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J Biol Chem, Vol. 273, Issue 35, 22498-22505, August 28, 1998


Parathyroid Hormone-Receptor Interactions Identified Directly by Photocross-linking and Molecular Modeling Studies*

Alessandro BiselloDagger , Amy E. AdamsDagger , Dale F. Mierke§, Maria Pellegrini, Michael RosenblattDagger , Larry J. SuvaDagger , and Michael ChorevDagger parallel

From the Dagger  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, the § Department of Pharmacology and Molecular Toxicology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, and the  Department of Chemistry, Clark University, Worcester, Massachusetts 01610

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Direct mapping of the interface between parathyroid hormone (PTH) and its receptor (hPTH1-Rc) was carried out by photoaffinity scanning studies. Photoreactive analogs of PTH singularly substituted with a p-benzoylphenylalanine (Bpa) at each of the first six N-terminal positions have been prepared. Among these, the analog [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]bPTH-(1-34)NH2 (Bpa1-PTH-(1-34)) displayed in vitro activity with potency similar to that of PTH-(1-34). The radioiodinated analog 125I-Bpa1-PTH-(1-34) cross-linked specifically to the hPTH1-Rc stably expressed in human embryonic kidney cells. A series of chemical and enzymatic digestions of the hPTH1-Rc-125I-Bpa1-PTH-(1-34) conjugate suggested that a methionine residue (either Met414 or Met425) within the contact domain hPTH1-Rc-(409-437), which includes the transmembrane helix 6 and part of the third extracellular loop, as the putative contact point. Site-directed mutagenesis (M414L or M425L) identified Met425 as the putative contact point. Molecular modeling of the hPTH1-Rc together with the NMR-derived high resolution structure of hPTH-(1-34), guided by the cross-linking data, strongly supports Met425, at the extracellular end of transmembrane helix 6, as the residue interacting with the N-terminal residue of the hPTH-(1-34). The photocross-linking and molecular modeling studies provide insight into the topologic arrangement of the receptor-ligand complex.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Parathyroid hormone (PTH)1 is the major regulator of calcium levels in blood and plays a role in the regulation of bone remodeling (1). Given intermittently, PTH displays anabolic activity in bone and, therefore, has considerable therapeutic potential (2). PTH and PTH-related protein exert their actions via a seven-transmembrane (TM) domain-containing receptor (PTH1-Rc) (3) belonging to a subfamily of related G protein-coupled receptors (4-11). The PTH1-Rc is coupled to both adenylyl cyclase/cyclic AMP and phospholipase C/inositol 1,4,5-trisphosphate/cytosolic calcium intracellular signaling pathways (12-15).

Understanding the molecular mechanism of ligand recognition and signal transduction by the PTH1-Rc may identify new directions for the design of novel hormone analogs for the treatment of diseases such as osteoporosis, hypercalcemia of malignancy and hyperparathyroidism (16). In order to directly identify the structural elements involved in PTH-PTH1-Rc interactions, we employed a photoaffinity scanning approach (17). The generation of covalently linked ligand-receptor conjugates and the identification of the cross-linked domains allows mapping of the interface between hormone and receptor. Photoaffinity cross-linking has been successfully applied in defining interactions between small peptides, such as substance P (18-20), cholecystokinin (21), and vasopressin (22), and their receptors. Recently, we used this general approach to identify directly the interaction between position 13 of PTH and a 17-amino acid domain (residues 173-189) of the hPTH1-Rc (17).

