Evidence for a Ligand Interaction Site at the Amino-Terminus of the Parathyroid Hormone (PTH)/PTH-related Protein Receptor from Cross-linking and Mutational Studies*

Michael Mannstadt, Michael D. Luck, Thomas J. Gardella, and Harald JüppnerDagger

From the Endocrine Unit, Department of Medicine and Children's Service, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Low resolution mutational studies have indicated that the amino-terminal extracellular domain of the rat parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (rP1R) interacts with the carboxyl-terminal portion of PTH-(1-34) or PTHrP-(1-36). To further define ligand-receptor interactions, we prepared a fully functional photoreactive analog of PTHrP, [Ile5,Bpa23,Tyr36]PTHrP-(1-36)-amide ([Bpa23]PTHrP, where Bpa is p-benzoyl-L-phenylalanine). Upon photolysis, radioiodinated [Bpa23]PTHrP covalently and specifically bound to the rP1R. CNBr cleavage of the broad approx 80-kDa complex yielded a radiolabeled approx 9-kDa non-glycosylated protein band that could potentially be assigned to rP1R residues 23-63, Tyr23 being the presumed amino-terminus of the receptor. This assignment was confirmed using a mutant rP1R (rP1R-M63I) that yielded, upon photoligand binding and CNBr digestion, a broad protein band of approx 46 kDa, which was reduced to a sharp band of approx 20 kDa upon deglycosylation. CNBr digestion of complexes formed with two additional rP1R double mutants (rP1R-M63I/L40M and rP1R-M63I/L41M) yielded non-glycosylated protein bands that were approx 6 kDa in size, indicating that [Bpa23]PTHrP cross-links to amino acids 23-40 of the rP1R. This segment overlaps a receptor region previously identified by deletion mapping to be important for ligand binding. Alanine scanning of this region revealed two residues, Thr33 and Gln37, as being functionally involved in ligand binding. Thus, the convergence of photoaffinity cross-linking and mutational data demonstrates that the extreme amino-terminus of the rP1R participates in ligand binding.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The PTH1/PTHrP receptor (P1R) mediates the biological actions of PTH and PTHrP (1, 2); both peptides bind to this common receptor with similar affinities and stimulate the formation of cAMP and inositol phosphates with similar efficacies (3-5). In contrast, the recently isolated PTH2 receptor (P2R) (6) is activated fully by PTH and only poorly by PTHrP (6-9).

Current information suggests that PTH and PTHrP interact with the P1R through multiple sites and that these are dispersed throughout the extracellular surface of the receptor and some portions of the transmembrane helices (1). Studies with chimeras formed between P1Rs from different species or different receptor subtypes (P1R or P2R) indicate that there are interactions between the amino-terminal extracellular domain of the receptor and region 15-34 of the ligand and between the core region of the receptor and the amino-terminal portion of the ligand (9-12). Furthermore, observations from studies on other members of this peptide hormone receptor family (13, 14), and particularly with chimeras between the P1R and the calcitonin receptor (15), suggest that this general orientation of ligand-receptor interaction may apply to all members of this family of G protein-coupled receptors.

In addition to mutagenesis approaches, affinity cross-linking methods can provide valuable information on the location of ligand-receptor interactions sites in peptide hormone receptors (16, 17). For the P1R, Zhou et al. (18) recently showed that a PTH-(1-34) analog containing a photoderivatized lysine 13 cross-linked to a 17-amino acid segment of the amino-terminal extracellular receptor domain that mapped close to the junction with the first membrane-spanning helix. In related experiments, this group showed that another PTH-(1-34) analog, which contains p-benzoyl-L-phenylalanine (Bpa) (19) at position 1, cross-linked to a region of the P1R containing transmembrane helix 6 and extracellular loop 3 (20). The results of these physicochemical analyses are in agreement with previous mutational studies that functionally identified similar regions of the P1R as candidate ligand-interaction sites (12, 21-23). In addition to these two putative ligand contact regions, mutational studies also identified segments within the large (approx 190 amino acids) amino-terminal domain of the receptor that appear to interact with region 15-34 of the ligand (10, 24).

