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Originally published In Press as doi:10.1074/jbc.M408270200 on November 17, 2004

J. Biol. Chem., Vol. 280, Issue 3, 1797-1807, January 21, 2005
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Novel Parathyroid Hormone (PTH) Antagonists That Bind to the Juxtamembrane Portion of the PTH/PTH-related Protein Receptor*

Naoto Shimizu, Thomas Dean, Janet C. Tsang, Ashok Khatri, John T Potts, Jr, and Thomas J. Gardella{ddagger}

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

Received for publication, July 21, 2004 , and in revised form, November 1, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Current antagonists for the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (PTHR) are N-terminally truncated or N-terminally modified analogs of PTH(1–34) or PTHrP(1–34) and are thought to bind predominantly to the N-terminal extracellular (N) domain of the receptor. We hypothesized that ligands that bind only to PTHR region comprised of the extracellular loops and seven transmembrane helices (the juxtamembrane or J domain) could also antagonize the PTHR. To test this, we started with the J domain-selective agonists [Gln10,Ala12,Har11,Trp14,Arg19 (M)]PTH(1–21), [M]PTH(1–15), and [M]PTH(1–14), and introduced substitutions at positions 1–3 that were predicted to dissociate PTHR binding and cAMP signaling activities. Strong dissociation was observed with the tri-residue sequence diethylglycine (Deg)1-para-benzoyl-L-phenylalanine (Bpa)2-Deg3. In HKRK-B7 cells, which express the cloned human PTHR, [Deg1,3,Bpa2,M]PTH(1–21), [Deg1,3,Bpa2,M]PTH(1–15), and [Deg1,3,Bpa2,M]PTH(1–14) fully inhibited (IC50s = 100–700 nM) the binding of 125I-[{alpha}-aminoisobutyric acid1,3,M]PTH(1–15) and were severely defective for stimulating cAMP accumulation. In ROS 17/2.8 cells, which express the native rat PTHR, [Deg1,3,Bpa2,M]PTH(1–21) and [Deg1,3,Bpa2,M]PTH(1–15) antagonized the cAMP-agonist action of PTH(1–34), as did PTHrP(5–36) (IC50s = 0.7 µM, 2.6 µM, and 36 nM, respectively). In COS-7 cells expressing PTHR-delNt, which lacks the N domain of the receptor, [Deg1,3,Bpa2, M]PTH(1–21) and [Deg1,3,Bpa2,M]PTH(1–15) inhibited the agonist actions of [{alpha}-aminoisobutyric acid1,3]PTH(1–34) and [M]PTH(1–14) (IC50s ~1 µM), whereas PTHrP(5–36) failed to inhibit. [Deg1,3,Bpa2,M]PTH(1–14) inhibited the constitutive cAMP-signaling activity of PTHR-tether-PTH(1–9), in which the PTH(1–9) sequence is covalently linked to the PTHR J domain, as well as that of PTHRcamH223R. Thus, the J-domain-selective N-terminal PTH fragment analogs can function as antagonists as well as inverse agonists for the PTHR. The new ligands described should be useful for further studies of the ligand binding and activation mechanisms that operate in the critical PTHR J domain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Parathyroid hormone (PTH)1 is a major regulator of ionized calcium and phosphate concentrations in the blood and extracellular fluids, and PTH-related protein (PTHrP) is a vital developmental morphogen (1, 2). These two peptide ligands mediate their actions by binding to the same receptor, the PTH/PTHrP receptor or PTHR (or PTH receptor subtype 1). The PTHR is abundantly expressed in bone and kidney, the principal target organs of PTH, and in a variety of developing tissues (e.g. skeleton, heart, and mammary glands), where it mediates the actions of PTHrP. Excessive circulating levels of PTH, as occurs in cases of hyperparathyroidism, or PTHrP, as frequently occurs in cancer because of secretion by malignant tumors, produces a hypercalcemic state, which can be severely debilitating and potentially fatal (1, 2). For both PTH and PTHrP, the first 34 amino acids encode all of the information needed for binding to and activating the PTHR.

The PTHR is a class 2 G protein-coupled receptor that activates the adenylyl cyclase/cAMP signaling pathway. Like all class 2 G protein-coupled receptors that bind peptide hormones, the PTHR has a relatively large (~160 amino acid) N-terminal extracellular domain, herein termed the N domain (3), that plays a major role in hormone binding (4, 5). The portion of the PTHR comprised of the extracellular loops and seven helical transmembrane domains (TMs), herein termed the juxtamembrane or J domain (3), is thought to mediate most, or all, of the ligand interactions that are involved in receptor activation and G protein coupling (5–8). The molecular mechanism by which PTH (and PTHrP) binds to the PTHR has been extensively analyzed through the approaches of receptor mutagenesis (4–6, 9–11), photochemical cross-linking (12–14), and molecular modeling (15–17). These studies have led to a "two-site" model for the ligand-receptor interaction mechanism, according to which the C-terminal portion of PTH(1–34) (i.e. residues in the 23–34 region) binds to the N domain of the receptor to provide the majority of the binding energy to the complex, and the N-terminal portion of the ligand interacts with a lower affinity with the J domain of the receptor to induce activation (3, 5, 18). A similar two-site binding mechanism appears to be used by other class 2 G protein-coupled receptors that bind peptide hormones (19–27).

As part of our investigations into the ligand binding and activation mechanisms used by the PTHR, we have employed N-terminal PTH fragment analogs as functional probes of the bimolecular complex. In particular, we have used the PTH(1–14) fragment as a scaffold peptide for performing substitution analysis of the principal signaling domain of the hormone. Native PTH(1–14) binds only poorly to the PTHR and is a very weak agonist (EC50 for stimulating cAMP formation = ~1 x 10–4 M) (6). A number of affinity-enhancing substitutions in the PTH(1–14) scaffold have been identified that, when combined, increase the cAMP-stimulating potency of the peptide by as much as 100,000-fold (7, 28–30). These substitutions include Ser1 -> Ac5c (aminocyclopentane-1-carboxylic acid), Ser3 -> Aib ({alpha}-aminoisobutyric acid), Asn10 -> Gln, Leu11 -> Har(homoarginine), Gly12 -> Ala and His14 -> Trp, and have resulted in potent analogs such as [Ac5c1,Aib3,Gln10,Har11,Ala12,Trp14]-PTH(1–14)amide (30), herein termed [Ac5c1,Aib3,M]PTH(1–14), where M refers to the combined modifications of Gln10,Har11, Ala12,Trp14 (as well as Arg19 in the [M]PTH(1–20) and [M]PTH(1–21) analogs described below). In cell-based assays of cAMP formation, the potency of [Ac5c1,Aib3,M]PTH(1–14) is in the low nanomolar range and thus similar to that seen for PTH(1–34) (30). The modified PTH(1–14) analogs resulting from these studies have helped to define the minimum N-terminal PTH agonist pharmacophore, which currently appears to reside within the first 9 or 10 amino acids of the hormone (7, 28, 29, 31). Each of the PTH(1–14) analogs studied so far maintains the same potency on a PTHR construct (PTHR-delNt) that lacks the N domain of the receptor as it does on the intact PTHR; in contrast, PTH(1–34) is ~100-fold less potent on PTHR-delNt than it is on the intact PTHR (7, 28–30). The PTH(1–14) domain, therefore, appears not to utilize N domain interactions to achieve binding affinity and signaling potency. Furthermore, the PTHR J domain, as defined by PTHR-delNt, can be sufficient to mediate transmembrane signaling and G protein coupling. Recent functional (11) and photo-affinity cross-linking (32) studies suggest that the region of PTH that contributes to the J domain interaction extends C-terminally to position 20. Structural studies performed on isolated PTH peptides indicate that the 1–20 region of PTH can adopt an {alpha}-helical conformation (17, 33).