We now report the evaluation of a series of photoreactive analogs obtained by a "p-benzoylphenylalanine (Bpa) scan" of the principal receptor activation domain (residues 1-6) of PTH-(1-34). A radiolabeled analog containing a photoreactive moiety at the N terminus, 125I-[Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]bPTH-(1-34)NH2 (125I-Bpa1-PTH-(1-34)), maintained full potency and led to the identification of a second "contact domain" between PTH and hPTH1-Rc. This information allows us to create, for the first time, a model describing interactions of hPTH-(1-34) with its receptor based on direct identification of the interacting regions.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Boc-protected amino acids, N-hydroxybenzotriazole, N,N'-dicyclohexylcarbodiimide, and p-methylbenzydrylamine resin were purchased from Applied Biosystems (Foster City, CA). Boc-(3-iodo)tyrosine[O-(3-BrBz)] was 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 was from Aldrich. Na125I was obtained from Amersham Pharmacia Biotech. Endoglycosidase F/N-glycosidase F (Endo-F) and lysyl endopeptidase (Lys-C) were purchased from Boehringer Mannheim. D-MEM, fetal bovine serum, trypsin, and PBS 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 (RP-HPLC) (23). Purity and structure of the peptides were confirmed by analytical RP-HPLC, amino acid analysis, and electron spray mass spectrometry (see Table I). Radioiodinations of [Nle8,18,Tyr34]bPTH-(1-34)NH2 and [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]bPTH-(1-34)-NH2, and RP-HPLC purifications were performed as described previously (24).

                              
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Table I
Physicochemical characterization of the Bpa-containing PTH-(1-34) analogs

Cell Culture-- HEK-293 cells and HEK-293/C-21 cells stably expressing hPTH1-Rc (~400,000 receptors/cell) were cultured in D-MEM supplemented with 10% fetal bovine serum as described (25).

PTH1-Rc Binding-- HEK293/C-21 cells were subcultured in polylysine-coated 24-well plates and grown to confluence. Radioreceptor assays were carried out as described previously (23).

Adenylyl Cyclase Activity-- HEK293/C-21 cells were subcultured in 24-well plates and grown to near confluence. COS-7 cells transiently expressing mutant receptors were subcultured 24 h following transfection at a density of 2 × 105/well in 24-well plates and assayed for adenylyl cyclase activity 72 h after transfection. Activation of adenylyl cyclase by PTH analogs was determined as described (23).

Intracellular Calcium Determinations-- The stimulation of increases in intracellular calcium levels following treatment by PTH-(1-34) and the Bpa-containing analogs was assessed spectroscopically in Fura-2-loaded HEK-293/C-21 cells as described (26).

Photoaffinity Cross-linking, Membrane Protein Preparation, and SDS-PAGE Purification-- Photoaffinity cross-linking of 125I-[Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]bPTH-(1-34)NH2 was carried out as described (17). Briefly, confluent HEK-293/C-21 cells were harvested with 0.5 mM EDTA, washed twice with PBS, and resuspended in D-MEM at a density of ~60 × 106 cells/ml. This suspension was incubated at room temperature for 30 min in the presence of 0.3 mCi (~0.5 nmol) of 125I-Bpa1-PTH-(1-34) after which cells were placed on ice in a Stratalinker (Stratagene) at a distance of ~10 cm from six 15-watt 365 nm UV lamps and irradiated for 1 h. Cells were then washed five times with PBS, resuspended in 50 mM Tris, pH 8.5, and lysed by five cycles of freezing and thawing. Membranes were obtained by centrifugation at 45,000 rpm for 2 h at 4 °C. Membranes were solubilized in 25 mM Tris, pH 8.5, containing Triton X-100 (2% v/v) at room temperature for 2 h.