We have now performed cross-linking studies with a PTHrP analog that contains photoreactive Bpa at position 23 in place of the native phenylalanine, a residue recently shown to be involved in the ligand binding specificity of the PTH2 receptor (7, 11). This new photoreactive ligand, [Ile5,Bpa23,Tyr36]PTHrP-(1-36)-amide, cross-linked to a short segment between residue 40 and the amino-terminus, which is predicted to be Tyr-23. We also confirmed the importance of this amino-terminal receptor region by mutational methods and have identified two amino acid residues that contribute to ligand binding affinity.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- [Nle8,21,Tyr34]rPTH-(1-34)-amide (rNlePTH), [Nle8,18,Tyr34]bPTH-(1-34)-amide (bNlePTH), [Ile5,Bpa23,Tyr36]hPTHrP-(1-36)-amide ([Bpa23]PTHrP), [Tyr36]PTHrP-(1-36)-amide (PTHrP), and [Leu11,D-Trp12]PTHrP-(7-34)-amide (PTHrP-(7-34)) were synthesized by the Protein and Peptide Core Facility at Massachusetts General Hospital (Boston, MA) by solid-phase method on Perkin-Elmer Model 430A and 431A synthesizers. The Fastmoc version of Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry was utilized, and peptides were purified by reversed-phase chromatography.

Na125I (specific activity of 2000 Ci/mmol) and radioiodinated anti-mouse IgG Fab (Nex162) were purchased from NEN Life Science Products. Dulbecco's modified Eagle's medium, Ham's F-12 medium, trypsin/EDTA, penicillin G/streptomycin, and horse serum were from Life Technologies, Inc.. Fetal bovine serum, 3-isobutyl-1-methylxanthine, bovine serum albumin, Tricine, and Me2SO were from Sigma. Trifluoroacetic acid was from Pierce, and CNBr was from Serva Fine Chemicals/Boehringer Ingelheim (Heidelberg, Germany). 14C-Methylated protein molecular mass markers for SDS-PAGE were purchased from Amersham Pharmacia Biotech, and peptide N-glycosidase F was from New England Biolabs Inc. (Beverly, MA). DEAE-dextran was from Pharmacia (Uppsala, Sweden), and X-Omat AR films for autoradiography were from Eastman Kodak Co. The monoclonal antibody 12CA5 was purchased from Berkeley Antibodies (Berkeley, CA).

Mutagenesis of the Rat PTH/PTHrP Receptors-- Mutations were introduced into single-strand plasmid DNA encoding the wild-type rat PTH/PTHrP receptor (rP1R) (4) or, for cassette replacements and alanine point mutations, into the P1R with a hemagglutinin (HA) epitope in the receptor's E2 domain (rP1R-HA) (24) by oligonucleotide-directed site-specific mutagenesis as described (11, 24, 25). The oligonucleotide primers were synthesized on an Applied Biosystems Model 380A DNA synthesizer. Positive mutants were verified by nucleotide sequence analysis of plasmid DNA.