The use of antagonist ligands contributes importantly to the analysis of ligand binding and signal transduction mechanisms used by G protein-coupled receptors. For the PTHR, all current antagonists are PTH or PTHrP analogs that extend C-terminally to at least residue 34 and are thought to depend strongly, although probably not completely (11), on interactions with the N domain of the receptor to achieve high binding affinity and, hence, inhibitory potency. The lack of signaling activity in these antagonist can be attributed to either the deletion, or modification, of N-terminal residues of the ligand, which are well known to play a critical role in PTHR activation (34–36). The most potent PTHR antagonists to date are based on the PTH(7–34) scaffold peptide (36); these "classical" PTHR antagonist include [Leu11,D-Trp12]PTHrP(7–34) (37) and [Ile5,Trp23, Tyr36]PTHrP(5–36) (38). The N- and C-terminally intact antagonists include PTH(1–34) or PTHrP(1–36) analogs that have either valine 2 substituted by arginine (39), tryptophan or para-benzoyl-L-phenylalanine (Bpa) (38, 40), or serine 3 substituted by Tyr or Phe (41). Several of the previous PTHR antagonists, including [Leu11,D-Trp12]PTHrP(7–34), [Leu11,D-Trp12]PTHrP(5–36), and [Bpa2]PTHrP(1–36), function as inverse agonists on the constitutively active mutant PTHRs, PTHRcamT410P, and/or PTHRcamH223R (42, 43).

In the current studies, we hypothesized that a ligand that binds exclusively to the PTHR J domain could, if appropriately modified, function as a PTHR antagonist. Such an antagonist would differ mechanistically from the previous, C-terminally extended PTHR antagonists and, as such, would represent a novel class of PTHR ligands that could potentially be useful in analyzing the ligand binding and activation mechanisms used by the PTHR. As an initial test of this hypothesis, we sought to confer antagonist activity to the recently described N-terminal PTH agonist analogs. We thus started with [Ac5c1,Aib3,M]PTH(1–14) (30) and introduced into this scaffold substitutions at positions 1–3 (30, 35, 38, 40) that were predicted to dissociate binding affinity and signaling activity. Most of these substitutions had the predicted effects. We thus identified several new analogs of [M]PTH(1–14), [M]PTH(1–15), and [M]PTH(1–21) that function as antagonists and, in some cases, inverse agonists for the PTHR. We show that these new antagonists indeed bind predominantly, if not exclusively, to the PTHR J domain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptides—The peptides used in the study are described in Table I and were synthesized using Fmoc chemistry and either a small-scale, multiple peptide synthesizer (Advanced Chemtech Model 396 MBS) with HBTU/HOBt/DIEA (O-benzotriazol-1-yl-N,N,N'N,'-tetramethyluronium hexafluorophosphate/1-hydroxybenzotriazole/N,N-diisopropylethylamine; 1:1:2 molar ratio) chemistry, a 10-fold molar excess relative to substrate of Fmoc amino acid, and double couplings, or a large-scale synthesizer (Applied Biosystems model 431A) with DCC/HOBt chemistry, a 10–40-fold molar excess of Fmoc amino acid, and extended (3-h) coupling cycles. Peptides were desalted (C18 Sep-Pak), purified by reverse-phase HPLC, and verified by analytical HPLC, matrix-assisted laser desorption/ionization mass spectrometry, and amino acid analysis. The lyophilized peptides were reconstituted as stock solutions in 10 mM acetic acid; the purity, identity and exact peptide concentration of each stock solution (~3 x 10–3 M) was secured by amino acid analysis. The stock solutions were stored at –80 °C. The radioligand 125I-[Aibl,3,Nle8,Gln10,Har11,Ala12,Trp14,Tyr15]hPTH(1–15)-NH2 (125I-[Aib1,3,M]PTH(1–15)) was prepared using 125I-Na and the chloramine-T-based oxidation reaction followed by HPLC purification.


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  		TABLE I
PTH peptide analogs utilized

 
Cell Culture and DNA Transfection—The cell lines HKRK-B28 and HKRK-B7 are clonal derivatives of the porcine kidney cell line, LLC-PK1, and were transfected with pCDNA1-based plasmid DNA to stably express recombinant PTH receptors at surface densities of ~280,000 and ~950,000 receptors/cell, respectively (44). HKRK-B28 cells express a fully functional PTHR chimera comprised of the opossum PTHR from the N terminus to the middle of TM3 and the rat PTHR from the middle of TM3 to the C terminus (11); HKRK-B7 cells express the wild-type human PTHR (44). COS-7 cells were transfected to transiently express the PTHR using the FuGENE 6 (Roche Applied Science) reagent and CsCl-purified, pCDNA1-based plasmid DNA (200 ng/well of a 24-well plate) encoding either the wild-type human PTHR (45) or a human PTHR derivative, PTHR-delNt (6, 7), PTHRcamH223R (46), or PTHR-tether-PTH(1–9). The PTHR-tether-PTH(1–9) construct is similar to constructs described by us previously (31) and consists of the following contiguous (N to C terminus) segments: the prepro sequence of native human PTH, residues 1–9 of native rat PTH (Ala-Val-Ser-Glu-Ile-Gln-Leu-Met-His), a tetraglycine linker, and the hPTHR sequence Glu182–Met593, which extends from the extracellular terminus of TM1 to the C terminus of the receptor. Upon expression and processing in COS-7 cells, the prepro-PTH sequence is removed so as to leave alanine 1 of the PTH sequence as the free N terminus of the protein (confirmed by N-terminal sequence analysis of the purified protein).2

