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Endocrinology Vol. 140, No. 11 4972-4981
Copyright © 1999 by The Endocrine Society


ARTICLES

Studies of the N-Terminal Region of a Parathyroid Hormone-Related Peptide(1–36) Analog: Receptor Subtype-Selective Agonists, Antagonists, and Photochemical Cross-Linking Agents1

Percy H. Carter, Harald Jüppner and Thomas J. Gardella

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

Address all correspondence and requests for reprints to: Thomas J. Gardella, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114. E-mail: gardella{at}helix.mgh.harvard.edu


    Abstract
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The N-terminal regions of PTH and PTH-related peptide (PTHrP) are involved in receptor-mediated signaling and subtype selectivity. To better understand the molecular basis for these processes, we first prepared a series of [I5,W23,Y36]-PTHrP(1–36)NH2 analogs having stepwise deletions of residues 1–4 and characterized them with the human (h)PTH-1 and hPTH-2 receptor subtypes stably transfected in LLC-PK1 cells. Deletions beyond residue 2 caused progressive and severe losses in cAMP-signaling efficacy without dramatically diminishing receptor-binding affinity; consistent with this, [I5,W23]-PTHrP(5–36) was a potent antagonist for both PTH receptor subtypes. We then prepared and characterized photolabile analogs of [I5,W23,Y36]-PTHrP(1–36)NH2 that were singly modified with para-benzoyl-L-phenylalanine (Bpa) along the first six residues. These full-length analogs exhibited receptor subtype-selective agonism, antagonism, and photochemical cross-linking profiles. In particular, the [Bpa2]- and [Bpa4]-substituted analogs selectively antagonized and preferentially cross-linked to the PTH-1 receptor and PTH-2 receptor, respectively. These results demonstrate that the 1–5 region of [I5,W23]-PTHrP(1–36) is critical for activating the PTH-1 and PTH-2 receptors and suggest that the individual residues in this region play distinct roles in modulating the activation states of the two receptors. The cross-linking of both agonist and antagonist ligands to these PTH receptors lays the groundwork for identifying critical signaling determinants in the ligand binding pocket of the receptor.


    Introduction
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
THE PTH/PTH-RELATED peptide receptor (PTH-1 receptor) is a member of the class II subfamily of G protein-coupled seven-transmembrane domain receptors that includes the receptors for secretin, calcitonin, and glucagon (1, 2, 3, 4). The PTH-1 receptor is well expressed in the kidney, bone, and growth-plate cartilage and, together with PTH and PTH-related peptide (PTHrP), serves important endocrine and paracrine functions that are essential to the maintenance of blood mineral ion homeostasis and skeletal development (5). Another member of the PTH/secretin/calcitonin family of receptors was recently identified, the PTH-2 receptor, the physiological role of which is not known (6). Unlike the PTH-1 receptor, the PTH-2 receptor responds to PTH but not to PTHrP (7).

For the PTH-1 receptor, the agonist peptides PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) bind with nanomolar affinity and stimulate robust increases (>20-fold) in intracellular cAMP. Previous studies using the PTH-1 receptor have shown that there are two distinct regions of functionality within these ligands; residues 15–34 contribute the majority of the binding energy (8, 9, 10), and residues 1–14 impart most of the stimulatory capacity to the molecule (11, 12). The regions of the PTH-1 receptor that are responsible for these effects have been broadly defined using receptor chimeras (13).

A separate line of work has investigated the molecular basis for the ligand-selectivity of the PTH-2 receptor. With this receptor, ligand residues 5 (Ile in PTH and His in PTHrP) (14) and 23 (Trp in PTH and Phe in PTHrP) were identified as important determinants for signaling and binding selectivity, respectively (15). Subsequent mutational studies have revealed several sites in the PTH-2 receptor that vary from the PTH-1 receptor sequence and play a role in determining ligand selectivity (16, 17, 18). Particularly relevant to the present study is the prior demonstration that the PTHrP analog [Ile5,Trp23,Tyr36]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)NH2 is a potent agonist for both receptor subtypes, exhibiting binding and signaling properties equal to those of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (15).

