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J Biol Chem, Vol. 275, Issue 1, 9-17, January 7, 2000

Photoaffinity Cross-linking Identifies Differences in the Interactions of an Agonist and an Antagonist with the Parathyroid Hormone/Parathyroid Hormone-related Protein Receptor^*

Vered Behar, Alessandro Bisello, Gal Bitan, Michael Rosenblatt, and Michael Chorev^§

From the Division of Bone and Mineral Metabolism, Charles A. Dana and Thorndike Laboratories, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Analogs of parathyroid hormone (PTH)-related protein (PTHrP), singularly substituted with a photoreactive L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in human embryonic kidney cells stably expressing the recombinant human PTH/PTHrP receptor. Two of these analogs, in which the photoreactive residue is either in position 1 or 2 (Bpa¹- and Bpa²-PTHrP, respectively) displayed high affinity binding. Bpa¹-PTHrP also displayed high efficacy for the stimulation of increased cAMP levels. Surprisingly, Bpa²-PTHrP was found to be a potent antagonist, despite the presence of the principal activation domain (sequence 1-6). Analysis of the digestion profiles of the ligand-receptor photoconjugates revealed that both the agonist and the antagonist cross-link to the S-CH₃ group of Met⁴²⁵ in transmembrane domain 6 of the human PTH/PTHrP receptor. However, the antagonist Bpa²-PTHrP also cross-links to a proximal site within the receptor domain Pro⁴¹⁵-Met⁴²⁵. Unlike the antagonist Bpa²-PTHrP, the potent agonist Bpa²-PTH, also bearing the Bpa residue in position 2, cross-links only to the S-CH₃ group of Met⁴²⁵ (similar to Bpa¹-PTHrP and Bpa¹-PTH). Taken together, these results suggest that the antagonist Bpa²-PTHrP is able to distinguish between two distinct conformations of the receptor. The comparison between PTHrP analogs substituted by Bpa at two consecutive positions and across PTH and PTHrP reveals insights into the PTH/PTHrP ligand-receptor bimolecular interaction at the level of a single amino acid.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The parathyroid hormone (PTH)¹/PTH-related protein (PTHrP) receptor (PTH1R) is a G protein-coupled, seven-transmembrane domain-containing receptor (GPCR) activated equipotently by two distinct hormones, PTH and PTHrP (1, 2). Although acting through the same receptor, these hormones have different physiological functions: PTH is a regulator of blood calcium levels (3), whereas PTHrP is an autocrine/paracrine factor involved in skeletal and cartilage development (4, 5). The PTH1R is a member of the glucagon/secretin/calcitonin subfamily of GPCRs (2). It is coupled to both adenylyl cyclase (AC)/cyclic AMP and phospholipase C/inositol 1,4,5-trisphosphate/cytosolic calcium intracellular signaling pathways (6-8).

Extensive efforts have been focused on investigating the structural basis for hormone recognition by PTH1R. These lines of research have identified regions in both the ligands and the receptor required for binding and signaling. On one hand, the mutagenesis approach, generating chimeric, mutated, or truncated receptors, has established that multiple receptor domains are involved in the complex interaction with the ligands, including the N-terminal extracellular tail, extracellular loops, and transmembrane domains (TMDs) (9-11). On the other hand, structure-function studies of the hormones and their analogs have revealed structural determinants required for interaction with the receptor. These studies have identified the C-terminal region of PTH-(1-34) and PTHrP-(1-34) as the principal binding domain (12-14), which is also required for activation of protein kinase C (15, 16). The N-terminal 1-6 sequence of either ligand was found to function as the principal activation domain (Ref. 17 and references therein).

The complementary biochemical approach of photoaffinity cross-linking has been utilized to examine directly ligand-receptor bimolecular interactions through the generation of covalently linked radiolabeled ligand-receptor photoconjugates. These studies identified three contact sites in PTH1R: position 1 in PTH interacts with Met⁴²⁵ (18), position 13 in PTH interacts with the sequence [Glu¹⁸²-Met¹⁸⁹] (19, 20), and position 23 in PTHrP interacts with the sequence 23-40 (21). Thus, the N-terminal part of PTH interacts with TMD 6 of the receptor near the cell surface and extracellular loop 3, the mid-region (position 13) with the receptor at the N-terminal extracellular tail-juxtamembrane region, and position 23 of PTHrP with the extreme N terminus of the receptor. Further data generated by this approach holds the promise of developing an experimentally based model of the hormone-receptor interface (18).

Here, we report the evaluation of a series of photoreactive analogs of PTHrP in order to probe the nature of receptor interaction with the principal activation domain of the hormone (residues 1-6). The receptor "contact domains" for two p-benzoylphenylalanine (Bpa)-containing radiolabeled PTHrP analogs, one an agonist and the other a potent antagonist, were identified and compared with the cross-linking sites of the corresponding PTH analogs. The data provide a unique opportunity to obtain insights into potential differences between the modes of interaction of agonists versus antagonists with the PTH1R.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Boc-protected amino acids, N-hydroxybenzotriazole, N,N'-dicyclohexylcarbodiimide, and p-methylbenzhydrylamine resin were purchased from Applied Biosystems (Foster City, CA). Boc-(3-iodo)tyrosine[O-(3-BrBz)] was purchased from Peninsula Laboratories (Belmont, CA). B&J brand dichloromethane, N-methylpyrrolidone and acetonitrile were obtained from Baxter (McGraw Park, IL). Iodogen® and 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole) were purchased from Pierce. Cyanogen bromide (CNBr) and Vydac 218TP C₁₈ silica were from Aldrich. Na¹²⁵I was obtained from Amersham Pharmacia Biotech. Endoglycosidase F/N-glycosidase F (Endo-F), lysyl endopeptidase (Lys-C) and FuGENE^TM 6 transfection reagent were purchased from Roche Molecular Biochemicals. Dulbecco's modified Eagle's medium, fetal bovine serum, Opti-MEM® I, and phosphate-buffered saline were obtained from Life Technologies, Inc. Tissue culture disposables and plasticware were obtained from Corning (Corning, NY). All other reagents were purchased from Sigma.