Proteins were precipitated by adding five volumes of cold acetone and redissolved in 25 mM Tris, pH 8.5, containing SDS (2% w/v). Proteins were reduced with 100 mM dithiothreitol for 2 h at 37 °C and alkylated with 200 mM iodoacetamide for 15 min at room temperature. The solution was desalted and concentrated on Centricon 50 (Amicon), diluted with reducing Laemmli sample buffer, and loaded on a 7.5% (v/v) SDS-PAGE. After autoradiography, the radioactive 125I-Bpa1-PTH-(1-34)-Rc conjugate was excised from the gel, passively eluted in 100 mM NH4HCO3/SDS (0.01% v/v), pH 7.5, and submitted to concentration and buffer exchange on Centricon 50 (Amicon) to 25 mM Tris, pH 8.5, containing Triton X-100 (0.1% v/v) and SDS (0.01% v/v). Small scale photoaffinity cross-linking of transiently transfected COS-7 cells, grown to overconfluence, was carried out in 24-well tissue culture plates. Cells were washed with D-MEM and were treated with 200 µl of D-MEM and either 25 µl of 10-5 M PTH-(1-34) in vehicle (PBS, 0.1% BSA), or vehicle alone. Reactions were incubated 15 min at room temperature, 1-2 × 106 cpm of 125I-Bpa1-PTH-(1-34) (total volume 25 µl) added to each well, and incubated an additional 15 min at room temperature. Plates were cross-linked in a Stratalinker for 30 min as described earlier. Each well was washed once with PBS, cells lysed with 0.5 ml of Laemmli sample buffer, shaken in dish for 10-30 min, and harvested into Eppendorf tubes. Tubes were incubated on a rotating platform at room temperature for 2-3 h, and analyzed by SDS-PAGE.

Enzymatic and Chemical Digestions of the 125I-Bpa1-PTH-(1-34)-Receptor Conjugate-- Batches of SDS-PAGE-purified radiolabeled hormone-receptor conjugate and fragments were prepared in small volumes (typically 10-20 µl) of 25 mM Tris-HCl, pH 7.4, 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 two 24-h treatments with 0.15 units (in 10 µl of water) at 37 °C. BNPS-skatole digestions were carried out with 2 mg/ml BNPS-skatole in 70% acetic acid at 37 °C for 24-48 h in the dark. CNBr digestions were performed with 50 mg/ml CNBr in 70% formic acid at 37 °C for 24 h in the dark. Samples were dried on Speed-Vac and dissolved in reducing sample buffer (27) prior to electrophoresis.

Electrophoresis and Autoradiography-- Electrophoretic analyses were performed with 7.5% SDS-PAGE for the hormone-receptor conjugates and 16.5% Tricine/SDS-PAGE for the cleavage products. Appropriate molecular weght markers (Amersham Pharmacia Biotech and Bio-Rad) were included in each gel. Gels were dried and exposed to x-ray films (X-Omat, Eastman Kodak Co.) with intensifying screens (XAR-5, Eastman Kodak Co.). Following autoradiography, the radioactive fragments were excised from the dried gels, extracted in 100 mM NH4HCO3, pH 7.5, SDS (0.01% w/v) and concentrated on Speed-Vac.

Receptor Mutagenesis-- Single mutations, M414L and M425L, were introduced into the hPTH1-Rc cDNA generating two mutated hPTH1[M414L] and hPTH1[M425L] Rcs, respectively. Primer pairs (sense and antisense) were prepared containing these amino acid modifications (Life Technologies, Inc. custom primers): sense M414L (5' to 3'): CCACGCTGGTGCTCCTGCCCCTCTTTGGCGTC; sense M425L (5' to 3'): CACTACATTGTCTTCCTGCCACACCATACACC. Primer pairs were used in the polymerase chain reaction-based Quik-Change site-directed mutagenesis kit (Stratagene), using the hPTH1-Rc (28) in the pZeoSV2 (Invitrogen) mammalian expression vector as a template. Individual polymerase chain reaction reactions were used to transform DH5alpha competent cells (Life Technologies, Inc.). Transformations were plated on bacteriologic agar containing Zeocin, colonies identified and selected for plasmid isolation (Miniprep Kit, Quiagen). Plasmid preparations were cycle sequenced (Genomyx, Foster City, CA) to confirm the fidelity of the mutations, using oligonucleotide primers located 5' to the regions of the hPTH1-Rc targeted for mutation. The entire mutant Rc was subsequently completely sequenced, on both strands, to ensure the single Met right-arrow Leu mutation.