Several laboratories are currently employing photochemical cross-linking methods to map the topology of the ligand-receptor interface between PTH/PTHrP ligands and the PTH-1 receptor (19, 20, 21). Recently, Behar et al. (22) reported the first mapping of a photochemical cross-link between a PTH ligand and the PTH-2 receptor, and they concluded from this study that photolabile residues at positions 1 and 13 in the ligand contact identical sites in the PTH-1 receptor and PTH-2 receptor.

To expand the current body of data on the ligand-receptor interaction mechanisms for both the PTH-1 receptor and PTH-2 receptor, we prepared a series of analogs of [I5,W23,Y36]-hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)NH2 and characterized their interactions with each receptor subtype. Given the importance of the N-terminal region of the ligand in receptor activation, we focused on this region and introduced either: 1) stepwise deletions of residues 1–4; or 2) single substitutions of the photoreactive amino acid para-benzoyl-L-phenylalanine (Bpa) (23) at positions one through six. The functional and biochemical properties of these peptides reveal ligand-dependent and receptor-dependent profiles of agonism, antagonism, and photochemical cross-linking reactivity, which suggest that the PTH-1 receptor and PTH-2 receptor accommodate large, hydrophobic sidechain substitutions in the N-terminal portion of the ligand in dramatically different ways.


    Materials and Methods
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Peptides
[Nle8,18,Tyr34]-bPTH(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)NH2 {PTH(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)} and [Leu11,D-Trp12]-hPTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)NH2 were purchased from Bachem California (Torrance, CA). [Nle8,21,Tyr34]-rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)NH2 {PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)}, [Leu11,D-Trp12, Trp23]-hPTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)NH2 {PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)}, [Ile5,Trp23,Tyr36]-hPTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)NH2 {[I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) or Parent}, and all of the amino-terminally truncated and Bpa-substituted analogs of [Ile5,Trp23,Tyr36]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)NH2 {[I5,W23]-PTHrP(X-36) and Bpa-1 through Bpa-6; cf. Table 1Go} used in this study were prepared on a model 430A peptide synthesizer (PE Applied Biosystems, Norwalk, CT) using N-(9-fluorenyl)methoxycarbonyl (Fmoc)-protecting group chemistry. After global deprotection, each of these peptides was purified by HPLC, lyophilized, reconstituted in 10 mM acetic acid, and stored at -80 C. The purity, identity, and stock concentration of each compound were secured by analytical HPLC, mass spectrometry, and amino acid analysis. Radiolabeling was performed using 125I-Na (2,200 Ci/mmol, DuPont NEN) and chloramine-T; the resultant [125I-Tyr]-ligand was purified by HPLC.


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Table 1. Peptides utilized in this study

 
Cell culture
Stably transfected derivatives of the porcine kidney cell line LLC-PK1 were used for all experiments. The HKRKB7 LLC-PK1 cell line expresses the hPTH-1 receptor at approximately 1 x 106 receptors/cell (24), and the hPR2–20 LLC-PK1 cell line expresses the hPTH-2 receptor at approximately 0.8 x 106 receptors/cell (provided by H. Takasu and F. R. Bringhurst, Endocrine Unit, Massachusetts General Hospital). Cells were cultured in T-75 flasks (75 mm2) in DMEM supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified atmosphere containing 95% air and 5% CO2. Cells were subcultured in 24-well plates and, when confluent, were treated with fresh media and shifted to 33 C for 12–24 h before the assay (16, 25). Under these conditions, cell densities at the time of assay were 2.2 ± 0.1 and 2.3 ± 0.2 million cells per single well of a 24-well plate for the HKRKB7 and hPR2–20 cell lines, respectively (mean ± SE, recorded in triplicate at the time of assay on four separate occasions).