Cell Culture and DNA Transfection-- COS-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified atmosphere containing 95% air and 5% CO2. Cells were seeded in 24-well plates (200,000 cells/well) for radioreceptor and cAMP assays and for preliminary cross-linking experiments; all other cross-linking experiments were performed in 150-mm dishes (6 × 106 cells). Once the cell monolayer reached 90-100% confluency, cells were transfected by the DEAE-dextran method as described (22) using 200 ng of plasmid DNA/well or 2 µg/150-mm dish. The medium was replaced daily, and 3 days after transfection, cells were used either for radioligand binding and cAMP accumulation assays or for cross-linking experiments. ROS 17/2.8 cells were maintained as described (26) in Ham's F-12 medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Radioligand-Receptor Binding Assays and cAMP Accumulation-- Radiolabeled rNlePTH, PTHrP, and [Bpa23]PTHrP were prepared by chloramine-T iodination, followed by high pressure liquid chromatography purification using a 30-50% acetonitrile in 0.1% trifluoroacetic acid gradient over 30 min. Radioligand-receptor binding assays were performed in 24-well plates as described (11). In brief, each well (final volume of 500 µl) contained binding buffer (50 mM Tris-HCl (pH 7.7), 100 mM NaCl, 5 mM KCl, 2 mM CaCl, 5% heat-inactivated horse serum, and 0.5% heat-inactivated fetal bovine serum), 125I-labeled radioligands (100,000-200,000 cpm), and varying concentrations of unlabeled peptide. After 4 h at 16 °C, the binding mixture was removed, and the cells were rinsed with cold binding buffer and lysed with 1 M NaOH. The entire lysate was counted for gamma -irradiation. Specific binding was determined after subtracting radioactivity bound in the presence of maximal concentrations of unlabeled competing peptide (10-6 M). Agonist-dependent accumulation of cAMP was determined by radioimmunoassay as described (11).

Cell-surface Expression of PTH/PTHrP Receptors-- Cell-surface expression was assessed as described (24) using antibody 12CA5 directed against the HA epitope in the rP1R-HA receptors and a second radiolabeled anti-mouse IgG Fab fragment. Relative specific binding of antibody to each mutant P1R was calculated by subtracting nonspecifically bound radioactivity (determined in mock-transfected COS cells; typically 0.1-0.2% of added radioactivity) from the total bound radioactivity divided by the radioactivity specifically bound to wild-type rP1R-HA (typically 1-2% of the added radioactivity).

Photoaffinity Labeling of PTH/PTHrP Receptors-- In preliminary experiments, COS-7 cells that were grown and transfected in 24-well plates were rinsed twice with 1 ml of cold binding buffer, and the cell monolayer was then incubated for 6 h at 4 °C with 125I-[Bpa23]PTHrP (1 × 106 cpm) diluted in 0.5 ml of binding buffer with or without unlabeled ligand (10-6 M bNlePTH or [Bpa23]PTHrP). After incubation, cells were rinsed three times with 1 ml of cold binding buffer before adding 200 µl of binding buffer and placing the dishes on ice under a UV light source for 20 min (Blak Ray long-wave lamp; 366 nm, 7000 microwatts/cm2; UV Products, San Gabriel, CA; source-to-cell distance of approx 5 cm). After photolysis, cells were rinsed once with cold phosphate-buffered saline, twice with a cold acidic buffer (0.05 M glycine and 0.15 M NaCl (pH 2.5)) to remove noncovalently bound radioligand, and twice with cold phosphate-buffered saline before solubilization with 0.5 ml of SDS-PAGE sample buffer (4% (w/v) SDS, 80 mM Tris-HCl (pH 6.8), 20% (v/v) glycerol, 0.2% bromphenol blue, and 100 mM dithiothreitol). The lysate was then passed six times through a 19-gauge needle to shear genomic DNA.

To prepare larger amounts of the cross-linked ligand-receptor complex, a similar protocol was followed using COS-7 cells that were grown and transfected in 150-mm dishes. For each rinsing step, 30 ml of cold binding buffer were used, and incubation with 125I-[Bpa23]PTHrP (2-4 × 107 cpm) was performed in a final volume of 20 ml of binding buffer. During UV light exposure, the cell monolayer was covered with 10 ml of binding buffer, and after photolysis and rinsing, cells were solubilized with 4 ml of SDS-PAGE sample buffer.

SDS-PAGE Analysis of the Ligand-Receptor Complex-- After heating to 70 °C, samples (and appropriate size markers) were either subjected to analytical SDS-PAGE analysis (5-20% acrylamide, 0.75-mm spacers) according to the method of Laemmli (27) or loaded onto a 16.5% (w/v) Tricine gel (0.75-mm spacers) according to the method of Schägger and von Jagow (28), with subsequent autoradiography of the dried gels (1-14 days at -80 °C with intensifying screens).