Peptide Synthesis-- All peptides were synthesized by solid-phase methodology with an Applied Biosystems 430A peptide synthesizer using Boc/N-hydroxybenzotriazole/N-methylpyrrolidone chemistry. After hydrogen fluoride cleavage, the peptides were purified by preparative reverse-phase high performance liquid chromatography (HPLC) (22). [Bpa²,Ile⁵,Arg^11,13,3-I-Tyr³⁶]PTHrP-(1-36)NH₂ (I-Bpa²-PTHrP) was synthesized by the same methodology using Boc-(3-iodo)tyrosine[O-(3-BrBz)]. Purity exceeded 97% as determined by analytical reverse-phase HPLC. Structural integrity of the peptides was confirmed by amino acid analysis and electrospray mass spectrometry.

Radioiodinations-- The peptides [Ile⁵,Arg^11,13,Tyr³⁶]PTHrP-(1-36)NH₂ [PTHrP-(1-36)], [Bpaⁿ,Ile⁵,Arg^11,13,Tyr³⁶]PTHrP-(1-36)NH₂ (n = 1, 2, 6) (Bpaⁿ-PTHrP), and [Bpa²,Nle^8,18,Arg^13,26,27,Nal²³,Tyr³⁴]bPTH-(1-34)NH₂ (Bpa²-PTH) were radioiodinated and purified by reverse-phase HPLC as described previously (23), but with the following modification: all iodination reactions were continued for 12 min. The iodination reactions of the PTHrP-based analogs were stopped by the addition of acetic acid (final concentration, 20% (v/v)), because the iodinated peptides were found to have low solubility in aqueous solution.

Cell Culture-- HEK-293 cells and HEK-293/C-21 cells stably expressing PTH1R (~400,000 receptors/cell) and COS-7 cells transiently expressing wild-type and mutant PTH1Rs were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum as described (24).

Radioreceptor Binding Assay-- HEK-293/C-21 cells were subcultured in 24-well plates and grown to confluency. Binding assays were carried out as described (22) using ¹²⁵I-[Ile⁵,Arg^11,13,Tyr³⁶]PTHrP-(1-36)NH₂ (¹²⁵I-PTHrP) as the radioligand. Radioreceptor binding assays with the radioiodinated ¹²⁵I-Bpaⁿ-PTHrP (n = 1, 2, or 6) were carried out in a similar fashion.

Adenylyl Cyclase Activity-- HEK-293/C-21 cells were subcultured in 24-well plates and grown to confluency. Activation of AC by PTHrP analogs was carried out as described (22). Antagonist activity was tested by measuring the inhibition of 50 nM PTHrP-stimulated AC activation following a preincubation period of 15 min with the antagonist at 37 °C.

Photoaffinity Cross-linking and Membrane Protein Preparation-- Photoaffinity cross-linking (preparative and analytical scales) and membrane protein preparation of ¹²⁵I-Bpa¹-PTHrP, ¹²⁵I-Bpa²-PTHrP, and ¹²⁵I-Bpa²-PTH photoconjugates with the PTH1R were carried out as described (18, 19).

Enzymatic and Chemical Digestions of the Ligand-Receptor Conjugates-- Samples of the isolated SDS-PAGE bands representing either the radiolabeled hormone-receptor conjugate or conjugated fragments were prepared in small volumes (typically 10-20 µl) of 25 mM Tris-HCl (pH 8.5) Triton X-100 (0.1% (v/v)), SDS (0.01% (w/v)). Endo-F digestions were carried out at 37 °C for 24 h, according to the manufacturer's procedure. Lys-C digestions were performed by treatments with 0.15 units of enzyme (in 10 µl water) in 25 mM Tris-HCl (pH 8.5), Triton X-100 (0.1% (v/v)), SDS (0.01% (w/v)) at 37 °C for 24 h. BNPS-skatole cleavage was carried out with 100 µl of 1 mg/ml (final concentration) in 70% acetic acid at room temperature for 24 h in the dark under N₂. Samples were dried on a Speed-Vac and dissolved in reducing sample buffer (25) prior to PAGE analysis.

CNBr digestions were performed either in solution or on a solid support (26). In solution, digestions were performed with a small crystal of CNBr in 70% formic acid at 37 °C for 24 h in the dark under N₂ (19). On a solid support, samples of the isolated SDS-PAGE bands representing the receptor conjugates were adsorbed onto C₁₈-derivatized silica gel (approximately 5000 cpm/10 mg of silica) that was prewashed with 0.1% trifluoroacetic acid in acetonitrile (v/v) (solvent B) followed by 0.1% trifluoroacetic acid in water (v/v) (solvent A). After loading the radiolabeled ligand-receptor conjugate, the silica gel was washed consecutively with 10 gel volumes of solvent A, solvent B, and solvent A. The silica gel was then equilibrated by washing twice with 10 gel volumes of 0.1 M HCl followed by the addition of 2 gel volumes of CNBr to a final concentration of 80 mg/ml in 0.1 M HCl. After overnight incubation at 37 °C in the dark under N₂, the silica gel was washed twice with 10 gel volumes of solvent A to remove the CNBr. CNBr-generated fragments were eluted from the silica gel with 40-50% solvent B in A, monitoring the elution profile by the release of the radioactivity from the column.

Electrophoresis and Autoradiography-- Electrophoretic analyses were performed using 7.5% (w/v) SDS-PAGE for the intact and deglycosylated hormone-receptor conjugates and 16.5% (w/v) Tricine/SDS-PAGE for the cleavage products (19). Appropriate molecular weight markers (Amersham Pharmacia Biotech) were included in each gel. Gels were dried and exposed to x-ray films (X-omat, Eastman Kodak Co.) with intensifying screens (XAR-5, Kodak). Following autoradiography, the radioactive bands were excised from the dried gels, electroeluted (Bio-Rad, Electroeluter model 422) in SDS-PAGE running buffer, and concentrated on a Speed-Vac.