Binding assays
Binding reactions were performed with stably transfected LLC-PK1 cells in 24-well plates. Cells were rinsed with 0.5 ml binding buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 2 mM CaCl2, 5% heat-inactivated horse serum, 0.5% FBS, adjusted to pH 7.7 with HCl), and treated successively with 100 µl binding buffer, 100 µl binding buffer containing various amounts of unlabeled competitor ligand, and 100 µl binding buffer containing ca. 100,000 cpm of 125I-tracer (ca. 26 fmol; final vol = 300 µl). Incubations were at room temperature for 2 h, except for homologous binding experiments, which were performed at 4 C for 6 h to minimize effects of G-protein coupling and receptor internalization. Cells were then placed on ice, the binding medium was removed, and the monolayer was rinsed three times with 0.5 ml cold binding buffer. The cells were subsequently lysed with 0.5 ml 5N NaOH and counted for radioactivity. The nonspecific binding for each experiment was determined by competition with a 1-µM dose of unlabeled rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) or [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36). The maximum specific binding (B0) was the total radioactivity bound in the absence of unlabeled PTH ligand, corrected for nonspecific binding. Binding IC50 values were determined using nonlinear regression (see below).

cAMP stimulation
Stimulation of stably transfected LLC-PK1 cells was performed in 24-well plates. Cells were rinsed with 0.5 ml binding buffer and treated with 200 µl cAMP assay buffer (DMEM containing 2 mM 3-isobutyl-1-methylxanthine, 1 mg/ml BSA, 35 mM HEPES-NaOH, pH 7.4) and 100 µl of binding buffer containing varying amounts of peptide analog (final vol = 300 µl). The medium was removed after incubation for 1 h at room temperature, and the cells were frozen (-80 C), lysed with 0.5 ml 50 mM HCl, and refrozen (-80 C). The cAMP content of the diluted lysate was determined by RIA. Where possible, cAMP EC50 values were determined using nonlinear regression (see below).

Antagonism studies
The cAMP stimulation protocol described above was used for antagonist studies, with some minor modifications. Cells were rinsed with 0.5 ml binding buffer and treated successively with 100 µl binding buffer containing varying doses of the candidate antagonist peptide, 100 µl cAMP assay buffer, and 100 µl cAMP assay buffer containing varying doses of rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (final vol = 300 µl). Cells were incubated for 30 min at room temperature and processed as above. The [L11,D-W12,W23]-PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) control antagonist inhibited PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) agonist action with both PTH receptors (see Fig. 5Go), and with potency similar to that of the antagonist [L11, D-W12]-PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) described by Nutt et al. (26) (data not shown). The dose of an antagonist that inhibited the PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)-mediated cAMP response by 50% (IC50A) was calculated as described below.



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Figure 5. Analysis of Bpa-substituted [I5, W23]-PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ) analogs as candidate antagonists. The various Bpa-substituted [I5, W23]-PTHrP analogs indicated on the graph axis (each at 1-µM dose) were screened for their ability to antagonize a 3.3-nM dose of rPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ). A 1-µM dose of [L11, D-W12, W23]-PTHrP (7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ) was used as a positive control for antagonism. Stimulation of the PTH-1 receptor (A) and PTH-2 receptor (B) by PTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) was performed in the presence of the candidate antagonist peptides, as described in Materials and Methods. Shown are data (mean ± SE) combined from three individual experiments, each of which was performed in duplicate on a separate day. The basal cAMP values from unstimulated cells were less than 5 pmol/well and were not subtracted. The asterisks indicate that the PTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 )-induced response observed in the presence of an analog was significantly lower than the PTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 )-induced response observed in the absence of an added analog (none): *, P < 0.08; **, P = 0.001.