Competition Binding Assays—Binding studies were performed using 125I-[Aib1,3,M]PTH(1–15) as the tracer radioligand and varying concentrations of unlabeled test peptides as competitors, as described previously (29, 30). In brief, cells in 24-well plates were rinsed in binding buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 2 mM CaCl2, 5% heat-inactivated horse serum, 0.5% fetal bovine serum, adjusted to pH 7.7 with HCl),and the following three components were added to the well successively, 100 µl of binding buffer, 100 µl of binding buffer containing an unlabeled test peptide, and 100 µl binding buffer containing 125I-[Aib1,3,M]PTH(1–15) (~100,000 cpm/well). All solutions were ice-cold, and the plates were embedded in a tray of ice during the addition phase. The plates were then incubated in a 15 °C water bath for 4 h, after which the well contents were removed by aspiration, and the cell monolayers were rinsed three times by adding and rapidly removing 0.5 ml of binding buffer. Then, 0.5 ml of 5 N NaOH was added to lyse the monolayers followed by the addition of 2.0 ml of H20, and the entire lysate was counted for radioactivity. Nonspecific binding was determined in wells containing an excess (1 x 10–5 M) of an unlabeled high affinity ligand, and the total specific binding at each competing ligand concentration was calculated as a percentile of the total specific binding observed in the absence of competitor. The data were then analyzed by non-linear regression analysis to obtain fitted curves and IC50 values (see below).

cAMP Stimulation Assays—Assays were performed in whole cells in 24-well plates, in which the cells had formed confluent monolayers 2–4 days prior to assay and had attained densities of 200,000–300,000 cells/well, as described previously (29, 30). The assays utilized binding buffer containing 2 mM IBMX (3-isobutyl-1-methylxanthine). For cAMP response assays involving the addition of a single peptide, cells were rinsed in IBMX-binding buffer, and the following two components were added to the wells successively, 200 µl of IBMX-binding buffer and 100 µl of IBMX-binding buffer containing the test peptide. For assays involving the addition of two peptides (agonist plus antagonist), cells were rinsed with IBMX-binding buffer and the following three components were added to the wells successively, 100 µl of IBMX-binding buffer, 100 µl of IBMX-binding buffer containing an antagonist peptide, and 100 µl of IBMX-binding buffer containing an agonist peptide. Following these additions, the plates were incubated at room temperature for 30 min. The buffer was then rapidly removed, the plates were embedded in powdered dry ice to freeze the monolayer, and 0.5 ml of 50 mM HCl was added. After freezing and thawing the contents, 2.0 ml of H20 was added to each well and an aliquot (0.5–20 µl) was withdrawn and was processed by a radioimmunoassay to quantify the concentration of (intracellular) cAMP.

Inositol Phosphate (IP) Stimulation Assays—COS-7 cells in 24-well plates were transiently transfected as above with the human PTHR and 3–4 days later were treated with serum-free, inositol-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin and [3H]myo-inositol (PerkinElmer Life Sciences) (2 µCi/ml) for 16 h. The cells were then rinsed with binding buffer containing LiCl (30 mM), and the following three components were added to the wells successively, 200 µl of LiCl-binding buffer, 40 µl of LiCl-binding buffer containing or lacking a test antagonist peptide, and 10 µl of LiCl-binding buffer containing or lacking an agonist peptide. The cells were then incubated at 37 °C for 40 min, after which the buffer was removed and replaced by 0.5 ml of ice-cold 5% trichloroacetic acid solution. After 2 h on ice, the lysate was collected, extracted twice with diethyl ether, applied to an ion exchange column (Dowex AG1-X8 resin, 0.5-ml bed volume), and total inositol phosphates were eluted as described previously (47) and counted in liquid scintillation mixture for radioactivity.

Data Calculations—All calculations were performed using the Excel software package (Microsoft Corp., Redmond, WA). Curves were fit to data points using a four parameter logistic equation: y = min + (max – min)/(1 + (IC50/x)n), where y is the observed response (specific binding of radioligand or cAMP value), min and max are the minimum and maximum response values, respectively, x is ligand concentration (nM), IC50 (or EC50 in cAMP assays) is the ligand concentration (nM) at which 50% of the maximum response occurs, and n is the slope of the plot of y versus x and the Excel Solver function to optimize the parameters by nonlinear regression analysis (48). Paired data sets were statistically compared using the Student's t test (two-tailed) assuming unequal variances for the two sets.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PTHR Binding and cAMP Signaling Properties of PTH Analogs in LLC-PK1 Cells—Starting with the potent agonist [Ac5c1,Aib3,M]PTH(1–14) (30) as a parent scaffold peptide, we first introduced at positions 1 or 2 single-residue modifications that were predicted to dissociate PTHR binding affinity and cAMP-signaling capacity. These modifications included the removal of the N-terminal amino function (35), as provided by des-NH2-Ac5c1 and des-NH2-Aib1 (Aib is structurally related to Ac5c and when substituted at position 1 results in activity profiles that are nearly indistinguishable from those seen with Ac5c1 (30)) and the substitution of valine 2 by arginine (39), tryptophan, or Bpa (38, 40) (Table I). The resulting analogs were tested for PTHR binding affinity and cAMP-stimulating potency using HKRK-B28 cells, an LLC-PK1-derived cell line in which a rat/opossum chimeric PTHR is expressed at a density of ~280,000 receptors/cell (44). As summarized in Table II, each of the single-residue modifications resulted in some loss of binding affinity, as assessed in competition experiments performed with 125I-[Aib1,3,M]PTH(1–15) tracer radioligand, with the strongest (830-fold) reduction occurring with the Arg2 substitution and the mildest (25-fold) reduction occurring with the Trp2 substitution. In the cAMP formation assays we used a peptide concentration of 10 µM, at which most, if not all, of the analogs could be predicted to attain at least half-maximum receptor occupancy. Each of the substituted analogs stimulated no more than 25% of the maximum cAMP response (Emax) observed with the parental peptide [Ac5c1, Aib3,M]PTH(1–14), and the Arg2-, Bpa2-, and Trp2-substituted analogs stimulated no more than 10% of the parental response.


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  		TABLE II
Binding properties of PTH analogs in HKRK-B28 cells

Values are means (±S.E.) of data compiled from three or four experiments (n), each performed in duplicate.