Transient Transfection-- COS-7 cells were plated at 5-7.5 × 105 cells/10-cm dish 24 h prior to transient transfections. Ten µg of either mutant or native receptor construct were co-transfected with 10 µg carrier DNA using calcium/phosphate (Life Technologies, Inc.). For adenylyl cyclase assay and photoaffinity cross-linking, transiently transfected cells were subcultured as described above.

Molecular Modeling-- The molecular model of the hPTH1-Rc was developed using the topological arrangement of the TM helices of rhodopsin (29). To identify the location of the TM portions of the hPTH1-Rc, assumed to be alpha -helices, a hydrophobicity profile (30) was calculated for the hPTH1-Rc sequence. Each of the initially identified transmembrane domains, expanded by approximately 15 amino acids on each side, was submitted to a BLAST search (31). The results helped to refine the location of the TM helices; there was good agreement with respect to the location of the helices from these two methods. After placing the identified helices of the hPTH1-Rc onto the rhodopsin template, the helices were rotated about their long axis to orient the hydrophobic moment toward the membrane environment. These orientations were then refined, requiring minor adjustments, following the substitution-table methodology reported by Donelly and co-workers (32). The loops connecting the TM helices were added to complete the model. In an attempt to develop the conformational preferences of the ectopic N-terminal portion of the receptor, the corresponding sequences were submitted to a BLAST search (31). There were numerous sequences of significantly high homology in the protein data bank, especially for a region of the ectopic N-terminal tail of the hPTH1-Rc contiguous to the TM1 helix. The homologous regions of each of these protein structures were analyzed for secondary structural features and then incorporated into the molecular model.

To refine the molecular model, molecular dynamics (MD) simulations and energy minimization were carried out with the CVFF91 force field within the Discover program (Biosym/MSI). To mimic the environment of the membrane, a two-phase simulation cell consisting of H2O and CCl4 was utilized. The explicit solvent simulations were carried out following previously published procedures (33). All molecular modeling was carried out with the Insight II program (Biosym/MSI).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Characterization of Bpa-containing PTH-(1-34) Analogs-- Binding affinities for the hPTH1-Rc stably expressed in HEK-293 cells (clone C-21) were measured by competition with 125I-[Nle8,18,Tyr34]bPTH-(1-34)NH2 (125I-PTH-(1-34)) (Fig. 1A). Agonist activity (stimulation of adenylyl cyclase and increase in intracellular calcium levels) was determined in HEK-293/C-21 cells (25) (Fig. 1, B and C). The substitution of Ala1 with Bpa in PTH-(1-34) generated Bpa1-PTH-(1-34), which displays a pharmacological profile similar to that of the parent peptide PTH-(1-34) (IC50 ~4.5 nM; EC50 ~2 nM and [Ca2+]i = 130 nM at 10-7 M ligand and IC50 ~25 nM, EC50 ~0.8 nM and [Ca2+]i = 100 nM at 10-7 M ligand for PTH-(1-34) and Bpa1-PTH-(1-34), respectively). Bpa substitution of Val2 caused a 17-fold reduction in binding affinity accompanied by a 10-fold reduction in adenylyl cyclase activity and only 50% mobilization of intracellular calcium relative to PTH-(1-34). Despite a 60-fold reduction in binding affinity, Bpa6-PTH-(1-34) displayed full potencies for the stimulation of both adenylyl cyclase and intracellular calcium transients (Fig. 1). Substitution at positions 3, 4, and 5 led to analogs with very weak binding affinity, weak stimulation of adenylyl cyclase, and no effect on [Ca2+]i levels (Fig. 1). Since [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]bPTH-(1-34)NH2 (Bpa1-PTH-(1-34)) displayed a biological profile similar to that of the parent peptide and its N-terminal photoreactive residue (Bpa) is located at a strategic site, it was selected for the photoaffinity labeling studies described below.