 
Data calculation
All calculations were performed using Excel (Microsoft Corp., Redmond, WA). Nonlinear regression analysis of binding and cAMP stimulation data were performed using four parameters, defined as the minimum (Min), maximum (Max), midpoint (IC50), and slope of the response curve. The predicted response (yp) for a given dose (x) of peptide was calculated using the following equation: yp = Min + [(Max - Min)/(1 + (IC50/x)slope)]. The initial parameter values were estimated from the primary data, and the Excel Solver function was then used to vary the four parameters to minimize the differences between the predicted and actual responses (least-squares method) (27). For cAMP EC50 calculations, the Max was not constrained to the maximum response observed (Maxobs), but was constrained to <=100. Hill coefficients were derived from homologous binding data by extracting the slope from the linear portion of the plot of log[B/(B0-B)] vs. log[competitor]. Antagonist IC50A values were calculated from the intercept of the linear portion of a plot of log[E/(Emax-E)] vs. log[Antagonist], where E and Emax are the cAMP responses observed in the presence and absence of antagonist, respectively. The statistical significance between two data sets was determined using a one-tailed Student’s t test, assuming unequal variances for the two sets.

Photochemical cross-linking
Photochemical cross-linking was carried out with stably transfected LLC-PK1 cell lines, which had been subcultured in 6-well plates; all manipulations were executed on ice using chilled reagents. Cells were rinsed with 2.0 ml binding buffer, treated with 2 ml binding buffer containing ca. 4 x 106 cpm (ca. 1 pmol) of a 125I-peptide, and incubated at 4 C for 6 h. The medium was removed, and the cells were rinsed twice with 1 ml binding buffer before being covered with 800 µl binding buffer. Cross-linking was induced by irradiation with a Black-Ray UV lamp (366 nM, 700 µW/cm2, source-to-cell = 4.5 cm) for 50 min on ice in a cold room (4 C). The medium was withdrawn, and the cells were rinsed twice with 2 ml of an acidic buffer (50 mM glycine, 150 mM NaCl, adjusted to pH 2.5 with HCl) and twice with 2 ml binding buffer. The cells were lysed with 250 µl of a Triton buffer (50 mM Tris-HCl, 10% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.05 mg/ml Bacitracin, 150 mM NaCl, pH 7.8) for 45 min at 0 C before being harvested (50 µl rinse with the Triton buffer) and centrifuged at 2000 x g for 20 min at 4 C. The supernatant was collected and stored at -20 C. Analysis of equivolume aliquots from these cross-linking reactions was accomplished using 5–20% SDS-PAGE (28) with subsequent autoradiography of the dried gel at -80 C with an intensifying screen.


    Results
 Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
N-terminal deletions
To compare the importance of the N-terminal region of an agonist peptide in mediating signal transduction with the PTH-1 receptor and PTH-2 receptor, we synthesized analogs of [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), which encompassed stepwise deletions from the N-terminus (Table 1Go). The excision of residues 1–4 did not have a pronounced deleterious effect on the ability of these analogs to inhibit the binding of 125I-rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) to either the PTH-1 or PTH-2 receptor stably transfected in LLC-PK1 cells (Fig. 1Go, A and B; Table 2Go). Indeed, the truncated analog [I5,W23]-PTHrP(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) exhibited a 2.3-fold higher apparent affinity for the PTH-1 receptor than did the Parent peptide (P = 0.0006), and [I5,W23]-PTHrP(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) showed a 1.5-fold higher apparent affinity for the PTH-2 receptor than did the full-length control (P = 0.007).



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Figure 1. Binding and cAMP-stimulation properties of [I5, W23]-PTHrP(X-36) analogs. A and B, Competition binding of the [I5, W23]-PTHrP(X-36) analogs (peptide lengths are indicated in the symbol key) to the PTH-1 receptor (A) and the PTH-2 receptor (B) stably expressed in LLC-PK1 cells was performed in the presence of 125I-rPTH(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ), as described in Materials and Methods. Shown are data (mean ± SE) combined from three individual experiments, each of which was performed in duplicate on a separate day. C and D, Stimulation of intracellular cAMP accumulation in LLC-PK1 cells expressing the PTH-1 receptor (C) and PTH-2 receptor (D) was performed as described in Materials and Methods. Shown are data (mean ± SE) combined from three individual experiments, each of which was performed in duplicate on a separate day. For some data points, the error bars are smaller than the symbols.