Receptor Mutagenesis-- The generation and biological characterization (binding and AC assays) of two single mutations in the PTH1R cDNA sequence, M414L and M425L, was described previously (18).

Transient Transfection-- COS-7 cells were plated at 65,000 cells/well in 24-well dishes, 24 h prior to transient transfections. Six hundred ng of empty vector, mutant, or wild-type receptor cDNA constructs were transfected using 1.8 µl of FuGENE^TM 6 (Roche Molecular Biochemicals) transfection reagent per well. Photoaffinity cross-linking, radioreceptor binding, and AC activity assays were carried out 48 h after transfection, as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of Bpa-containing PTHrP-(1-36) Analogs-- A series of photoreactive analogs of PTHrP-(1-36), singularly substituted with a Bpa at each of the first six N-terminal positions was prepared (Fig. 1A). Lys¹¹ and Lys¹³ in these analogs were replaced by Arg residues to render the ligands resistant to enzymatic cleavage by Lys-C, as required by the mapping scheme. In addition, analogs Bpa^1-4- and Bpa⁶-PTHrP carry the modification His⁵  Ile, which markedly enhances PTHrP interaction with the type 2 PTH receptor (PTH2R) subtype (27, 28). Preliminary experiments established that the parent compound of this series, [Ile⁵,Arg^11,13,Tyr³⁶]PTHrP-(1-36)NH₂ [PTHrP-(1-36)], was equipotent with [Tyr³⁶]PTHrP-(1-36)NH₂, as well as [Nle^8,18,Tyr³⁴]bPTH-(1-34)NH₂ in receptor binding and AC activation assays (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   A, schematic presentation of PTHrP and PTH and their Bpa-containing analogs. The position of Bpa in each of the analogs is shaded. All peptides have a free N terminus and a carboxamide at the C terminus. B, Bpa; X, norleucine; Z, 2-naphtylalanine; Ag, agonist; Ant, antagonist. B-D, in vitro characterization of the Bpa-containing PTHrP-(1-36) analogs in HEK-293/C-21 cells stably expressing the recombinant human PTH1R. Shown are competition for 125I-[Ile5,Arg11,13,Tyr36]PTHrP-(1-36)NH2 [125I-PTHrP-(1-36)] binding (B) and dose-response curves for the stimulation of AC activity (C) in PTH1R-expressing cells by PTHrP-(1-36) (), Bpa1-PTHrP (), Bpa2-PTHrP (×), Bpa3-PTHrP (), Bpa4-PTHrP (), Bpa5-PTHrP () and Bpa6-PTHrP (). Note that × (Bpa2-PTHrP) is obscured by the open triangles (Bpa6-PTHrP) or the open circles (Bpa1-PTHrP) in B. Data are expressed as the percentage of total binding (B) or maximal stimulation (C) relative to the control PTHrP-(1-36) peptide. D, antagonistic activity of Bpa2-PTHrP () and I-Bpa2-PTHrP () of PTHrP-stimulated AC activation. Data are presented as percentage of inhibition of PTHrP-stimulated AC activation at a dose of 50 nM PTHrP-(1-36). Data are the mean ± S.E. of triplicate wells from one experiment. Similar results were obtained in two additional independent experiments.

The Bpa-containing PTHrP analogs were pharmacologically evaluated in HEK-293 cells stably expressing the PTH1R (~400,000 receptors/cell, HEK-293/C-21) (24). Binding affinity was measured by competition with ¹²⁵I-[Ile⁵,Arg^11,13,Tyr³⁶] PTHrP-(1-36)NH₂ (¹²⁵I-PTHrP) (Fig. 1B). Agonist activity (stimulation of AC) was determined in HEK-293/C-21 cells (Fig. 1C). The binding affinities of Bpa¹-, Bpa²-, and Bpa⁶-PTHrP were similar to that of the parent compound (IC₅₀ = 41 nM for PTHrP-(1-36) and Bpa¹-PTHrP and 32 nM for Bpa²- and Bpa⁶-PTHrP) (Fig. 1B). Bpa¹-PTHrP was equipotent to PTHrP-(1-36) in stimulating AC activation (EC₅₀ = 4 and 8 nM for PTHrP and Bpa¹-PTHrP, respectively) (Fig. 1C). The analogs Bpa⁶- and Bpa²-PTHrP, despite of having high affinity binding, displayed weak and negligible efficacies, respectively: at 1 µM, Bpa⁶-PTHrP achieved only ~30% AC stimulation compared with the parent peptide, and Bpa²-PTHrP reached a maximal response of <10%. The high affinity and negligible efficacy of Bpa²-PTHrP suggested that it might be an effective antagonist of PTH1R. Indeed, Bpa²-PTHrP was found to be a potent antagonist of PTHrP-stimulated AC activation for PTH1R (IC₅₀ = 10 nM) on HEK-293/C-21 cells (Fig. 1D).

Substitution at position 3 led to an analog with a markedly reduced binding affinity (IC₅₀ = 310 nM), whereas substitution at positions 4 and 5 essentially abrogated binding.

The high affinity analogs, Bpa¹-, Bpa²-, and Bpa⁶-PTHrP, were further evaluated. Following radioiodination, ¹²⁵I-Bpa¹- and ¹²⁵I-Bpa²-PTHrP exhibited full binding capacity, whereas ¹²⁵I-Bpa⁶-PTHrP lost more than 50% of its total binding to PTH1R compared with ¹²⁵I-PTHrP-(1-36) (not shown). The ability of nonradioactive iodinated Bpa²-PTHrP to antagonize PTHrP-stimulated AC activation was also examined. Fig. 1D shows that I-Bpa²-PTHrP indeed maintains antagonistic effects similar to the noniodinated Bpa²-PTHrP.