 
Because we recently found, and confirmed in the studies reported in Table II, that introducing the paired dialkyl amino acid substitution of Deg1,3 in the [M]PTH(1–14) scaffold confers partial agonist behavior to the peptide (30), we combined the Deg1,3 substitutions with Bpa2 to obtain [Deg1,3,Bpa2,M]PTH(1–14). This analog exhibited the strongest dissociation of binding affinity and cAMP signaling efficacy (IC50 = ~840 nM, Emax = ~3% of the parental response, Table II) of any analog tested in the [M]PTH(1–14) series. In the LLC-PK1-derived cell line HKRK-B7 cells, in which the wild-type human PTHR is expressed at a surface density of ~950,000 receptors/cell (44), [Deg1,3,Bpa2,M]PTH(1–14) again showed a strong dissociation of binding affinity and cAMP-signaling efficacy (IC50 = 700 nM, Emax = ~6% of the response seen with [Ac5c1,Aib3,M]PTH(1–14), Fig. 1, A and B and Table III). The dissociation of binding and cAMP signaling caused by the Deg1-Bpa2-Deg3 substitutions were confirmed in an independently synthesized N-terminal PTH fragment analog, [Deg1,3,Bpa2,M]PTH(1–15),3 which exhibited functional properties identical to those of [Deg1,3,Bpa2,M]PTH(1–14) (Fig. 1, A and B and Table III). In an attempt to further improve PTHR binding affinity without increasing signaling efficacy, we extended [Deg1,3,Bpa2,M]PTH(1–15) to position 21 and included the Arg19 substitution, as we recently showed that residues in the 15–20 region of the ligand, and particularly the Arg19 modification, improve the binding affinity on the PTHR J domain by at least severalfold (11, 33). The resulting analog [Deg1,3,Bpa2,M]PTH(1–21) exhibited a 6-fold higher apparent binding affinity in HKRK-B7 cells, as compared with the binding of [Deg1,3,Bpa2,M]PTH(1–15) (IC50s = 110 versus 710 nM, p = 0.02), and exhibited little or no increase in cAMP-signaling capacity (Emax = 8% of that seen for [Ac5c1,Aib3,M]PTH(1–14), Fig. 1, A and B, Table III). We then assessed the antagonist properties of the Deg1-Bpa2-Deg3-containing N-terminal peptides in PTHR-expressing cells, as discussed below.



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  		FIG. 1.
cAMP and binding properties of agonist and antagonist analogs in HKRK-B7 cells. Competition binding (A) and stimulatory cAMP response (B) assays were conducted in HKRK-B7 cells with the peptide ligands identified in the symbol key. Shown are combined data (means ± S.E.) from three to five experiments, each performed in duplicate. Competition binding studies were performed with the 125I-[Aib1,3,M]PTH(1–15) tracer radioligand and varying concentrations of the unlabeled competing ligand indicated in the figure key. Binding data show the amount of radioligand specifically bound (SB) calculated as a percentile of the total radioligand specifically bound in the absence of competitor (B0). Curves were fit to the data points using non-linear regression analysis; the corresponding IC50 and EC50 values are reported in Table III. The cAMP data were calculated as a percentile of the maximum response observed in each assay with PTH(1–34), the average of which was 394 ± 14 pmol/well. The corresponding basal cAMP value was 5.9 ± 0.7 pmol/well and was not subtracted in the calculations.

 


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  		TABLE III
Functional properties of PTH analogs in HKRK-B7 cells

 

Antagonism of Agonist-induced cAMP Responses in ROS 17/2.8 Cells—We first examined the capacity of [Deg1,3,Bpa2,M]PTH(1–15) and [Deg1,3,Bpa2,M]PTH(1–21) to antagonize PTH agonist analogs in ROS 17/2.8 cells. These rat osteosarcoma-derived cells endogenously express the rat PTHR at a surface density of ~50,000 receptors/cell (49) and were found to give more discernable antagonist responses to PTH analog peptides than did HKRK-B7 and HKRK-B28 cells (data not shown), presumably because of their lower level of receptor expression. As agonist analogs in these assays, we used PTH(1–34) to represent a two-site mode of agonist binding (see Introduction) and [Ac5c1,Aib3,M]PTH(1–14) to represent a J domain-selective mode of agonist binding. These two agonists are approximately equipotent for stimulating cAMP formation in ROS 17/2.8 cells; thus the 1 nM concentration of each peptide used in these experiments resulted in a 40–50-fold increase in cAMP accumulation, relative to the basal cAMP level (Fig. 2). Both [Deg1,3,Bpa2,M]PTH(1–21) and [Deg1,3,Bpa2,M]PTH(1–15) inhibited to near completion (89 and 76%, respectively) the cAMP response induced by PTH(1–34). This maximal level of inhibition was similar to that attained with PTHrP(5–36) (94%), although 70- and 20-fold higher concentrations of the two N-terminal peptides were required to achieve 50% inhibition than was required for PTHrP(5–36) (IC50s = 2.6 µM, 0.7 µM, and 36 nM, respectively, Fig. 2A). The 3–4-fold higher inhibitory potency of [Deg1,3,Bpa2,M]PTH(1–21), as compared with that of [Deg1,3,Bpa2,M]PTH(1–15), is consistent with the 6-fold higher apparent binding affinity that the longer analog exhibited in HKRK-B7 cells (Table III).



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  		FIG. 2.
Inhibition of PTH analog-induced cAMP responses in ROS 17/2.8 cells. ROS 17/2.8 cells, which endogenously express the rat PTHR, were used to assess antagonism of PTH agonist analog-induced cAMP responses. The cells were treated with an agonist, PTH(1–34) (A) or [Ac5c1,Aib3,M]PTH(1–14) (B), each at a concentration (1 nM) that elicited an approximate half-maximum stimulatory cAMP response, either in the absence of an additional peptide (0 on the abscissa) or in the presence of a candidate antagonist, PTHrP(5–36), [Deg1,3,Bpa2,M]PTH(1–21), or [Deg1,3,Bpa2,M]PTH(1–15), at the indicated concentrations for 30 min, and intracellular cAMP was measured. Shown are combined data (means ± S.E.) from three experiments, each performed in duplicate. In each experiment, and for each of the two agonists, the cAMP level observed in the presence of a test antagonist was calculated as a percentile of the cAMP level observed in cells treated with that agonist alone, the mean values of which were 51 ± 4 and 41 ± 5 pmol/well for the experiments shown in A and B, respectively. The corresponding basal cAMP value was 1.1 ± 0.2 pmol/well and was not subtracted in the calculation.

 
When [Ac5c1,Aib3,M]PTH(1–14) was used as the agonist, [Deg1,3,Bpa2,M]PTH(1–15), [Deg1,3,Bpa2,M]PTH(1–21), and PTHrP(5–36) each again achieved ~90% inhibition, and the IC50s for the two N-terminal peptides were similar to each other and ~100-fold higher than that observed for PTHrP(5–36) (1.3 µM, 0.9 µM, and 11 nM, respectively, Fig. 2B). These data show that, although weaker than PTHrP(5–36), the modified PTH(1–15) and PTH(1–21) analogs can clearly function as antagonists in these osteoblast-derived cells that expresses the intact PTHR.