 

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Table 2. Competition binding and cAMP stimulation data for N-terminally truncated [Ile5,Trp23]-PTHrP analogs

 
In contrast to its effect on binding, stepwise removal of residues 1–4 progressively diminished the cAMP stimulation potency and efficacy of the [I5,W23]-PTHrP(X-36) analogs with both receptors (Fig. 1Go, C and D; Table 2Go). With the PTH-1 receptor, [I5,W23]-PTHrP (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) and [I5,W23]-PTHrP (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) showed 2.2-fold and 3.5-fold reductions in potency relative to the Parent peptide, respectively (P < 0.07 for each analog vs. Parent). The efficacy of [I5,W23]-PTHrP (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) was indistinguishable from that of the Parent peptide, whereas [I5,W23]- PTHrP (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) was a partial agonist with this receptor (Maxobs = 32% Parent, P = 0.003). Similar effects were observed with the PTH-2 receptor for deletion of ligand residues 1 and 2 (Fig. 1Go D). The shorter-length peptides, [I5,W23]-PTHrP (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) and [I5,W23]-PTHrP (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) elicited maximal cAMP responses with each receptor subtype that were at least 10-fold lower than that produced by [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36).

Bpa substitutions
To probe further the importance of the N-terminal region of [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) in mediating receptor activation, we prepared a series of photolabile analogs of [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) that were singly modified with Bpa along the first six residues (Table 1Go) and examined their functional and biochemical properties with both PTH receptors in LLC-PK1 cells. In competition binding assays conducted with the partial agonist 125I-bPTH(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) and the PTH-1 receptor, all of the Bpa-substituted peptides bound more weakly than did the Parent(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) (P < 0.03 for each analog IC50 vs. Parent IC50), and more than 20-fold reductions in apparent binding affinity were observed with Bpa-4 and Bpa-5 (Fig. 2AGo and Table 3Go). Smaller effects on binding potencies were seen with the PTH-2 receptor, as Bpa-1 and Bpa-5 bound with potencies that were not significantly different (P > 0.25) from that of the Parent ligand, and only Bpa-3 and Bpa-4 exhibited more than 2-fold reductions in apparent affinity, relative to the control peptide (Fig. 2BGo and Table 3Go). When the agonist radioligand 125I-rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) was used as the tracer, the binding profiles observed for these analogs with either receptor were similar to those seen in the competition studies performed with 125I-bPTH (3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), in that Bpa-4 and Bpa-5 exhibited the lowest apparent binding affinity for the PTH-1 receptor (IC50’s > 1000 nM, still representing a >20-fold shift from the Parent), and Bpa-3 and Bpa-4 exhibited the lowest apparent binding affinity for the PTH-2 receptor (but were still only 2-fold weaker than the Parent peptide) (Table 3Go). The binding IC50 values obtained with the agonist tracer tended to be higher than those measured with the partial agonist tracer.



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Figure 2. Binding properties of BPA-substituted [I5, W23]-PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ) analogs. A and B, Competition binding of the Bpa-substituted [I5, W23]-PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ) analogs to the PTH-1 receptor (A) and PTH-2 receptor (B) in the presence of 125I-bPTH (3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 ) was performed as described in Materials and Methods. Shown are data (mean ± SE) combined from three individual experiments, each of which was performed in duplicate on a separate day. C and D, Each curve represents homologous competition binding of a 125I-labeled Bpa-analog and the same noniodinated analog to the PTH-1 receptor (C) and PTH-2 receptor (D), as described in Materials and Methods. Shown are data (mean; SE < 10%) combined from three independent experiments.