Photoaffinity Labeling of PTH1R with ¹²⁵I-Bpa-containing Analogs of PTHrP-(1-36)-- Photocross-linking of either ¹²⁵I-Bpa¹- or ¹²⁵I-Bpa²-PTHrP to PTH1R generated a single diffuse band migrating at ~90 kDa, as analyzed by 7.5% SDS-PAGE (Fig. 2, lanes 2 and 6 for ¹²⁵I-Bpa¹- and ¹²⁵I-Bpa²-PTHrP, respectively). However, radioiodination of Bpa⁶-PTHrP resulted in a substantially reduced photocross-linking efficiency (Fig. 2, lane 10), comparable to the loss in receptor binding following the radioiodination. The ligand-receptor photoconjugates represented by the ~90-kDa bands are receptor-specific, as they are not observed in nontransfected parental HEK-293 cells (Fig. 2, lanes 1, 5, and 9 for ¹²⁵I-Bpaⁿ-PTHrP, n = 1, 2 and 6, respectively). Moreover, formation of the ligand-receptor conjugates was inhibited by the presence of excess (1 µM) unlabeled agonist PTH-(1-34) (Fig. 2, lanes 3, 7, and 11 for ¹²⁵I-Bpaⁿ-PTHrP, n = 1, 2 and 6, respectively) or PTHrP-(1-36) (Fig. 2, lanes 4, 8, and 12 for ¹²⁵I-Bpaⁿ-PTHrP, n = 1, 2 and 6, respectively). These data suggested that the agonist Bpa¹-PTHrP and the antagonist Bpa²-PTHrP should be good candidates for detailed analysis of their cross-linking contact sites on PTH1R.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Autoradiography of photoaffinity cross-linking of 125I-Bpan- PTHrP analogs (n = 1, 2 or 6) to HEK-293/C-21 cells stably expressing the recombinant PTH1R. Nontransfected HEK-293 cells (lanes 1, 5, and 9 for 125I-Bpan-PTHrP, n = 1, 2, and 6, respectively) or PTH1R-expressing HEK-293/C-21 cells were photolabeled with 125I-Bpan-PTHrP (lanes 2, 6, and 10 for n = 1, 2, and 6, respectively) and in the presence of 1 µM PTH-(1-34) (lanes 3, 7, and 11 for n = 1, 2, and 6, respectively) or PTHrP-(1-36) (lanes 4, 8, and 12 for n = 1, 2, and 6, respectively). The arrow indicates the position of the ~90-kDa bands, representing the photolabeled ligand-receptor conjugates. The band at ~66 kDa corresponds to variable degrees of nonspecific cross-linking, probably to bovine serum albumin, and was observed in all experiments. Samples were analyzed by 7.5% (w/v) SDS-PAGE. Molecular weight markers are shown.

Identification of the Cross-linking Contact Sites of the Agonist ¹²⁵I-Bpa¹- and the Antagonist ¹²⁵I-Bpa²-PTHrP on PTH1R-- The ~90-kDa bands corresponding to either ¹²⁵I-Bpa¹- or ¹²⁵I-Bpa²-PTHrP-PTH1R conjugates were isolated from 7.5% SDS-PAGE and subjected to a series of chemical and enzymatic cleavages. One digestion pathway consisted of enzymatic digestion with Lys-C at the carboxyl side of lysyl residues, followed by chemical cleavage with BNPS-skatole at the carboxyl side of tryptophanyl residues. Exhaustive Lys-C treatment of the ~90-kDa ligand-receptor conjugates of either ¹²⁵I-Bpa¹- or ¹²⁵I-Bpa²-PTHrP yielded single radiolabeled bands with similar apparent molecular masses of ~12 kDa, as analyzed by 16.5% Tricine/SDS-PAGE (Fig. 3, A, lane 1, and C, lane 1, for ¹²⁵I-Bpa¹- and ¹²⁵I-Bpa²-PTHrP, respectively). Similar treatment of the deglycosylated conjugates (~60 kDa) yielded fragments with the same apparent molecular masses (not shown), indicating the absence of glycosylation sites within the Lys-C-generated fragment. BNPS-skatole treatment of the excised and eluted ~12-kDa bands produced a single band for each radioligand with similar apparent masses of ~7-8 kDa (Fig. 3, A, lane 2, and C, lane 2, for ¹²⁵I-Bpa¹- and ¹²⁵I-Bpa²-PTHrP, respectively).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Autoradiographs of the chemical and enzymatic digestions of 125I-Bpa1- and 125I-Bpa2-PTHrP-PTH1R conjugates. A, Lys-C treatment of the ~90-kDa 125I-Bpa1-PTHrP-PTH1R conjugate (lane 1). The excised and eluted Lys-C-derived ~12-kDa fragment was then treated with BNPS-skatole, yielding a ~7-8-kDa fragment (lane 2). The reciprocal treatment pathway is shown in lanes 3 and 4. BNPS-skatole treatment of the intact conjugate yielded a ~13-14-kDa fragment (lane 3). The excised and eluted BNPS-skatole-derived ~13-14-kDa fragment was then treated with Lys-C, yielding a ~7-8-kDa fragment (lane 4). B, CNBr digestion of the intact 125I-Bpa1-PTHrP-PTH1R conjugate (lane 1). Also shown is the 125I-Bpa1-PTHrP ligand (molecular weight, 4645) (lane 2). C, Lys-C treatment of the isolated ~90-kDa 125I-Bpa2-PTHrP-PTH1R conjugate (lane 1). The excised and eluted Lys-C-derived ~12-kDa fragment was then treated with BNPS-skatole, yielding a ~7-8-kDa fragment (lane 2). The reciprocal treatment pathway is shown in lanes 3 and 4. BNPS-skatole treatment of the intact conjugate yielded a ~13-14-kDa fragment (lane 3). The excised and eluted BNPS-skatole-derived ~13-14-kDa fragment was then treated with Lys-C, yielding a ~7-8-kDa fragment (lane 4). D, CNBr digestion of the intact 125I-Bpa2-PTHrP-PTH1R conjugate yielded two fragments, one at ~4.5 kDa, similar to the one obtained for 125I-Bpa1-PTHrP-receptor conjugate (B, lane 1), and the other at ~6 kDa. Samples were analyzed by 16.5% (w/v) Tricine/SDS-PAGE. Molecular masses of the fragments are indicated in kDa and represent the actual size of the digested conjugated fragments, including the ligand 125I-Bpa1- (molecular weight, 4645) or 125I-Bpa2-PTHrP (molecular weight, 4618). Size markers (in kDa) are shown on the side of each panel. Arrows indicate the position of the ligand-receptor conjugated fragments.