Inhibition and Binding Properties in COS-7 Cells Expressing PTHR-delNt—To examine the receptor domains used by the N-terminal PTH antagonist analogs, we used COS-7 cells transiently transfected with PTHR-delNt, a PTHR construct that lacks most of the N domain of the receptor. In COS-7 cells, PTHR-delNt is expressed at ~50% the level of the wild-type PTHR and couples efficiently to the cAMP-signaling pathway (6). As a PTH(1–34)-based agonist peptide in these studies we used [Aib1,3]PTH(1–34), which is ~100-fold more potent on PTHR-delNt than is unmodified PTH(1–34) (30) (the potency of the latter peptide on PTHR-delNt was too weak to permit use in these experiments). As a PTH(1–14)-based agonist peptide we again used [Ac5c1,Aib3,M]PTH(1–14), which exhibits nearly the same agonist potency on PTHR-delNt as it does on the wild-type PTHR (30). In the absence of antagonist, [Aib1,3]PTH(1–34) (100 nM) and [Ac5c1,Aib3,M]PTH(1–14) (1 nM) stimulated 11- and 21-fold increases in cAMP levels, respectively, relative to the cAMP levels in the untreated cells (Fig. 3, A and B). The cAMP response induced by [Aib1,3]PTH(1–34) was inhibited by ~70% by both [Deg1,3,Bpa2,M]PTH(1–21) and [Deg1,3,Bpa2,M]PTH(1–15), and the inhibitory potencies of the two antagonists (IC50s = ~4 µM, Fig. 3A) were again similar to each other and comparable with those seen for these two analogs when tested on the intact PTHR in ROS 17/2.8 cells using PTH(1–34) as an agonist (Fig. 2A). The cAMP response induced by [Ac5c1,Aib3,M]PTH(1–14) in COS-7 cells expressing PTHR-delNt was also inhibited by ~70% by each of the two N-terminal peptides, and the IC50s were again comparable to each other (~3 µM, Fig. 3B) and to those observed on the intact PTHR using the same PTH(1–14) agonist peptide (Fig. 2B). In contrast, PTHrP(5–36) failed to inhibit either agonist peptide on PTHR-delNt (Fig. 3, A and B). Consistent with the divergent capacities of these N- and C-truncated analogs to function as antagonists on PTHR-delNt, [Deg1,3, Bpa2,M]PTH(1–15) fully inhibited (IC50 ~ 0.8 µM) the binding of the 125I-[Aib1,3,M]PTH(1–15) radioligand to COS-7 cells expressing PTHR-delNt, whereas PTHrP(5–36) failed to inhibit this binding (Fig. 3C). These results established that the inhibitory action of both [Deg1,3,Bpa2,M]PTH(1–15) and [Deg1,3, Bpa2,M]PTH(1–21), but not that of PTHrP(5–36), is based on binding interactions that occur predominantly, if not exclusively, to the PTHR J domain.



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  		FIG. 3.
cAMP-inhibition and receptor binding properties of PTH antagonist analogs in COS-7 cells expressing PTHR-delNt. COS-7 cells transiently transfected to express the N-terminally truncated PTHR construct, PTHR-delNt, were treated with an agonist, [Aib1,3]PTH(1–34) (A) or [Ac5c1,Aib3,M]PTH(1–14) (B), at a concentration (100 nM in A and 1 nM in B) that elicited an approximate half-maximum stimulatory cAMP response, either in the absence of an additional peptide (0 on the abscissa) or in the presence of a candidate antagonist, PTHrP(5–36), [Deg1,3,Bpa2,M]PTH(1–21), or [Deg1,3,Bpa2,M]PTH(1–15). At the indicated concentrations and after a 30-min incubation, intracellular cAMP was measured. Shown are the resulting cAMP responses (means ± S.E.) from 5 to 10 experiments, each performed in duplicate. In each experiment and for each agonist, the cAMP level observed in the presence of a test antagonist was calculated as a percentile of the cAMP level observed in cells treated with that agonist alone, the mean values of which were 100 ± 7 and 187 ± 11 pmol/well, for the experiments shown in A and B, respectively. The corresponding basal cAMP values, indicated by the dashed lines in the graphs, were 8.8 ± 0.8 and 8.9 ± 1.1 pmol/well, respectively, and were not subtracted in the calculations. In C, COS-7 cells transiently transfected to express PTHR-delNt were used in competition binding studies performed with the 125I-[Aib1,3,M]PTH(1–15) tracer radioligand and varying concentrations of unlabeled PTHrP(5–36) or [Deg1,3,Bpa2,M]PTH(1–15). The binding data show the amount of radioligand specifically bound (SB) calculated as a percentile of the total radioligand specifically bound in the absence of competitor (B0). Shown are combined data (means ± S.E.) from three experiments, each performed in duplicate. The IC50 value obtained for the [Deg1,3,Bpa2,M]PTH(1–15) binding curve was 1400 nM. A curve was not fit to the data obtained with for PTHrP(5–36), because no response was observed.

 
We then examined the capacity of a single high concentration (10 µM) of [Deg1,3,Bpa2,M]PTH(1–15) to shift the cAMP dose-response curve obtained with PTH(1–34) in ROS 17/2.8 cells (Fig. 4A) and for that obtained with [Aib1,3]PTH(1–34) in COS-7 cells expressing PTHR-delNt (Fig. 4B). In ROS 17/2.8 cells, the addition of [Deg1,3,Bpa2,M]PTH(1–15) caused an ~4-fold rightward shift in the PTH(1–34) dose-response curve, relative to that obtained in the absence of the antagonist (EC50s = 1.2 and 0.3 nM, respectively). This shift occurred in a parallel fashion and resulted in approximately the same maximum cAMP level as that attained in the absence of antagonist (p = 0.5). Similarly, in COS-7 cells expressing PTHR-delNt, the addition of [Deg1,3,Bpa2,M]PTH(1–15) caused an ~8-fold rightward and parallel shift in the dose-response curve obtained for [Aib1,3]PTH (1–34), compared with that obtained in the absence of antagonist (EC50s = 560 and 70 nM, respectively) and caused little or no change in the Emax value (Fig. 4B, p = 0.1). The analog [Deg1,3,Bpa2,M]PTH(1–14) caused a similar ~10-fold rightward shift in the dose-response curve obtained for [Aib1,3]PTH(1–34) in COS-7 cells expressing PTHR-delNt (data not shown). These effects of the antagonists on the agonist potency curves (i.e. parallel, rightward shifts with same Emax values) are consistent with a competitive mechanism of inhibition (50).