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  		Fig. 1.   In vitro characterization of the Bpa-containing PTH-(1-34) analogs. Competition for 125I-PTH-(1-34) binding (A) and dose-response curves for the stimulation of adenylyl cyclase activity (B) in HEK-293/C-21 cells by PTH-(1-34) (bullet ), Bpa1-PTH-(1-34) (open circle ), Bpa2-PTH-(1-34) (×), Bpa3-PTH-(1-34) (black-square), Bpa4-PTH-(1-34) (), Bpa5-PTH-(1-34) (black-triangle), and Bpa6-PTH-(1-34) (triangle ) are shown. Experiments were carried out in triplicate. Curves in panels A and B show the mean ± S.E. of three independent experiments. C, stimulation of intracellular calcium release by 10-7 M PTH-(1-34) and Bpa-containing PTH-(1-34) analogs in Fura-2-loaded HEK-293/C-21 cells obtained in a single experiment. The numbers of the bars refer to the following compounds: 1, PTH-(1-34), 2, Bpa1-PTH-(1-34), 3, Bpa2-PTH-(1-34), 4, Bpa3-PTH-(1-34), 5, Bpa4-PTH-(1-34), 6, Bpa5-PTH-(1-34), and 7 Bpa6-PTH-(1-34). Similar results were obtained in two additional experiments.

Photoaffinity Labeling of the hPTH1-Rc with 125I-Bpa1-PTH-(1-34)-- The structural identity of the radioligand 125I-Bpa1-PTH-(1-34) was confirmed by its coelution with the non-radioactive [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,(3-iodo)-Tyr34]bPTH-(1-34)NH2 on analytical RP-HPLC (data not shown).

Photocross-linking of 125I-Bpa1-PTH-(1-34) to the hPTH1-Rc yielded a single diffuse band migrating at ~87 kDa on 7.5% SDS-PAGE (Fig. 2A, lane 2). This band is receptor-specific, since it is not observed in similar experiments in receptor-lacking parental HEK-293 cells (Fig. 2A, lane 1). Moreover, formation of the ligand-receptor conjugate was completely inhibited in the presence of excess (1 µM) unlabeled agonist PTH-(1-34) or antagonist PTH-(7-34) (Fig. 2A, lanes 3 and 4, respectively). The apparent molecular mass of the cross-linked band was similar to that observed for the conjugate obtained through photocross-linking of a position 13 benzophenone-containing PTH-(1-34) analog, [Nle8,18,Lys13(epsilon -pBz2),2-L- Nal23,Tyr34]bPTH-(1-34)NH2, to the same hPTH1-Rc-expressing cells (28). Endo-F-mediated deglycosylation of the 87-kDa band shifted the complex to ~70-kDa band (Fig. 2B, lane 2) as described previously (17, 34-36).


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  		Fig. 2.   Photoaffinity cross-linking of 125I-Bpa1-PTH-(1-34) to the recombinant hPTH1-Rc. A, autoradiography of non-transfected HEK-293 cells (lane 1) and hPTH1-Rc expressing HEK-293/C-21 cells photolabeled with 125I-Bpa1-PTH-(1-34) alone (lane 2) or in the presence of competition by 10-6 M PTH-(1-34) (lane 3) and PTH-(7-34) (lane 4). The arrow indicates the position of the ~87-kDa cross-linked hPTH1-Rc. B, Endo-F-mediated deglycosylation of the 125I-Bpa1-PTH-(1-34)-hPTH1-Rc conjugate. The ~87-kDa labeled conjugate was incubated in the absence (lane 1) or presence (lane 2) of endoglycosidase F/N-glycosidase F. The arrow indicates the position of the ~66-kDa deglycosylated labeled receptor. Samples were loaded on 7.5% (w/v) SDS-PAGE. Molecular mass markers are also shown. Similar results were obtained in two additional experiments.