Purification of [Bpa23]PTHrP·P1R Complexes-- To isolate larger amounts of radiolabeled ligand-receptor complexes from cells cultured in 150-mm dishes, we used preparative SDS-polyacrylamide gels (5-20% acrylamide, 3-mm spacers); the complexes were identified by autoradiography of the wet gels (exposure time of 4-12 h at room temperature) and electroeluted from an excised gel slice using a Model 422 electroeluter (Bio-Rad). The isolated radiolabeled ligand-receptor complex was stored at -20 °C in elution buffer (25 mM Tris, 192 mM glycine, and 0.02% SDS) before chemical/enzymatic treatment (see below).

CNBr Cleavage-- CNBr was dissolved in 100% trifluoroacetic acid and then added to the partially purified radiolabeled ligand-receptor complex to give a final concentration of 100 mM CNBr in 70% trifluoroacetic acid. After an overnight, light-protected incubation, the digest was reduced in volume under a stream of nitrogen and then repeatedly lyophilized to remove trifluoroacetic acid and CNBr. Once the apparent molecular mass of each CNBr-derived ligand-receptor fragment had been established, trifluoroacetic acid and CNBr were eliminated more efficiently by ultrafiltration using a Microcon 3 concentrator (Amicon, Inc., Beverly, MA) and repeated dilution of the retentate with H2O.

Peptide N-Glycosidase F Digestion-- The CNBr-cleaved and concentrated radioligand-receptor complex was treated with peptide N-glycosidase F (2500 units) for 1 h at 37 °C in 30 µl of 50 mM sodium phosphate (pH 7.5), 0.5% SDS, 1% beta -mercaptoethanol, and 1% Nonidet P-40 according to the protocol provided by the supplier.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Functional Characterization of [Bpa23]PTHrP-- [Bpa23]PTHrP was tested in competition binding studies performed with COS-7 cells expressing the native rP1R and was found to have an apparent binding affinity that is indistinguishable from that of bNlePTH and of other analogs of PTH and PTHrP (7, 10). The Bpa-containing PTHrP analog was also fully functional in cAMP accumulation assays and exhibited a potency that was indistinguishable from that of bNlePTH (data not shown).

Photoaffinity Labeling of Rat PTH/PTHrP Receptors-- After binding and photoactivation, the covalent complex formed between radioiodinated [Bpa23]PTHrP and the rP1R was visualized by analytical SDS-PAGE and subsequent autoradiography. The complex migrated as a single broad band corresponding to a glycosylated protein with a molecular mass of approx 80 kDa (Fig. 1, lane 1). This size of the ligand-receptor complex is comparable to that previously seen with other photoreactive PTH or PTHrP analogs using either cells expressing endogenous PTH/PTHrP receptors (29-32) or HEK-293 cells expressing the cloned P1R (18). Coincubation of transfected COS-7 cells with 125I-[Bpa23]PTHrP and unlabeled bPTH-(1-34) (10-6 M) or unlabeled [Bpa23]PTHrP (10-6 M) completely eliminated the formation of the radiolabeled ligand-receptor complex (data not shown).


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Fig. 1.   Analysis of the CNBr-digested [Bpa23]PTHrP·P1R complex using COS-7 cells expressing the wild-type rat PTH/PTHrP receptor and the M63I receptor mutant. As described under "Experimental Procedures," the partially purified complex of ligand and wild-type P1R (lanes 1 and 2) or the M63I receptor mutant (lanes 3 and 4) was incubated in 70% trifluoroacetic acid in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of CNBr (100 mM). After repeated lyophilization, samples were analyzed by SDS-PAGE with subsequent autoradiography (overnight at -80 °C). The positions of different size markers are indicated in kilodaltons.