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  		FIG. 4.
Effect of [Deg1,3,Bpa2,M]PTH(1–15) on the cAMP dose-response curve obtained for PTH(1–34) analogs in cells expressing the intact PTHR or PTHR-delNt. In A, ROS 17/2.8 cells were treated with varying concentrations of PTH(1–34) either in the absence (closed symbols) or presence (open symbols) of a single concentration (10 µM) of [Deg1,3,Bpa2,M]PTH(1–15), and after a 30-min incubation intracellular cAMP was measured. Shown are the resulting cAMP values (means ± S.E.) from four combined experiments, each performed in duplicate. In B, COS-7 cells transiently transfected to express PTHR-delNt were treated with varying concentrations of [Aib1,3]PTH(1–34), either in the absence (closed symbols) or presence (open symbols) of a single concentration (10 µM) of [Deg1,3,Bpa2,M]PTH(1–15), and after a 30-min incubation intracellular cAMP was measured. Shown are the resulting cAMP values (means ± S.E.) from three combined experiments, each performed in duplicate. The basal cAMP values (not subtracted) were 23 ± 5 and 15 ± 1 pmol/well, for the experiments shown in A and B, respectively.

 
Inhibition of Constitutively Active Mutant PTH Receptors in COS-7 Cells—We then investigated the capacity of the antagonist analogs to inhibit the basal cAMP-stimulating activity of two constitutively active mutant PTH receptor derivatives. The first of these derivatives was a "tethered" ligand-receptor construct in which the N-terminal domain of the PTH receptor is replaced by the (1–9) segment of native PTH, representing the minimum agonist pharmacophore of the ligand (31). In this construct, the ligand sequence is covalently joined to the body of the PTHR via a tetra-glycine linker between His9 of the ligand sequence and Glu182 of the receptor, which is located approximately nine residues N-terminal of the predicted extracellular end of TM1. When transiently transfected into COS-7 cells, this construct, in the absence of exogenously added ligand, results in basal cAMP levels that are comparable to those seen in COS-7 cells transfected with the wild-type PTHR and stimulated maximally with PTH(1–34). As shown in Fig. 5A, the addition of [Deg1,3,Bpa2,M]PTH(1–14) (10 µM) to such transfected COS-7 cells caused an ~40% diminishment in the basal cAMP levels. The analog PTHrP(5–36) resulted in a small (16%) yet significant (p = 0.02) reduction in basal signaling activity (Fig. 5A).



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  		FIG. 5.
Effects of antagonist peptides on basal cAMP signaling of constitutively active PTH receptors. COS-7 cells were transiently transfected with either PTHR-tether-PTH(1–9) (A), which has the PTH(1–9) sequence covalently joined to the PTHR J domain (31), or PTHRcamH223R (B), which contains an activating point mutation (His223 -> Arg) at the cytoplasmic terminus of TM2 (46). The transfected cells were either untreated (basal) or treated with a candidate inhibitory peptide, each used at a concentration of 10 µM for 30 min, and the resulting cAMP levels were measured. Each panel shows combined data (means ± S.E.) from three experiments, each performed in duplicate. In each experiment and for each receptor construct, the cAMP levels observed in the presence of a test peptide were calculated as the percentile of the corresponding basal cAMP level observed with that receptor in that experiment; the combined average basal cAMP levels, indicated by the dashed lines in the graphs, were 219 ± 34 and 78 ± 10 pmol/well for the experiments shown in A and B, respectively. The basal cAMP levels observed in COS-7 cells transfected in parallel plates with the wild-type PTHR were 13 ± 1 pmol/well. The p values indicate statistical analyses (Student's t test) of differences between ligand-treated and corresponding basal cAMP levels.

 
The second constitutively active mutant PTHR utilized was PTHRcamH223R. This intact mutant PTHR contains an activating point mutation (His223 -> Arg) at or near the cytoplasmic terminus of TM2 (46). We included in these studies [Deg1,3,Bpa2,M]PTH(1–14), [Ac5c1,Bpa2,Aib3,M]PTH(1–14), as well as two C-terminally extended PTHrP ligands known to function as inverse agonists on this constitutively active PTHR, [Leu11,D-Trp12]PTHrP(5–36) and [Bpa2]PTHrP(1–36) (43). Each of the tested analogs, applied at a concentration of 10 µM, reduced to at least some degree the intracellular cAMP levels in COS-7 cells expressing PTHRcamH223R (Fig. 5B). The strongest reduction (72%) occurred with [Ac5c1,Bpa2,Aib3,M]PTH(1–14)4 followed in rank-order by [Leu11,D-Trp12]PTHrP-(5–36) (49%), [Bpa2]PTHrP(1–36) (33%), and [Deg1,3,Bpa2,M]PTH(1–14) (28%).

Inhibition of Ligand-induced Inositol Phosphate Signaling in COS-7 Cells Expressing the Wild-type PTHR—Because the wild-type PTHR can also couple to the PLC/IP signaling pathway, we analyzed the capacity of the antagonist ligands to inhibit this mode of PTHR signaling. As shown in the experiments of Fig. 6A performed in COS-7 cells transfected with the hPTHR, PTHrP(5–36) and [Deg1,3,Bpa2,M]PTH(1–15), each at a concentration of 30 µM, did elicit an IP response and inhibited by more than 90% (p < 0.001) the IP response induced by [Ac5c1,Aib3,M]PTH(1–14) used at a concentration of 1 µM. Similarly, [Deg1,3,Bpa2,M]PTH(1–14) and [Deg1,3,Bpa2,M]PTH(1–21) lacked IP signaling activity and inhibited by more than 90% the response induced by [Ac5c1,Aib3,M]PTH(1–14) [(data not shown). As shown in the experiments of Fig. 6B, PTHrP(5–36) (30 µM) inhibited by more than 90% (p < 0.001) the IP response induced by PTH(1–34) (300 nM), whereas [Deg1,3,Bpa2,M]PTH(1–15) (30 µM) resulted in partial (22%) inhibition (p = 0.04). These IP antagonism results are fully consistent with the cAMP-antagonism data described above and are also consistent with the notion that mechanism of ligand-induced IP signaling in the PTHR is similar to the mechanism of ligand-induced cAMP signaling, in that each involves interactions between the N-terminal residues of the ligand and the juxtamembrane region of the receptor (28, 30, 51).