Identification of the Ligand Binding Domain-- The 87-kDa 125I-Bpa1-PTH-(1-34)-Rc conjugate was purified from 7.5% SDS-PAGE and subjected to a series of chemical and enzymatic cleavages. The first digestion pathway (I) consisted of enzymatic cleavage at the carboxyl side of lysyl residues with Lys-C, followed by chemical cleavage at the carboxyl side of tryptophanyl residues with BNPS-skatole. Exhaustive Lys-C treatment of the 87-kDa ligand-receptor conjugate yielded a single radiolabeled band with apparent molecular mass of ~11 kDa (Ia) (Fig. 3A, lane 2). Similar treatment of the deglycosylated conjugate (~66 kDa) yielded a band with the same apparent molecular mass (data not shown), confirming the absence of glycosylation within the Lys-C-generated fragment Ia. BNPS-skatole treatment of the excised and eluted 11-kDa (Ia) fragment produced a single band with apparent mass of ~7 kDa (Ib) (Fig. 3A, lane 3).


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  		Fig. 3.   Chemical and enzymatic digestions of the 125I-Bpa1-PTH-(1-34)-hPTH1-Rc conjugate. A, the SDS-PAGE purified ~87-kDa conjugate was incubated in the absence (lane 1) or presence (lane 2) of Lys-C. The excised and eluted Lys-C-derived 11-kDa (Ia) fragment was then treated with BNPS-skatole (Ib) (lane 3). Samples were loaded on 16.5% (w/v) Tricine/SDS-PAGE. Molecular mass markers are also shown. B, the SDS-PAGE purified ~87-kDa conjugate was incubated in the absence (lane 1) or presence of BNPS-skatole (lane 2). The excised and eluted ~14-kDa (IIa) band was then treated with Lys-C (IIb) (lane 3). Samples were loaded on 16.5% (w/v) Tricine/SDS-PAGE. Molecular mass markers are also shown. C, the SDS-PAGE of a mixture of the purified ~87-kDa conjugate in the presence of free ligand (lane 1), and the purified ~87-kDa conjugate incubated in the presence of cyanogen bromide (IIIa) (lane 2). Samples were loaded on 16.5% (w/v) Tricine/SDS-PAGE. Molecular mass markers are also shown. Similar results were obtained in three additional experiments.

A second digestion pathway (II), the reciprocal of I, initially yielded a single band migrating at ~14 kDa (IIa) (Fig. 3B, lane 2). Lys-C treatment yielded the final fragment migrating at ~7 kDa (IIb) (Fig. 3B, lane 3), similar to Ib obtained from pathway I.

Treatment with CNBr (III) of the purified intact ligand-receptor conjugate in 70% formic acid solution produced a band of very low apparent molecular mass (IIIa) (~4 kDa, Fig. 3C, lane 2), with electophoretic mobility similar, if not identical, to that of the free radioligand 125I-Bpa1-PTH-(1-34) (Fig. 3C). In addition, CNBr treatment of the 11-kDa (Ia) fragment obtained after Lys-C digestion produced a similar ~4-kDa band (data not shown).

Fig. 4 summarizes schematically the different fragmentation pathways employed in the analysis of hPTH1-Rc-125I-Bpa1-PTH-(1-34) conjugate.


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  		Fig. 4.   Schematic summary of the fragmentation pattern observed for the 125I-Bpa1-PTH-(1-34)-hPTH1-Rc following pathways I (------), II (- - -), and III (···). Endo-F, BNPS-skatole, Lys-C, and cyanogen bromide digestions were carried out as detailed under "Experimental Procedures." - · - indicates data not shown. Molecular masses of the fragments are indicated in kDa and represent the actual size of the digested conjugate fragments including the ligand 125I-Bpa1-PTH-(1-34) (molecular weight 4489).