To identify the region of the P1R that interacted with the Bpa23 moiety, we isolated larger amounts of the radiolabeled ligand-receptor complex using preparative SDS-PAGE, cleaved the partially purified complex with CNBr, and separated the cleavage products on analytical gels. After CNBr cleavage, most of the radioactivity migrated on SDS-PAGE as a single sharp protein band corresponding to a size of <14 kDa (Fig. 1, lane 2). A minor fraction migrated as a diffuse band at the 46-kDa size marker and probably corresponded to a partially cleaved glycosylated ligand-receptor complex. Tricine/SDS-PAGE analysis was used to achieve higher resolution in the low molecular mass range, and this suggested a molecular size of approx 9 kDa for the principal radiolabeled CNBr-generated fragment (see also Fig. 5, lane 1). Since [Bpa23]PTHrP has a molecular size of 4.286 kDa, the receptor fragment contributing to the complex was estimated to have a molecular size of approx 5 kDa. The same results were obtained when analyzing the complex formed between radiolabeled [Bpa23]PTHrP and the endogenous PTH/PTHrP receptor of ROS 17/2.8 cells (data not shown).

The above results suggested that Bpa at position 23 of PTHrP interacts with an approx 5-kDa non-glycosylated CNBr-generated portion of the receptor. Inspection of the amino acid sequence of the rP1R showed that several fragments delimited by methionine residues are within this molecular size range (Fig. 2). Because of the predicted overall architecture of ligand-receptor interaction (1, 11, 15), we considered the hypothesis that Bpa23 interacts with the receptor segment defined by Met63 and the amino-terminus, which is predicted to be Tyr23 by a recently developed algorithm (33). A mutant rP1R was generated, rP1R-M63I, in which methionine at position 63 was replaced by isoleucine (Fig. 3B); this mutant receptor had functional (data not shown) and cross-linking (Fig. 1, lane 3) properties that were indistinguishable from the wild-type rP1R. CNBr cleavage of the covalently labeled rP1R-M63I mutant yielded to a broad radioactive band comigrating with the 46-kDa marker (Fig. 1, lane 4, and Fig. 4, lane 1); this cleavage product was reduced to a sharp protein band of <20 kDa upon further digestion with peptide N-glycosidase F (Fig. 4, lane 2). The increase in size from approx 9 to approx 46 kDa of the CNBr-derived ligand-receptor complex that occurred as a result of the M63I mutation and the subsequent size reduction of this larger receptor fragment upon deglycosylation indicated that, in the mutant receptor, Bpa23 interacts with a receptor segment that extends from the amino-terminus to Met174. These results confirmed that cross-linking of [Bpa23]PTHrP to the wild-type P1R occurred between Met63 and the receptor's amino-terminal residue.


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Fig. 2.   Schematic model of the rat P1R showing the seven putative transmembrane domains and the locations of all 14 methionine residues in the mature protein. The amino-terminus is at the top. Potential N-linked glycosylation sites (psi ), the putative signal peptide, and the predicted first residue (Tyr23) of the mature receptor are shown. The sites for leucine (L) to methionine (M) and for methionine (M) to isoleucine (I) substitutions and the location of methionine residues (bullet ) are indicated (see also Fig. 3).


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Fig. 3.   Schematic drawing of the amino-terminus of the wild-type rat PTH/PTHrP receptor and two receptor mutants. The predicted signal sequence cleavage site (arrow), the sites for N-linked glycosylation (psi ), and the locations of methionine residues (bullet ) and the introduced isoleucine () are indicated. The calculated molecular sizes of CNBr fragments are indicated in daltons. A, wild-type P1R; B, P1R-M63I; C, P1R- M63I/L40M.


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Fig. 4.   Analysis of the CNBr-digested [Bpa23]PTHrP·P1R-M63I complex using COS-7 cells expressing the M63I receptor mutant. As described under "Experimental Procedures," the partially purified, CNBr-cleaved radioligand-receptor complex (lane 1) was digested with peptide N-glycosidase F (lane 2). Samples were analyzed by Tricine/SDS-PAGE with subsequent autoradiography (14 days at -80 °C). The positions of different size markers are indicated in kilodaltons.