The second digestion pathway, the reciprocal of the first one, yielded upon BNPS-skatole treatment of each of the intact hormone-receptor conjugate a band migrating at ~13-14 kDa (Fig. 3, A, lane 3, and C, lane 3, for ¹²⁵I-Bpa¹- and ¹²⁵I-Bpa²-PTHrP, respectively). Lys-C treatment of the isolated ~13-14-kDa bands yielded ~7-8-kDa fragments for either conjugate, similar to the conjugated fragments obtained from the first digestion pathway (Fig. 3, A, lane 4, and C, lane 4; compare with lane 2).

The isolated radioactive bands corresponding to the intact hormone-receptor conjugates were treated also with cyanogen bromide (CNBr). For ¹²⁵I-Bpa¹-PTHrP-PTH1R conjugate, this treatment generated a single band with an apparent mass of ~4.5 kDa (Fig. 3B, lane 1), similar to the gel mobility of the free ligand ¹²⁵I-Bpa¹-PTHrP (Fig. 3B, lane 2). CNBr treatment of the ¹²⁵I-Bpa²-PTHrP-PTH1R conjugate yielded two bands with apparent masses of ~6 and ~4.5 kDa (Fig. 3D). The later band had a gel mobility similar to the band obtained by CNBr treatment of the ¹²⁵I-Bpa¹-PTHrP-PTH1R conjugate. The size of the ~6-kDa band was not further reduced by retreatment with CNBr to achieve exhaustive digestion, suggesting that this band represents the minimal sized CNBr-generated fragment. In addition, subsequent treatment with either Lys-C or BNPS-skatole did not reduce the size of this fragment, suggesting the absence of cleavage sites for these reagents (not shown).

Characterization of Transfected COS-7 Cells Expressing Mutated PTH1R-- We used two single point-mutated PTH1Rs, Met⁴¹⁴  Leu (M414L) and Met⁴²⁵  Leu (M425L), which eliminate either one of two CNBr cleavage sites within the minimal cleavage restricted domain Ser⁴⁰⁹-Trp⁴³⁷ containing the putative contact site for Bpa¹-PTH (contact domain) (18). These mutated receptors maintain biological properties similar to the wild-type receptor when transiently expressed in COS-7 cells (18): the AC activation profiles of wild-type receptor, mutant M414L, and mutant M425L in response to PTHrP-(1-36) were comparable (Fig. 4A). The antagonistic activity of Bpa²-PTHrP was observed in transiently expressed native and mutated receptors (Fig. 4B); the dose-inhibition of PTHrP-(1-36)-stimulated AC curves were similar to those obtained in the HEK-293/C-21 cells stably expressing PTH1R (Fig. 1D).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Characterization of COS-7 cells transiently expressing native and single point-mutated PTH1R. A, stimulation of AC by PTHrP-(1-36) in COS-7 cells transiently expressing native (), M414L (), and M425L (×) receptors. B, antagonistic activity of Bpa2-PTHrP of PTHrP-stimulated AC activation in COS-7 cells transiently expressing native (), M414L (), and M425L (×) receptors. Data are presented as percentage of inhibition of PTHrP-stimulated AC activation at a dose of 50 nM PTHrP-(1-36). Data are the mean ± S.E. of triplicate wells from one experiment. Similar results were obtained in two additional independent experiments. C and D, autoradiographs of photoaffinity cross-linking of the wild-type PTH1R and the point-mutated receptors, M414L and M425L. 125I-Bpa1-PTHrP (C) and 125I-Bpa2-PTHrP (D) were photocross-linked to COS-7 cells transiently expressing the native PTH1R (lanes 1 and 2), mutated M414L receptor (lanes 3 and 4), and mutated M425L receptor (lanes 5 and 6), in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of 1 µM unlabeled PTHrP-(1-36). Size markers (in kDa) are shown on the side of each panel. Arrows indicate the position of the ligand-receptor conjugates.

The transiently expressed wild-type and mutated receptors were photocross-linked to either ¹²⁵I-Bpa¹- or ¹²⁵I-Bpa²-PTHrP (Fig. 4, C and D, respectively). Photoaffinity cross-linking of ¹²⁵I-Bpa¹-PTHrP to either wild-type or M414L mutant generated a single band migrating at the expected mass of ~90 kDa (Fig. 4C, lanes 1 and 3 for wild-type and M414L mutant, respectively), which was competed by 1 µM unlabeled PTHrP- (1-36) (Fig. 4C, lanes 2 and 4). These bands are not observed in mock-transfected parental COS-7 "control" cells (not shown). In contrast, the functional M425L receptor mutant failed to photocross-link to ¹²⁵I-Bpa¹-PTHrP (Fig. 4C, lanes 5 and 6).

Cross-linking of ¹²⁵I-Bpa²-PTHrP to wild-type or M414L also generated a band at ~90 kDa (Fig. 4D, lanes 1 and 3 for wild-type and M414L mutant, respectively), which was competed by 1 µM unlabeled PTHrP-(1-36) (Fig. 4D, lanes 2 and 4 for wild-type and M414L mutant, respectively). However, cross-linking of ¹²⁵I-Bpa²-PTHrP to M425L mutant, under identical conditions, generated a weak radioactive band (Fig. 4D, lane 5), which was competed by 1 µM unlabeled PTHrP-(1-36) (Fig. 4D, lane 6).