Characterization of Transiently Transfected COS-7 Cells Expressing Mutated hPTH1-Rc-- PTH-(1-34) stimulation of adenylyl cyclase in COS-7 cells transiently expressing either the hPTH1[M414L], hPTH1[M425L], or the native hPTH-Rc's resulted in very similar dose-response curves (Fig. 5A). Point mutation of either Met414 or Met425 to Leu does not alter the receptor's response to PTH-(1-34). However, the mutants differ in their ability to cross-link with 125I-Bpa1-PTH-(1-34) (Fig. 5B). The transiently transfected hPTH1[M414L] and native hPTH Rcs cross-link to 125I-Bpa1-PTH-(1-34) (Fig. 5B, lanes 1 and 3, respectively) generating the anticipated ~87-kDa band corresponding to the 125I-Bpa1-PTH-(1-34)-PTH1Rc conjugate. This cross-linking can be inhibited competitively by 10-6 M PTH-(1-34) (Fig. 5B, lanes 2 and 4). In contrast, the functional transiently transfected hPTH1[M425L] does not cross-link to 125I-Bpa1-PTH-(1-34) (Fig. 5B, lanes 5 and 6), suggesting that Met425 may be involved in the cross-linking of 125I-Bpa1-PTH-(1-34).


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  		Fig. 5.   Characterization of COS-7 cells transiently expressing native and point mutant hPTH1-Rc. A, stimulation of adenylyl cyclase (cAMP in pmol/well above basal) by PTH-(1-34) in COS-7 cells transiently expressing native hPTH1-Rc (bullet ), and receptor containing mutations M414L (open circle ) and M425L (×). B, SDS-PAGE analysis of 125I-Bpa1-PTH cross-linking to COS-7 cells transiently transfected with native hPTH1-Rc (lanes 1 and 2), and receptor containing mutations M414L (lanes 3 and 4), and M425L (lanes 5 and 6) in the absence (lanes 1, 3, and 5) or presence of 10-6 M PTH-(1-34) (lanes 2, 4, and 6). Size markers (in kDa) are also shown. The arrow to the right of lane 6 indicates the location of the 125I-Bpa1-PTH-PTH1-Rc conjugate (~87 kDa).

Molecular Modeling-- Results from the BLAST search of hPTH1-Rc-(172-189), consisting of the distal end of the ectopic N-terminal domain contiguous with TM1, indicate a high probability of alpha -helix encompassing residues Lys172-Arg186, and possibly a few additional residues on either side. The homologous regions identified in this search are listed in Table II. Equally important, in many of the protein structures examined, the topological location of the homologous helices was on the surface of the protein, dictated by the relative amphipathic nature of these alpha -helices. Given the apparent amphipathicity of the Lys172-Met189 helix, we assume it is lying on the surface of the membrane, with the hydrophilic amino acids projecting into the aqueous phase. This ectopic helix is extended by an extracellular loop consisting of a few amino acids, Leu187-Gly188-Met189. An important point to emphasize is that the BLAST results clearly suggest a conformational discontinuity between the TM1 helix and this ectopic helix.

                              
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Table II
Sequence homology analysis of the hPTH1-Rc-(172-189)

Although these data support the presence of an amphipathic alpha -helix, the orientation of this helix with respect to the bundle of TM helices is not clear. Molecular dynamics simulation of many different starting orientations of the helix were carried out using the two phase simulation cell (33). Throughout the molecular dynamics simulations, the ectopic helix always tended to move away from the bundle of TM helices. Therefore, for the purpose of docking the ligand hPTH-(1-34), a conformation of hPTH1-Rc with the ectopic amphipathic helix projecting away from the TM bundle was utilized.

Molecular modeling of hPTH-(1-34) with the hPTH1-Rc model was then performed, guided by the contact domain identified by previous photoaffinity cross-linking studies (17) (Fig. 6). The conformation of hPTH-(1-34) used in the molecular modeling was obtained from our high resolution NMR studies performed in different environments, including aqueous saline conditions and the presence of dodecylphosphocholine as a membrane mimetic (37).