To further define the site of cross-linking, we introduced methionine substitutions at either Leu40 or Leu41 in the rP1R-M63I mutant to yield the double mutants rP1R-M63I/L40M and rP1R-M63I/L41M, respectively (Fig. 3C). Like rP1R M63I, these two mutants had biological properties that were indistinguishable from those of the wild-type rP1R (data not shown). Leu40 and Leu41 were chosen because their substitution with methionine is a conservative replacement and because CNBr cleavage at these positions would yield ligand-receptor conjugates whose size upon SDS-PAGE analysis would easily distinguish between the two possible sites of interaction with [Bpa23]PTHrP. Thus, cross-linking to a site amino-terminal of Met40 or Met41 would yield non-glycosylated, low molecular mass complexes corresponding to receptor residues 23-40 and 23-41, respectively, whereas cross-linking to a site carboxyl-terminal of either mutation would yield glycosylated, high molecular mass complexes corresponding to residues 41-174 and 42-174, respectively. As shown in Fig. 5 (lane 3), CNBr cleavage of the affinity-labeledM63I/L40M mutant yielded a small radiolabeled, non-glycosylated complex of approx 6 kDa, as did cleavage of the M63I/L41M mutant (data not shown). This indicated that the covalent interaction between Bpa23 and the rP1R occurred between the receptor's amino-terminus and Leu40.


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Fig. 5.   Analysis of the CNBr-digested [Bpa23]PTHrP·P1R complex using COS-7 cells expressing the wild-type rat PTH/PTHrP receptor and two different receptor mutants. As described under "Experimental Procedures," the partially purified ligand-receptor complexes were incubated in 70% trifluoroacetic acid in the presence of CNBr. After repeated lyophilization, samples were analyzed by Tricine/SDS-PAGE with subsequent autoradiography (5 days at -80 °C). Lane 1, wild-type P1R; lane 2, P1R-M63I; lane 3, P1R-M63I/L40M. The positions of different size markers (in kilodaltons) and of 125I-[Bpa23]PTHrP (arrowhead) are indicated.   

Effects of Point Mutations in the Amino-terminal Extracellular Domain of the PTH/PTHrP Receptor on Ligand Binding-- The amino-terminal receptor fragment identified by the above physicochemical approach overlaps a P1R region previously shown by functional studies to be important for ligand binding (24). Two mutant receptors with deletions of residues 26-60 (the E1 region) or 31-47 (E1a) were shown to have only moderately reduced receptor expression levels in COS-7 cells (22 ± 1 and 36 ± 3% of the wild type, respectively) and little or no capacity to bind radiolabeled PTH (24). To further examine the functional importance of residues in this amino-terminal E1a region, we first made four cassette mutant receptors, termed E1a-1 through E1a-4, in which four or five adjacent residues were replaced by either alanine or valine (Fig. 6, A and B). Each mutant receptor was adequately expressed on the surface of COS-7 cells (>35% of the wild-type) (Fig. 6C). The two mutants in which residues 31-35 and 36-39 were altered displayed diminished 125I-rNlePTH binding capacity, whereas the two mutants with substitutions of residues 40-43 and 44-47, respectively, maintained high levels of PTH binding (Fig. 6D).


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Fig. 6.   Cassette mutagenesis of the E1a region of the rat PTH/PTHrP receptor. The location of the E1a region, previously identified by deletion analysis as a ligand-binding site (24), is shown in A. The four cassette substitutions used to further divide the E1a region are shown in B. The effects of each cassette mutation on surface expression as assessed by antibody binding to the HA epitope in the E2 domain of each receptor and 125I-[Nle8,21,Tyr34]rPTH-(1-34)-NH2 binding are shown in C and D, respectively. Included as controls are COS-7 cells transfected with a mutant P1R in which the entire E1a region was deleted (del.E1a) and mock-transfected COS-7 cells. Data are the means ± S.E. of triplicate values from one of two equivalent experiments. WT, wild-type.   