 

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Table 3. Competition binding data for Bpa-substituted [Ile5,Trp23]-PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ) analogs

 
We also conducted binding experiments under more homologous conditions, wherein each radiolabeled analog was bound in the presence of varying doses of the same noniodinated peptide (Fig. 2Go, C and D). For the PTH-1 receptor, lower binding IC50 values were observed in the homologous binding experiments, compared with the values observed in the 125I-PTH(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)-based experiments, for Bpa-1 (2.7-fold, P = 0.002), Bpa-4 (2.8-fold, P = 0.006), and Bpa-5 (24-fold, P = 0.03). For the PTH-2 receptor, lower IC50 values were again noted for Bpa-1 (2.6-fold, P = 0.07) and Bpa-5 (2.8-fold, P = 0.06). The improvements in the apparent binding potencies of Bpa-1 and Bpa-5 with either receptor were even more pronounced when the homologous competition data were compared with the corresponding results obtained with 125I-rPTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Table 3Go). Finally, we noted that the slopes of the homologous binding curves obtained with the PTH-1 receptor were nonparallel, and in particular, Bpa-5 exhibited a shallower slope than did peptides of comparable affinity (e.g. Bpa-1 and Bpa-2; Fig. 2CGo). The Hill coefficient derived for Bpa-5 (0.9 ± 0.2) was significantly different from that of Bpa-1 (1.7 ± 0.03, P = 0.02) or Bpa-2 (1.5 ± 0.2, P = 0.04), perhaps indicating a change in binding cooperativity (29). In contrast, the slopes observed with the PTH-2 receptor were parallel (Fig. 2Go D), and the Hill coefficients ranged from 1.0–1.2.

The Bpa-substituted analogs exhibited striking differences in their ability to stimulate cAMP generation (Fig. 3Go and Table 4Go). Bpa-1 was a potent and efficacious agonist with both the PTH-1 receptor (EC50 = 8 ± 1 nM, Maxobs = 100% Parent) and PTH-2 receptor (EC50 = 17 ± 11 nM, Maxobs = 80% Parent). In contrast, Bpa-3 and Bpa-4 were weak partial agonists with both receptors (EC50 values > 85 nM and Maxobs < 35% Parent for each receptor). The remaining analogs exhibited receptor subtype- selective agonist properties. Bpa-5 was a ca. 4-fold more potent and 5-fold more efficacious agonist, relative to Parent, for the PTH-1 receptor than for the PTH-2 receptor, despite having a binding profile that dramatically favored the latter, particularly when assayed by heterologous competition (see Discussion). In contrast, Bpa-2 was an agonist/partial agonist with the PTH-2 receptor but was nearly inactive in the cAMP assays performed with the PTH-1 receptor (Maxobs = 10% Parent, P < 10-5). Bpa-6 was 2-fold more potent as an agonist with the PTH-2 receptor than with the PTH-1 receptor (EC50 values = 15 ± 10 and 34 ± 1 nM, respectively, P = 0.05), and elicited only a partial response with the latter receptor (Maxobs = 50% Parent, P < 10-5).



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Figure 3. cAMP stimulation with Bpa-substituted-[I5, W23]-PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ) analogs. Each panel shows the response of LLC-PK1 cells stably expressing either the PTH-1 receptor (•) or PTH-2 receptor ({triangleup}) to varying doses of a [I5, W23]-PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ) analog containing the Bpa substitution indicated above the graph. Assays were performed as described in Materials and Methods. Data shown (mean ± SE) are combined from three individual experiments, which were performed in duplicate on separate days. The ordinate values are expressed as a percent of the maximum cAMP response observed in parallel cells treated with [I5, W23]-PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ) (100 nM); for the three combined experiments, these maximum cAMP values were 129 ± 22 pmol/well for the PTH-1 receptor and 294 ± 39 pmol/well for the PTH-2 receptor. The corresponding basal cAMP values (not subtracted) were 1.5 ± 0.8 and 2.1 ± 1.0 pmol/well.

