cells utilizing
radiolabeled I-[Nle,Tyr]bPTH-(1-34)NH as a tracer. Data are the average ± S.E. of three
experiments, each performed in triplicate.
with Arg
in PTH-(1-34) resulted in a slight enhancement in receptor
binding affinity, as the binding of
[Arg]PTH(1-34) was 3-fold stronger than
that of PTH-(1-34) (Table 2). This result is not
surprising, because the high binding affinity of hybrid-2 previously
demonstrated that an arginine at position 19 is compatible with the
1-14 sequence of PTH.
Amino-terminal Incompatibility Site
The severe
binding defect displayed by hybrid-1 suggests that one or more of the
six divergent sites in the 1-14 region of PTHrP is incompatible
with the 15-34 region of PTH. These differences occur at
positions 1, 5, 8, 10, 11, and 14. Previous structure-activity studies
indicated that the Ser-to-Ala substitution at position 1 or the
Asn-to-Asp and Leu-to-Lys changes at positions 10 and 11, respectively,
were not likely to be responsible for the binding defect of
hybrid-1(17, 18) . Because of the sharp reduction in
biological activity that accompanies oxidation of Met
in
PTH(19) , we considered the possibility that the Met-for-Leu
divergence at this site accounts for the binding defect of hybrid-1.
However, we found that replacing Leu of hybrid-1 with Met
did not alter the hybrid's binding properties (data not shown). of hybrid-1 was replaced by Ile, then receptor binding affinity
dramatically improved; in fact, Ile-hybrid-1 bound to the
rat PTH/PTHrP receptor with an affinity that was comparable with that
of the two parent ligands (Table 1). These results suggest that
the histidine at position 5 plays a major role in determining the
binding defect of hybrid-1 and that this residue is incompatible with
the 15-34 region of PTH. This interpretation is supported by the
severe reduction in binding affinity that occurred when Ile of PTH-(1-34) was replaced by His (Table 2).Effects of Combining the Amino- and Carboxyl-terminal
Modifications
The ability of the Ile modification to
rescue the binding defect of hybrid-1 and the apparent role of
positions 19 and 21 in the binding defect of hybrid-1 led us to
investigate whether the Ile modification could also rescue
the binding defects caused by the Glu and Val modifications in PTHrP-(1-34). This was found to be the
case, because
[Ile,Glu]PTHrP-(1-34) bound to
AR-C40 cells with an affinity that was 140-fold greater than that of
[Glu]PTHrP-(1-34) and nearly equal to that
of PTHrP-(1-34) itself (Table 2). The Ile modification could also rectify the effects of the Val modification and the effects of the combined
Glu/Val modification (Table 2). These
results are thus consistent with the observations made with the hybrid
peptides. The affinityenhancing effect of the Ile modification was not limited to analogs with changes at positions
19 and 21, as the Ile substitution slightly improved the
binding affinity of PTHrP(1-34) itself, as well as the affinity
of [Glu
]PTHrP-(1-34), an analog with very
weak binding affinity due to the substitution of glutamic acid for the
invariant leucine at position 24 (Table 2).-to-Arg
modification partially reversed the binding defect caused by the
Ile-to-His change (Table 2).Effect of Mutations at Positions 19 and 21 on the Binding
Properties of 15-34 Fragments
The ability of mutations in
the 1-14 or 15-34 portions of PTH and PTHrP to dramatically
modify the effects of mutations in the reciprocal domain suggests that
the effects of the mutations are based on long range tertiary
interactions involving the two domains rather than on more local
effects, such as changes in direct receptor interaction or local
secondary structure. To test for any possible local effects, we
investigated whether the carboxyl-terminal mutations altered the
receptor binding properties of the 15-34 fragments of PTH and
PTHrP. As shown in Fig. 4A, neither the Glu or Val substitution had any effect on the ability of
the PTHrP-(15-34) fragment to bind to AR-C40 cells. These results
contrast with the severe effects that the same mutations had on the
binding of full-length PTHrP-(1-34) (Fig. 3) and suggest
that the mutations do not cause severe local disruptions of the
15-34 domain or change the interaction of this domain with the
receptor.
I-[Nle,Tyr]bPTH(1-34)NH to the cloned rat PTH/PTHrP receptor expressed in AR-C40 cells (A) or the cloned opossum PTH/PTHrP receptor expressed in
AOK-B50 cells (B) is shown. A, ,
[Tyr]hPTHrP-(1-34)NH; ,
[Tyr]hPTHrP-(15-34)NH; ,
[Tyr,Val]hPTHrP-(15-34)NH;
,
[Tyr,Glu]hPTHrP-(15-34)NH. B, ,
[Tyr]hPTH-(1-34)NH; ,
[Tyr]hPTH-(15-34)NH; ,
[Arg,Tyr]hPTH-(15-34)NH.
Data are the average ± S.E. of three experiments, each performed
in duplicate or triplicate.
-to-Arg modification was incorporated into
PTH-(15-34), a slight enhancement in receptor binding affinity
occurred (Fig. 4B); this effect was relatively small
when compared with the more substantial effect that the Arg modification had on the binding of
[His]PTH-(1-34) (Table 2).
> 1,000 nM). The severe binding defect associated
with the His/Glu combination could be relieved
by replacing either the histidine with isoleucine or the glutamic acid
with arginine. The combination of isoleucine at position 5 and arginine
at position 19 resulted in peptides that displayed the highest apparent
binding affinities (IC < 8 nM). That these
relationships were maintained in the PTH and PTHrP analogs as well as
in the various hybrid peptides is consistent with the notion that
PTH-(1-34) and PTHrP-(1-34) adopt similar conformations
when binding to the receptor(9) .]bPTH-(7-34) and
[D-Trp
]PTHrP-(7-34)(21) .
That neither of these hybrids displayed the severe receptor binding
defect exhibited by our hybrid-1 would be expected from our findings,
because residue 5 was deleted. modification in PTH-(1-34) is based solely on local
effects, because the same modification had a much smaller effect when
introduced into hybrid-2, which has the 1-14 sequence of PTH. 
-turn structure near the midregion of the molecule. Such
structural features might enable long range interactions between
non-adjacent residues. It is possible, however, that such tertiary
interactions in PTH and PTHrP are too unstable to be detected in the
solvent conditions employed in spectroscopic analyses and that the
active conformations may need to be induced or stabilized by the
receptor. In this case, the determination of the ligand's
three-dimensional structure might require the co-crystallization of the
ligand-receptor complex, as has been achieved for human growth hormone
and the soluble fragment of its receptor(29) .
)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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