(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, 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.
PTH ()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) .
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]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.
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 values are the average ± S.E. of three or more
experiments, each performed in triplicate.
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 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.
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 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.
In contrast to
the severe effect of the position 19 change on receptor binding
affinity, the corresponding substitution of Glu 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.
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 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).
Functional
interactions caused by changes in residues 5 and 19 were also apparent
in the context of PTH-(1-34). Thus, the Glu-to-Arg
modification partially reversed the binding defect caused by the
Ile
-to-His change (Table 2).
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 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.
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-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).
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 > 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) .
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]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.
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 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.
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
-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) .