 

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Table 4. cAMP stimulation data for Bpa-substituted [Ile5,Trp23]-PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ) analogs

 
Photochemical cross-linking
The ability of each of the radioiodinated photolabile analogs to cross-link to the two PTH receptor subtypes was analyzed in stably transfected LLC-PK1 cells (Fig. 4Go). In most cases, a single band that migrated with the mobility expected for the glycosylated PTH-1 or PTH-2 receptor (~90,000 Da) was apparent by SDS-PAGE. This photoaffinity labeling was specific for the PTH receptors, as indicated by: 1) the ability of a 1-µM dose of noniodinated [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) to abolish each of the cross-links (data not shown); and 2) the difference in the mobilities of the bands obtained from the reactions performed with the PTH-1 receptor (593 amino acids) and its lower molecular weight PTH-2 receptor counterpart (550 amino acids). With the PTH-1 receptor, Bpa-1, Bpa-2, Bpa-3, and Bpa-5 each yielded strong band intensities; with the PTH-2 receptor, Bpa-1, Bpa-4, Bpa-5, and Bpa-6 yielded the strongest signals. Thus, as with the binding and cAMP signaling assays, receptor-selective effects were observed in the photochemical cross-linking of some of these analogs.



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Figure 4. Photochemical cross-linking of Bpa analogs to the PTH-1 and PTH-2 receptors. Shown is the cross-linking of the six radioiodinated Bpa-substituted [I5, W23]-PTHrP(1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 ) analogs to LLC-PK1 cells stably expressing either the PTH-1 or PTH-2 receptor. After radioligand binding, cells were collected and separated by SDS-PAGE (5–20% gradient), under reduced conditions, and visualized by autoradiography, as described in Materials and Methods. The position of the Bpa-substitution is indicated at the lane origins. The differential mobilities of the PTH-1 receptor and PTH-2 receptor complexes seen in the 97K region of the gel are consistent with the lower MW of the PTH-2 receptor (550 amino acids vs. 593 amino acids for the PTH-1 receptor). The autoradiograph shows a single experiment and is representative of two other experiments performed in similar fashion.

 
Antagonism of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)
The strong dissociation of apparent binding affinity and signaling potency observed for several of the Bpa-substituted [I5,W23]-PTHrP(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) analogs suggested that these compounds might act as competitive antagonists. This hypothesis was confirmed upon testing four of these analogs and a PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) control peptide (each at 1 µM) for the ability to inhibit the stimulation of the PTH-1 receptor or PTH-2 receptor by a half-maximal stimulatory dose (3.3 nM) of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (Fig. 5Go). This assay revealed that Bpa-2 and Bpa-4 were antagonists, but, in contrast to the truncated PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) analog, these full-length peptides exhibited signs of receptor-subtype selectivity. A similar analysis of [I5,W23]-PTHrP(3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), -(4–36), and -(5–36) revealed that [I5,W23]-PTHrP(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) more efficiently antagonized the actions of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) on both receptor subtypes than did the other two N-terminally truncated analogs (data not shown).

When tested against a full-range dose-response curve of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) with the PTH-1 receptor, a 1-µM dose of either Bpa-2 or [I5,W23]-PTHrP(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) increased the EC50 of the PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) response by 8- to 10-fold (Fig. 6AGo). Inhibition of the PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) response by the PTH-1 receptor partial agonist Bpa-4 was not detected with the PTH-1 receptor (Fig. 6AGo). The dose of peptide (IC50A) that achieved 50% inhibition of the response elicited by 3.3 nM PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) on the PTH-1 receptor was 0.32 ± 0.07 µM for Bpa-2 and 0.27 ± 0.08 µM for [I5,W23]-PTHrP(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) (Fig. 6CGo); these two peptides were both 10-fold more potent antagonists than [L11, D-W12, W23]-PTHrP(7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) (IC50A = 4.6 ± 1.0 µM, P = 0.02 vs. Bpa-2 or (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36), Fig. 6CGo). The maximum inhibition observed for Bpa-2 (65%, at 1 µM) differed significantly (P < 0.0001) from that attained by [I5,W23]-(5–36) (90% at 3 µM) (Fig. 6CGo). With the PTH-2 receptor, a 1-µM dose of either Bpa-4 or [I5,W23]-PTHrP(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) altered the response to only low doses of PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) (e.g. <10 nM) and did not alter the EC50 of the PTH(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34) dose-response curve (Fig. 6Go B). Although Bpa-2 was a weak or partial agonist with the PTH-2 receptor, it did not antagonize the actions of PTH(1, 2, 3, 4,