Characterization of the Cross-linking Contact Domain of ¹²⁵I-Bpa²-PTH with PTH1R-- Cross-linking of the PTHrP-derived agonist and antagonist indicates an overlapping band (~4.5 kDa), as well as a distinct band (~6 kDa) for the antagonist Bpa²-PTHrP. This result may be attributed to differences between the binding modes of the agonist (Bpa¹-PTHrP) and the antagonist (Bpa²-PTHrP) or between the two consecutive positions in the PTHrP sequence (Bpa¹ versus Bpa²). In order to distinguish between these two possibilities, we examined the analog [Bpa²,Nle^8,18,Arg^13,26,27,Nal²³,Tyr³⁴]bPTH-(1-34)NH₂ (Bpa²-PTH). Similarly to Bpa²-PTHrP, this PTH-derived analog carries the photoreactive amino acid in position 2; but unlike its PTHrP counterpart, it is a potent agonist of PTH1R (IC₅₀ = 75 nM, EC₅₀ = 20 nM) (18).

Cross-linking of ¹²⁵I-Bpa²-PTH to PTH1R generated a radiolabeled band migrating at ~90 kDa, corresponding to the intact hormone-receptor conjugate (not shown). Subsequent CNBr-cleavage yielded a single band migrating at ~4.5 kDa (Fig. 5A, lane 2), similar to the mobility of the ligand ¹²⁵I-Bpa²-PTH (molecular weight, 4460) (Fig. 5A, lane 3). This result therefore suggests that all three agonists, Bpa¹-PTHrP, Bpa¹- (18), and Bpa²-PTH, interact with PTH1R in a very similar manner. In order to analyze the receptor interactions of ¹²⁵I-Bpa²-PTH in greater details, we cross-linked it to the mutants M414L and M425L.

View larger version (65K): [in this window] [in a new window]   Fig. 5.   Characterization of cross-linking of 125I-Bpa2-PTH to PTH1R and mutated receptors. A, 125I-Bpa2-PTH was photocross-linked to HEK-293/C-21 cells stably expressing the PTH1R. The intact 125I-Bpa2-PTH-PTH1R conjugate was isolated and then incubated in the absence (lane 1) or presence (lane 2) of CNBr. Also shown is the 125I-Bpa2-PTH ligand (molecular weight, 4460) (lane 3). B, photoaffinity cross-linking of wild-type PTH1R and point mutated receptors M414L and M425L, transiently expressed in COS-7 cells, to 125I-Bpa2-PTH. 125I-Bpa2-PTH was cross-linked to COS-7 cells transiently transfected with an empty vector (lane 9), the wild-type PTH1R (lanes 1 and 2), mutated M414L receptor (lanes 3 and 4), and mutated M425L receptor (lanes 5, and 6) in the absence (lanes 1, 3, 5, and 9) or presence (lanes 2, 4, and 6) of 1 µM unlabeled PTH-(1-34). The receptor mutant M425L was also cross-linked to 125I-K13 (lanes 7 and 8), a photoreactive analog of PTH that contacts a distinct site in the receptor (see text), in the absence (lane 7) or presence (lane 8) of 1 µM unlabeled PTH-(1-34). Samples were analyzed by 7.5% SDS-PAGE. Size markers (in kDa) are shown on the side of each panel. Arrows indicate the position of the ligand-receptor conjugate or conjugated fragment.

Characterization of Transfected COS-7 Cells Expressing Mutated PTH1R with ¹²⁵I-Bpa²-PTH-- The transiently expressed wild-type and mutated receptors were photocross-linked to ¹²⁵I-Bpa²-PTH (Fig. 5B). Photoaffinity cross-linking of ¹²⁵I-Bpa²-PTH to either wild-type or M414L mutant generated a single diffuse band migrating at the expected mass of ~90 kDa (Fig. 5B, lanes 1 and 3 for wild-type and M414L mutant, respectively), which was inhibited competitively by 1 µM unlabeled PTH-(1-34) (Fig. 5B, lanes 2 and 4). This band was not observed in mock-transfected parental COS-7 cells (Fig. 5B, lane 9). Similarly to ¹²⁵I-Bpa¹-PTHrP, ¹²⁵I-Bpa²-PTH also failed to photocross-link to the M425L receptor mutant (Fig. 5B, lanes 5 and 6). However, ¹²⁵I-K13, a PTH analog modified with a photoreactive moiety at position 13, and known to cross-link to the N-terminal extracellular domain of the wild-type receptor (19, 20), did cross-link to M425L, generating the ~90-kDa band that corresponds to the ligand-receptor conjugate (Fig. 5B, lane 7). This cross-linking was inhibited competitively by 1 µM unlabeled PTH-(1-34) (Fig. 5B, lane 8).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Understanding the nature of the ligand-receptor interface is of fundamental importance for elucidating the principles of molecular recognition and the molecular mechanisms underlying receptor selectivity and activation. In this study, the cross-linking sites to the PTH1R of two photoreactive PTHrP analogs (substituted with Bpa in positions 1 and 2) and one PTH analog (substituted in position 2) were examined. In a preliminary step, a series of analogs of PTHrP-(1-36), containing Bpa substituted at positions in the N-terminal "activation" domain and homologous to the series previously described for PTH-(1-34) (18), was prepared and characterized for their biological activity. Intriguingly, Bpa²-PTHrP is a potent antagonist (IC₅₀ = 10 nM) of PTHrP-stimulated cAMP accumulation. Notably, very few analogs containing the activation domain of PTH-(1-34) or PTHrP-(1-34) (sequence positions 1-6) have been reported to exhibit antagonistic activity (29-31). One example is the compound [Arg²]hPTH-(1-34), which was reported to be a weak partial agonist of the rat PTH1R but a full agonist of the opossum PTH1R (29, 30). In human osteosarcoma Saos-2/B-10 cells, this analog is a potent antagonist (22). Additionally, [Phe³]- and [Phe⁶]bPTH-(1-34) have been shown to be weak partial agonists (31). Little is known, however, about the binding modes of such antagonists in comparison to agonists.

