©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Parathyroid Hormone (PTH)-PTH-related Peptide Hybrid Peptides Reveal Functional Interactions between the 114 and 1534 Domains of the Ligand (*)

(Received for publication, August 8, 1994; and in revised form, October 27, 1994)

Thomas J. Gardella (§) Michael D. Luck Andrew K. Wilson Henry T. Keutmann Samuel R. Nussbaum John T. Potts Jr. Henry M. Kronenberg

From the Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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(2), 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(2), 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^5-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.


INTRODUCTION

PTH (^1)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.


EXPERIMENTAL PROCEDURES

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(2) in 250-ml culture flasks (75 cm^2) 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) (2.2 times 10^6 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(2) and no unlabeled PTH, respectively. IC values were obtained from the midpoints of competition binding curves.


RESULTS

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) .


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(2) (circle) and hybrid-2, [Tyr]hPTH-(1-14)-hPTHrP-(15-34)NH(2) (bullet). Radioreceptor binding assays using I-[Nle,Tyr]bPTH-(1-34)NH(2) 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.


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. bullet, [Tyr] hPTHrP-(1-34)NH(2); box, [Val,Tyr]hPTHrP-(1-34); circle, [Glu,Tyr] hPTHrP-(1-34)NH(2); , [Glu,Val,Tyr]hPTHrP-(1-34)NH(2). Competition binding assays were performed with AR-C40 cells utilizing radiolabeled I-[Nle,Tyr]bPTH-(1-34)NH(2) 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.



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^8 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^8 of hybrid-1 with Met did not alter the hybrid's binding properties (data not shown).

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^5 of hybrid-1 was replaced by Ile, then receptor binding affinity dramatically improved; in fact, Ile^5-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^5 of PTH-(1-34) was replaced by His (Table 2).

Effects of Combining the Amino- and Carboxyl-terminal Modifications

The ability of the Ile^5 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^5 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^5,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^5 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^5 modification was not limited to analogs with changes at positions 19 and 21, as the Ile^5 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).

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^5-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.


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(2) 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, bullet, [Tyr]hPTHrP-(1-34)NH(2); circle, [Tyr]hPTHrP-(15-34)NH(2); , [Tyr,Val]hPTHrP-(15-34)NH(2); , [Tyr,Glu]hPTHrP-(15-34)NH(2). B, bullet, [Tyr]hPTH-(1-34)NH(2); circle, [Tyr]hPTH-(15-34)NH(2); , [Arg,Tyr]hPTH-(15-34)NH(2). 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^5]PTH-(1-34) (Table 2).


DISCUSSION

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^5/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^5 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 beta-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) .


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel: 617-726-3966; Fax: 617-726-7543.

(^1)
The abbreviations used are: PTH, parathyroid hormone; PTHrP, parathyroid hormone-related peptide; hPTH, human PTH; hPTHrP, human PTHrP; bPTH, bovine PTH; ROS, rat osteosarcoma cells; HPLC, high pressure liquid chromatography.


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