To further localize candidate binding residues within region 31-39 (E1a-1 and E1a-2), an alanine-scanning approach was used. Several of the individual alanine substitutions in this region, which had little or no effect on cell surface expression (Fig. 7A), resulted in small reductions in 125I-rNlePTH binding capacity (Fig. 7B). A reduction in PTH binding of >25% occurred with two substitutions, T33A and Q37A (Fig. 7B). In addition, each of these two point mutations had a more severe effect on 125I-PTHrP binding than on 125I-rNlePTH binding (Fig. 7C). In competition binding studies with 125I-rNlePTH as tracer radioligand, the apparent binding affinity of rNlePTH for wild-type and mutant P1Rs was comparable (Fig. 8A). The apparent binding affinity of bNlePTH for the T33A and Q37A mutant receptors was 5.0- and 2.3-fold weaker, respectively, than it was for the wild-type receptor (Fig. 8B). Consistent with the reduced maximal binding of radiolabeled PTHrP, the apparent binding affinity of PTHrP-(1-36) for these two mutant receptors was 14- and 48-fold weaker, respectively, than it was for the wild-type receptor (Fig. 8C). Both receptor mutations also abolished binding of an amino-terminally truncated PTHrP analog, [Leu11,D-Trp12]PTHrP-(7-34)-amide, indicating that Thr33 and Gln37 affect interactions with region 7-34 of the ligand rather than with region 1-6 (Fig. 8D and Table I).


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Fig. 7.   Alanine-scanning mutagenesis of residues 31-39 of the rat PTH/PTHrP receptor. Individual residues in the receptor regions defined by the E1a-1 and E1a-2 cassette mutations (see Fig. 6) were replaced by alanine; the resulting mutants were transfected into COS-7 cells and tested for surface expression (A), binding of 125I-[Nle8,21,Tyr34]rPTH-(1-34)-NH2 (B), and binding of 125I-[Tyr36]PTHrP-(1-36)-NH2 (C). Data are the means ± S.E. of three experiments, each performed in triplicate. WT, wild-type.   


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Fig. 8.   Effects of alanine substitution of Thr33 or Gln37 in the PTH/PTHrP receptor on ligand binding. The rP1R (wild type (WT); bullet ) and the T33A (triangle ) and Q37A (black-square) mutants were expressed in COS-7 cells and evaluated for binding PTH or PTHrP analogs. Competition binding analyses were performed using 125I-[Nle8,21,Tyr34]rPTH-(1-34)-NH2 as a tracer radioligand and [Nle8,21,Tyr34]rPTH-(1-34)-NH2 (A), [Nle8,18,Tyr34]bPTH-(1-34)-NH2 (B), [Tyr36]hPTHrP-(1-36)-NH2 (C), or [Leu11,D-Trp12]hPTHrP-(7-34)-NH2 (D) as an unlabeled competitor ligand. Each graph shows data (mean ± S.E.) from four or five experiments, each performed in duplicate.   

                              
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Table I
Peptide analog binding to wild-type and mutant PTH/PTHrP receptors
Competition binding studies were performed with 125I-[Nle8,21, Tyr34]rPTH(1-34)-NH2 and the indicated unlabeled peptides as described under "Experimental Procedures" and in the legend to Fig. 8. Values are the means ± S.E.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Previous studies have suggested that the amino-terminal extracellular region of the PTH/PTHrP receptor interacts with carboxyl-terminal region 15-34 of either PTH or PTHrP; a similar architecture of ligand-receptor interaction may well apply to other members of this family of G protein-coupled receptors (9-15). In this study, we confirmed and extended these predictions for the P1R with a PTHrP analog containing photoreactive Bpa at position 23, a residue with apparent functional significance based on its ability to determine ligand binding specificity in the P2R (7, 11). After CNBr digestion of [Bpa23]PTHrP·P1R complexes, an approx 9-kDa radiolabeled protein was detected upon Tricine/SDS-PAGE analysis. This fragment was likely to represent 125I-[Bpa23]PTHrP covalently coupled to a receptor fragment extending from Tyr23, the first residue after the predicted cleavage site for the signal peptide (33), to Met63, the first methionine in the mature receptor sequence. We confirmed this assignment and refined the mapping further by using site-directed mutagenesis to introduce or remove methionines at strategic sites in the receptor. First, the rP1R- M63I mutant was generated and shown to be fully functional. When the ligand-receptor complex formed with this receptor was cleaved with CNBr, the approx 9-kDa band was replaced by an approx 46-kDa glycosylated band corresponding to the receptor fragment extending from the amino-terminus to Met174. This receptor segment contains three of the four potential N-linked glycosylation sites, and glycosylation is consistent with the broadness of the approx 46-kDa complex on SDS-PAGE and its reduction to a smaller, non-glycosylated protein band by peptide N-glycosidase F treatment. These results confirmed that cross-linking between BPA23 and the rat PTH/PTHrP receptor involved residues that are located between the amino-terminus and Met63. The M63I mutation allowed us to exclude other CNBr-derived receptor fragments of similar size, such as Ala426-Met450. Two additional, fully functional receptor double mutants, rP1R-M63I/L40M and rP1R-M63I/L41M, were prepared to further refine the cross-linking site. Both mutants contained the M63I mutation to eliminate the natural CNBr cleavage site at position 63. CNBr cleavage of the complexes formed between 125I-[Bpa23]PTHrP and either of these two mutant receptors resulted in low molecular mass radiolabeled protein conjugates (Fig. 5). This result established that Bpa23 cross-linked to a side amino-terminal to Met41 in the rP1R and clearly excluded segment 41-174 as the site of interaction.