Photoaffinity cross-linking of radioiodinated Bpa¹- and Bpa²-PTHrP to PTH1R followed by consecutive and reciprocal treatments with Lys-C and BNPS-skatole generated similar digestion patterns for the two ligand-receptor conjugates. These treatments yielded bands with similar electrophoretic mobilities at ~7-8 kDa that are not affected by Endo-F treatment (Fig. 3, A and C). The molecular weights of ¹²⁵I-Bpa¹- and ¹²⁵I-Bpa²-PTHrP are 4645 and 4618, respectively, and therefore, the receptor fragment contributing to the final ~7-8 kDa of the photoconjugated fragments is approximately 3.5 kDa. Examination of the theoretical Lys-C and BNPS-skatole digestion maps of PTH1R reveals only one possible nonglycosylated fragment consistent with the observed fragmentation. This fragment corresponds to Ser⁴⁰⁹-Trp⁴³⁷, which includes most of TMD 6 and part of extracellular loop 3 (Fig. 6A).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   A, schematic representation of a portion of the PTH1R. The putative minimal digestion restricted domain containing the contact site for 125I-Bpa1- and 125I-Bpa2-PTHrP. Both ends of the domain Ser409-Trp437, which was obtained by consecutive and reciprocal Lys-C and BNPS-skatole cleavages, are indicated by shaded circles. Met425 and Met414 are indicated by black circles.  represents putative glycosylation sites. B, schematic summary of the potential cleavage sites in the region of TMD 6-extracellular loop 3 of PTH1R. The cleavage sites for Lys-C, BNPS-skatole, and CNBr are indicated by the appropriate residue numbers. The predicted receptor fragment size is indicated in Da.

CNBr digestion of the ¹²⁵I-Bpa¹-PTHrP-PTH1R conjugate (Fig. 3B) suggests that cross-linking occurred at the -methyl of a Met residue. In such a case, CNBr treatment would yield a conjugated fragment that is simply a "CH₂SCN"-modified ligand represented by the ~4.5-kDa band and therefore indistinguishable from ligand alone by SDS-PAGE (18, 26). CNBr digestion of the ¹²⁵I-Bpa²-PTHrP-PTH1R (antagonist) conjugate suggests that cross-linking occurred at two distinct sites (Fig. 3D). One site is the -methyl of a Met residue, similar to Bpa¹-PTHrP cross-linking, and the other is a different site, as evidenced by the additional CNBr-generated fragment of ~6 kDa (Fig. 3D). The additional site could be either a different amino acid or the -CH₂ within the same Met, which would result in the generation of a fragment of ~6 kDa. The receptor fragment contributes 1-2 kDa to the ~6-kDa fragment conjugate, which corresponds to either Pro⁴¹⁵-Met⁴²⁵ or Ala⁴²⁶-Met⁴⁴¹. The finding that the size of the ~6-kDa fragment is not further reduced following treatment with either Lys-C or BNPS-skatole indicates that Pro⁴¹⁵-Met⁴²⁵ is the minimal digestion-restricted domain that includes the photoinsertion site for ¹²⁵I-Bpa²-PTHrP (Fig. 6B).

The contact domain identified by Lys-C and BNPS-skatole digestions contains 2 methionine residues, Met⁴¹⁴ and Met⁴²⁵, either of which could be the putative "contact point" for ¹²⁵I-Bpa¹-PTHrP or ¹²⁵I-Bpa²-PTHrP. Photoaffinity cross-linking to single point-mutated receptors (M414L and M425L) suggests that the benzophenone moiety in both the agonist Bpa¹- and the antagonist Bpa²-PTHrP contacts Met⁴²⁵ in TMD 6 of PTH1R. Cross-linking of ¹²⁵I-Bpa²-PTHrP to M425L mutant receptor yielded a very weak but specifically labeled radioactive band at ~90 kDa (Fig. 4D, lane 5), whereas no cross-linking was detected with radioiodinated Bpa¹-PTHrP. This observation suggests that cross-linking of the antagonist to the M425L mutant does occur, but with a markedly reduced efficiency. The low level of incorporation into the M425L receptor mutant precludes detailed analysis of the actual contact domain. This finding, however, emphasizes a distinct difference between the cross-linking of ¹²⁵I-Bpa¹- and ¹²⁵I-Bpa²-PTHrP to PTH1R and corroborates the CNBr digestion data.

The results may reflect either differences between the binding modes of the agonist and the antagonist or differences in the interaction between the two consecutive positions in the PTHrP-(1-36) sequence and PTH1R. In an attempt to distinguish between these two possibilities, we utilized the agonist analog Bpa²-PTH, which carries the same photoreactive moiety at the same position as the antagonist Bpa²-PTHrP. Analysis of ¹²⁵I-Bpa²-PTH photoconjugates with wild-type, M414L, and M425L mutated PTH1R indicates that this ligand cross-links only to the -methyl of Met⁴²⁵ (Fig. 5), similar to Bpa¹-PTHrP (Fig. 3, A and B, and Fig. 4C) and to Bpa¹-PTH cross-linking (18). These results, therefore, provide strong support for the hypothesis that the differences observed between the cross-linking of ¹²⁵I-Bpa¹- and ¹²⁵I-Bpa²-PTHrP may reflect different interaction modes of an agonist versus an antagonist with the receptor.

One of the models of GPCR activation suggests that in the absence of a ligand, the receptor is in equilibrium among several conformational states, spanning the transition between resting and activated conformations (32). According to this model, antagonists bind the receptor without affecting this equilibrium, whereas agonist interaction shifts the equilibrium to the activated conformation. One possible interpretation of our results is that in the "agonist-bound" conformation (activated), the photoinsertion site for either Bpa¹- or Bpa²-PTHrP is the -methyl of Met⁴²⁵. In the presence of antagonist, where a more heterogeneous population of antagonist-receptor complexes prevails, there are receptor conformations in which the photophore at position 2 is positioned to allow photoinsertion to an alternate site or several other distinct sites in proximity to Met⁴²⁵. Hence, the additional cross-linking site of the antagonist Bpa²-PTHrP (represented by the ~6-kDa band) is probably obtained through interaction with receptors in a conformation different from the one obtained in the presence of the agonist.