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  		FIG. 6.
Effects of antagonist peptides on inositol phosphate signaling in COS-7 cells. COS-7 cells transiently transfected with the hPTHR were used to assess inositol phosphate signaling by [Deg1,3,Bpa2,M]PTH(1–15) and [Deg1,3, Bpa2,M]PTH(1–21), each at 10 µM, and the capacities of these analogs to antagonize the inositol phosphate response induced by either [Ac5c1,Aib3,M]PTH(1–14) at a concentration of 1 µM (A) or PTH(1–34) at a concentration of 300 nM (B). Shown are data combined from three (A) and two (B) experiments, each performed in duplicate. The p values indicate statistical analyses (Student's t test) of differences between the responses seen in cells treated with both agonist and antagonist and cells treated with the agonist alone.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we explored whether a PTHR ligand that binds exclusively to the portion of the receptor comprised of the extracellular loops and seven transmembrane helices (the J region) could function as a PTHR antagonist. Our results showed that this is indeed possible, as PTH(1–14), PTH(1–15), and PTH(1–21) agonist analogs, previously shown to interact mainly, if not exclusively, with the PTHR J domain (7, 11, 29, 30), when modified with activity-lowering substitutions at positions 1–3, exhibit antagonist properties on both the intact PTHR, as well as on PTHR-delNt, which lacks most of the N-terminal extracellular domain (N domain) of the receptor. These new antagonist ligands differ structurally and mechanistically from all previous PTHR antagonists as the latter analogs are C-terminally extended to at least position 34 and depend strongly on interactions with the N domain of the receptor to achieve their binding affinity and, hence, inhibitory potency (4, 5, 52). The modifications that conferred antagonist properties to the new N-terminal PTH analogs included the removal of the N-terminal amino group, the substitution of valine 2 by arginine, tryptophan, or Bpa, and the substitution of residues 1 (normally serine or alanine) and 3 (normally serine) by Deg. The activity-diminishing effects observed for these substitutions in the context of the modified N-terminal PTH fragment analogs are fully consistent with the known importance of the first few residues of native PTH(1–34) in inducing PTHR activation (34–36,41), particularly that of valine2 (9, 38, 40). In the current analogs, the strongest dissociation of binding and signaling activities occurred with the valine 2 -> Bpa substitution; combining Bpa2 with the Deg1,3 substitutions led to analogs with little or no cAMP or IP agonist activity, even when present at concentrations that would nearly saturate the receptor binding sites (Table II, Fig. 6). The tri-residue sequence, Deg1-Bpa2-Deg3, thus constitutes a strong antagonist pharmacophore for the PTHR and, as such, was incorporated into the affinity-enhanced [M]PTH(1–14), [M]PTH(1–15), and [M]PTH(1–21) analog scaffolds for subsequent functional studies. The functional properties of these three analogs were found to be very similar; the slightly (6-fold) higher binding affinity of the longer PTH(1–21) analog, compared with the PTH(1–14) and PTH(1–15) analogs (Table III) resulted in slightly higher inhibitory potencies, as seen in some of the antagonism assays (e.g. Fig. 2A), reflecting mostly the contribution of residue 19 to binding affinity (11).

One goal of these studies was to obtain new antagonist ligands that could be used to further explore the ligand binding and activation mechanisms that operate in the PTHR. In this regard, the results of our studies are generally consistent with and extend the two-site model that has been proposed for the PTH/PTHR interaction mechanism (3–6). The principal postulates of this model are depicted in Fig. 7 and in our current studies are perhaps best demonstrated by the observations that [Deg1,3,Bpa2,M]PTH(1–15) and [Deg1,3,Bpa2,M]PTH(1–21) maintained approximately the same inhibitory potency on PTHR-delNt as they did on the intact PTHR, whereas in contrast, PTHrP(5–36), a highly effective antagonist on the intact PTHR, exhibited little or no inhibitory capacity on the N-truncated receptor. These divergent functional activities observed for the N- and C-truncated antagonists on PTHR-delNt provided the principal support for our conclusion that the new N-terminal antagonist analogs bind purely to the J domain of the receptor, and thus differ mechanistically from the previous C-terminally extended antagonists, including the classical N-terminally truncated antagonists (36–38), which derive the major portion of their binding energy from interactions with the N domain of the receptor (4, 5, 52). Our findings with PTHR-delNt also demonstrate that interactions with the N domain of the receptor are not necessary for antagonizing the PTHR, at least in the case where the antagonist binds with high affinity to the J domain, as do the new N-terminal PTH antagonist analogs described here.



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  		FIG. 7.
Binding modes used by agonist and antagonist ligands of the PTH receptor. The schematic illustrates hypothetical binding mechanisms used by PTH(1–34) agonist peptides (A), "N" domain-selective antagonist peptides (B), and "J" domain-selective antagonists (C). The agonist PTH(1–34) utilizes a two-site binding mechanism (3, 5) that involves the putatively helical N- and C-terminal domains of the ligand (depicted as white cylinders) interacting with the N-terminal extracellular (N) domain of the receptor to provide the major component of binding energy to the complex and the juxtamembrane (J) region of the receptor to induce receptor activation (depicted by a shift in the positioning of the seven transmembrane helices), respectively. Classical, N-terminally truncated antagonists, represented in this study by PTHrP(5–36), bind mainly to the N domain with some overlap in the J domain. The new PTH(1–14)-derived antagonists described in this study bind mainly, if not exclusively, to the PTHR J domain. These N-terminal antagonists are effective inhibitors of J domain-specific agonists, such as [Ac5c1,Aib3,M]PTH(1–14), but not of two-site agonists, such as PTH(1–34).