Earlier mutagenesis studies had indicated that deletion of a portion of the rP1R that included 17 residues (the E1a region) close to the amino-terminus of the mature receptor abolished binding of radiolabeled PTH or PTHrP, with only moderate effects on receptor expression (24). To further map functional binding residues in this region, we constructed four "cassette" mutants in which four or five adjacent amino acids were replaced by alanine or valine. Two of these cassette mutants, E1a-1 and E1a-2, showed normal cell-surface expression, but little or no binding of radiolabeled PTH or PTHrP. These results suggested that residues within segments 31-35 and 36-39 contribute to ligand interaction. The replacement of each of these nine residues with individual alanine substitutions confirmed this hypothesis. Two mutants, rP1R T33A and rP1R Q37A, exhibited the weakest capacity to bind the radioligand. Interestingly, the effects of these mutations on ligand binding were more pronounced with PTHrP than with PTH; this pattern might be attributable to the divergence in region 15-34 of these two ligands, a hypothesis supported by the observation that the mutations also impaired PTHrP-(7-34) binding.

In summary, our physicochemical observations indicate that Bpa23 (and presumably Phe23 in the native PTHrP molecule) interacts with residues at the extreme amino-terminus of the PTH/PTHrP receptor. Mutational analysis of this receptor region supported this conclusion and identified two amino acid residues, Thr33 and Gln37, as possible sites for ligand interaction. The combined use of the two techniques, photoaffinity cross-linking and receptor mutagenesis, should enable the definition of other receptor segments that comprise contact points for PTH and PTHrP.

    ACKNOWLEDGEMENTS

We thank Drs. Henry T. Keutmann, Ashok Khatri, Abdul-Badi Abou-Samra, Henry M. Kronenberg, and John T. Potts, Jr. for continuous support and helpful discussions and John H. Davies, Cindy W. Su, and Peter Lyons for technical assistance.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK-11794 and by a Deutsche Akademische Austauschdienst fellowship (to M. M.).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.

Dagger To whom correspondence should be addressed: Endocrine Unit, Wellman 503, Massachusetts General Hospital, 50 Blossom St., Boston, MA 02114. Tel.: 617-726-3966; Fax: 617-726-7543; E-mail: jueppner{at}helix.mgh.harvard.edu.

1 The abbreviations used are: PTH, parathyroid hormone; rPTH, rat PTH; bPTH, bovine PTH; PTHrP, PTH-related protein; hPTHrP, human PTHrP; P1R, PTH/PTHrP receptor; rP1R, rat P1R; P2R, PTH2 receptor; Bpa, p-benzoyl-L-phenylalanine; Nle, norleucine; Tricine, N-tris(hydroxymethyl)methylglycine; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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