Several publications have discussed the binding modes of agonists versus antagonists in the GPCR superfamily for the following receptors in particular: neurokinin 1 (33-35), cholecystokinin B/gastrin (36, 37), angiotensin-1 (38), corticotropin-releasing factor (39), B₂ bradykinin (40), neuromedin B (41), and gonadotropin-releasing hormone (42). These studies have concluded that the binding sites of peptide agonists are distinct from those of peptide or nonpeptidic antagonists. In contrast, a few other investigators have found that peptide agonists and peptide or nonpeptidic antagonists have overlapping binding sites on their receptors, including the B₂ bradykinin (43), the -adrenergic (44), the cholecystokinin (45), and the neuropeptide Y Y1 receptors (46). These differences may be attributed to elucidation of only a subset out of multiple interaction sites between ligand and receptor, especially in the case of intermediate-size peptide ligands. It is conceivable that some interaction sites are distinct whereas some others overlap, particularly, as presented here, if the agonist and antagonist are structurally very similar.

The finding that both residues 1 and 2 in both PTH-(1-34) and PTHrP-(1-36) cross-link to Met⁴²⁵ (or a larger segment in TMD 6) is consistent with the idea that this region of the receptor is essential for activation. TMD 6 is contiguous with intracellular loop 3, which is hypothesized to be coupled to G_s (47, 48). Although the molecular details of activation of GPCRs are not fully understood, it has been postulated that, upon agonist binding, conformational changes are induced in the receptor that affect the intracellular loop domains, such as intracellular loop 3, to increase affinity for G proteins (guanyl nucleotide-binding proteins). Using site-selective fluorescently labeled 2 adrenergic receptor, experimental evidence suggested that movements of TMD 3 and 6 underlie the activation of the receptor (49). Our observations are in line with this hypothesis and suggest that TMD 6 is involved in activation of PTH1R, and probably of the entire PTH/secretin subfamily.

The finding that Bpa²-PTHrP is an antagonist, whereas Bpa²-PTH is an agonist, suggests that PTH-(1-34) and PTHrP-(1-36) have some distinct sets of interactions with their common receptor. Moreover, PTH1R is able to distinguish between PTH-(1-34) and PTHrP-(1-36) analogs substituted at position 2 by Bpa (and presumably other amino acids). The nonequivalence of PTH and PTHrP is demonstrated by the selective interaction of PTH-(1-34) with the PTH2R, which does not recognize PTHrP-(1-36) (50). Replacement of His⁵ by Ile in PTHrP-(1-36) overcomes this distinction and converts the analog into a potent agonist (27, 28). Interestingly, we found that Bpa²-PTHrP the analog is a full agonist for the PTH2 receptor.² This observation suggests that Bpa²-PTHrP is able to distinguish between two highly homologous receptor subtypes and emphasizes the key role of the interaction between position 2 and the receptor. The critical role of this position is also illustrated by the [Arg²]hPTH-(1-34) analog, which interacts differently with PTH1R from different species (30).

In conclusion, this study examined the cross-linking sites of peptide agonist and antagonist analogs of PTHrP. We found that both Bpa¹- and Bpa²-PTHrP cross-link to the -methyl of Met⁴²⁵ in TMD 6 of PTH1R. The antagonist Bpa²-PTHrP also cross-links to a distinct proximal region, probably within the domain Pro⁴¹⁵-Met⁴²⁵. These differences in the cross-linking sites are attributed to different interactions of peptide agonists and antagonists with the PTH1R and suggest that the antagonist Bpa²-PTHrP interacts with distinct populations of the receptor, one or more of which is distinct from the conformation recognized by the agonist.

	ACKNOWLEDGEMENT

We thank Dr. Amy E. Adams for providing the cDNA constructs for the receptor mutants.

	FOOTNOTES

^* This work was supported in part by Grant RO1-DK47940 (to M. R.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work is presented in partial fulfillment of the requirements of a Ph.D. thesis.

^§ To whom correspondence should be addressed: Division of Bone and Mineral Metabolism, Beth Israel Deaconess Medical Center, 330 Brookline Ave. (HIM 944), Boston, MA 02215. Tel.: 617-667-0901; Fax: 617-667-4432; E-mail: mchorev@warren.med.harvard.edu.

² V. Behar, unpublished results.

	ABBREVIATIONS

The abbreviations used are: PTH, parathyroid hormone; bPTH, bovine PTH; hPTH, human PTH; AC, adenylyl cyclase; BNPS-skatole, 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine; Bpa, L-p-benzoylphenylalanine; Bpaⁿ-PTHrP, [Bpaⁿ,Ile⁵,Arg^11,13,Tyr³⁶]PTHrP-(1-36)NH₂ (n = 1-6); Bpaⁿ-PTH, [Bpaⁿ,Nle^8,18,Arg^13,26,27,Nal²³,Tyr³⁴]bPTH-(1-34)NH₂ (n = 1-6); GPCR, G protein-coupled receptor; HEK, human embryonic kidney; I-Bpa²-PTHrP, [Bpa²,Ile⁵,Arg^11,13,3-I-Tyr³⁶]PTHrP-(1-36)NH₂; PTH1R, hPTH/PTHrP receptor; Lys-C, lysyl endopeptidase; PAGE, polyacrylamide gel electrophoresis; PTHrP, PTH-related protein; PTH-(1-34), [Nle^8,18,Tyr³⁴]bPTH-(1-34)NH₂; PTHrP-(1-36), [Ile⁵,Arg^11,13,Tyr³⁶]PTHrP-(1-36)NH₂; HPLC, high performance liquid chromatography; TMD, transmembrane domain; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

	REFERENCES

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