 
The overall results so far on the PTH/PTHR interaction mechanism achieved with multiple PTH ligand analogues, mutant PTH receptors, and photo-affinity cross-linking studies, provide an initial definition of the regions of interaction between the ligand and the receptor. Although the new antagonists of the current study bind exclusively to the J domain, we also found evidence to suggest that the classical, N-terminally truncated antagonists, represented here by one of the strongest antagonists, PTHrP(5–36) (38, 43), do not bind purely to the N domain of the receptor, a possibility that has been raised previously (3, 6). The new evidence for this includes the capacity of PTHrP(5–36) to fully inhibit the cAMP-stimulating activity of [Ac5c1,Aib3,M]PTH(1–14) (a J domain-specific agonist) on the PTHR (Fig. 2B), as well the capacity of PTHrP(5–36) to fully inhibit the binding of the 125I-[Aib1,3,M]PTH(1–15) radioligand to the intact receptor (Fig. 1A). These results thus suggest that some portion of PTHrP(5–36) directly interacts with the PTHR J domain. Consistent with this possibility, recent mutational and photo-affinity cross-linking data provide evidence for interactions or proximities between residues in the 5–27 region of the ligand and the PTHR J domain. Thus, the extension of PTH(1–14) peptides to position 20/21 and inclusion of the Glu19 -> Arg substitution enhances binding affinity and/or signaling potency on PTHR-delNt (the isolated J domain) by as much as 100-fold (11). In cross-linking studies, a PTH(1–34) analog modified with benzophenone at position 17 cross-linked to Leu261 in the first extracellular loop (53), a benzophenone 13-modified PTH(1–34) peptide cross-linked to Arg186 near the N domain/TM1 boundary (13), and [Bpa19]-PTHrP(1–36), as well as [Bpa19,M]PTH(1–21), each cross-linked to the extracellular terminus of TM2 (32). At least some such proximities in the PTHR J domain must contribute to the overall binding energy with which PTHrP(5–36) interacts with the receptor. In further support of this hypothesis we found that although PTHrP(5–36) failed to inhibit the binding of 125I-[Aib1,3,M]PTH(1–15) to PTHR-delNt or the cAMP-signaling activity of [Ac5c1,Aib3,M]PTH(1–14) and [Aib1,3]PTH(1–34) on this truncated receptor (emphasizing the critical dependence of the ligand on the presence of the N domain of the receptor) it did inhibit marginally, yet significantly, signaling by PTHR-tethered-PTH(1–9), which like PTHR-delNt lacks nearly all of the N domain of the receptor. Any interactions that occur between PTHrP(5–36) and the PTHR J domain would therefore be too weak by themselves to enable the inhibition of binding of N-terminal PTH (1–14) analogs modified with affinity-enhancing substitutions yet be strong enough to enable at least partial inhibition of the binding of the unmodified N-terminal PTH (1–9) sequence to the PTHR J domain.

Another possibility not entirely excluded by the present data is that PTHrP(5–36) inhibits the modified PTH(1–14) agonist actions on the intact PTHR via an allosteric mechanism, for example, by binding to the PTHR N domain in a way that interferes with movements in the J domain that are required for the formation of a PTHR active state (54) that is specifically recognized by the PTH(1–14) agonist. In this regard, it is worth noting that several non-peptidic antagonist compounds have been identified for other class 2 G protein-coupled receptors, and some of these appear to bind outside of the receptor region occupied by the agonist pharmacophore of the native ligand. Thus, a key binding determinant for a non-peptide antagonist for the calcitonin gene-related protein receptor maps to the receptor-associated-modifying protein that modulates the ligand-binding specificity of that receptor (55), and a binding determinant for a non-peptide antagonist of the glucagon-like-peptide-1 receptor was identified as a tryptophan residue located at position 23 near the N terminus of that receptor (56). On the other hand, Hoare and colleagues (27) have shown that several small molecule antagonists of the corticotropin-releasing factor (CRF) receptor bind directly to the isolated CRF receptor J domain, and residues required for the binding of such antagonists have been identified in the midregions of TM3 and TM6 of the intact CRF receptor and within ~10 amino acids of residues at the extracellular ends of the same helices that contribute to CRF binding (57). It would thus seem that these CRF receptor antagonists could inhibit CRF action via direct, competitive mechanisms. A small molecule antagonist for the glucagon receptor also binds within the J domain of that receptor and requires for this binding residues within the midregions of TM2 and TM3; in this case, however, the data point to a non-competitive mechanism of inhibition (58).

The mechanism by which PTHrP(5–36) inhibits the J domain-selective agonists on the PTHR is not at present clear. It is clear, however, that the PTH(1–14)-based antagonists bind to the J domain of the receptor and appear to function via a direct competitive mechanism (Fig. 4B). This mode of inhibition renders these new antagonists potentially useful probes of the region of the PTHR J domain that is involved in binding and responding to the PTH agonist pharmacophore. That the Bpa2 substitution in these antagonists produces approximately the same profile of antagonism and inverse agonism as that seen with Bpa2-modified PTH(1–34) or PTHrP(1–36) further suggests that the modified, N-terminal PTH antagonists interact with the PTHR in a fashion similar to that used by the N-terminal portion of the relatively unmodified, longer length ligands and thus do not mediate antagonism via long range, allosteric effects.

The overall results of our study show that it is possible to antagonize the PTH receptor by targeting ligands specifically to the PTHR J domain. The data provide support for and extend the current two-domain model of the PTH/PTHR binding and activation mechanisms. The principal determinants of the antagonist pharmacophore of the new analogs are the photolabile Bpa2 group and the conformationally constraining Deg1,3 substitutions. The ligands should therefore be useful for further mapping sites of interaction in the PTH/PTH receptor interface, specifically those that occur within the J domain of the receptor and are involved in activation (16), and potentially for the further design of ligands (peptidic or non-peptidic) that exhibit potent, agonist or antagonist properties on the PTH/PTHrP receptor.


   FOOTNOTES
 
* This work was supported by Grant DK-11794 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed. Tel.: 617-726-3966; Fax: 617-726-7543; E-mail: gardella{at}helix.mgh.harvard.edu.

1 The abbreviations used are: PTH, parathyroid hormone; PTHrP, PTH-related protein; PTHR, PTH receptor; TM, transmembrane domain; J, juxtamembrane; Ac5c, aminocyclopentane-1-carboxylic acid; Aib, {alpha}-aminoisobutyric acid; Har, homoarginine; M, Gln10,Ala12, Har11,Trp14,Arg19; Bpa, para-benzoyl-L-phenylalanine; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HPLC, high pressure liquid chromatography; IBMX, 3-isobutyl-1-methylxanthine; IP, inositol phosphate; Deg, diethylglycine; CRF, corticotropin-releasing factor; h, human. Back

2 G. Segre, Massachusetts General Hospital, Boston, MA, personal communication. Back

3 The C-terminal Tyr and Met8 -> Nle (norleucine) modifications in this analog and in the [Deg1,3,Bpa2,M]PTH(1–21) analog were introduced to enable oxidative radioiodination as part of our still ongoing efforts to develop an N-terminal PTH antagonist radioligand. Back

4 In the experiments of Fig. 5A, this analog reduced cAMP signaling by PTHR-tether-PTH(1–9) by 30%, p < 0.0001 (data not shown). Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Juppner, H., Gardella, T., Kronenberg, H., and Potts, J., Jr. (2001) in Endocrinology (DeGroot, L., and Jameson, J., eds) Vol. 2, pp. 969–1053, W. B. Saunders Co., Philadelphia
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  5. Bergwitz, C., Gardella, T. J., Flannery, M. R., Potts, J. T., Jr., Kronenberg, H. M., Goldring, S. R., and Jüppner, H. (1996) J. Biol. Chem. 271, 26469–26472[Abstract/Free Full Text]
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