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  		Fig. 6.   Model for the binding of hPTH-(1-34) to hPTH1-Rc. For clarity, only portions of the TM helices, N terminus, and the third extracellular loop are shown in blue (non-cross-linked domains) and green (contact domains 173-189 and 409-437) (A, side view; B, top view). The amphipathic alpha -helix of the extracellular N terminus of the receptor is projecting to the right, lying on the surface of the membrane. The high resolution, low energy structure of hPTH-(1-34) determined by NMR in a micellar environment is presented in pink. Residues in cross-linking positions 1 and 13 of the hPTH-(1-34) are denoted in yellow. The C-terminal amphipathic alpha -helix of hPTH-(1-34) is aligned in a antiparallel arrangement with the amphipathic alpha -helix of the extracellular N terminus (173-189), contiguous with TM1 and encompassing the 17 amino acid contact domain (in green), to optimize the hydrophilic interactions. Side chains of residue Met414 and Met425 within the "contact domain" TM6-third extracellular loop (hPTH1-Rc[Ser409-Trp437]) are shown in detail.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The characterization of the bimolecular interaction between the activation domain of PTH-(1-34) and the hPTH1-Rc is of fundamental importance for elucidating the molecular mechanism of signal transduction. To this end, photoaffinity cross-linking of bioactive analogs enables direct identification of "contact domains" and/or "contact points" between ligand and receptor (17-21, 38-42). The goal of this study was to identify the region of the hPTH1-Rc which is in direct contact with the principal "activation domain" of PTH-(1-34). Residues 1 through 6 (43), particularly positions 1 and 2, have been shown to be essential for full agonist activity. Stepwise deletion of N-terminal residues from PTH-(1-34) yields the antagonist/partial agonist PTH-(3-34) and the antagonist PTH-(7-34) (16, 44) with little diminishment of binding affinity, but with progressive loss of agonist bioactivity.

The photoreactive [Bpa1-6]PTH-(1-34) analogs were specifically designed for this study. Both Met8 and Met18 were replaced by the isosteric Nle residue, rendering the ligand resistant to cyanogen bromide treatment. Replacement of Trp23 with 2-naphthylalanine (2-Nal) introduces stability toward digestion by Trp-specific reagents. Replacement of all Lys residues with Arg provides resistance to Lys-C-mediated cleavage, and replacement of Phe34 with Tyr generates a reactive site for incorporation of radioiodine. These modifications were introduced singularly and found to be well-tolerated (23, 43). As demonstrated previously, the combination of all the modifications is, in many cases, also well tolerated (17, 23).

Based on the protein sequence of hPTH1-Rc, an exhaustive Lys-C digestion of the 125I-Bpa1-PTH-(1-34)-hPTH1-Rc cross-linked conjugate should generate three fragments with molecular masses of approximately 6.5 kDa: hPTH1-Rc-(487-539), -(409-471), and -(173-240) (5,410.1, 7,291.5, and 8,031.5 Da, respectively). The generation of the Ia fragment (Fig. 4) from the deglycosylated receptor eliminates hPTH1-Rc-(173-240), which contains a documented glycosylation site at Asn176 (17). The hPTH1-Rc-(487-539) fragment does not contain a tryptophan and therefore will not be cleaved when treated with BNPS-skatole. Therefore, hPTH1-Rc-(409-471) represents the region in the receptor cross-linked to the ligand in conjugate Ia (Figs. 4 and 3A, lane 2).

The smallest and sole overlapping sequence among the BNPS-skatole-generated fragments from both reciprocal digestion pathways (pathway I: hPTH1-Rc-(409-437) and -(438-471), and pathway II: hPTH1-Rc-(1-69), -(362-437), and -(478-589)) (Fig. 4) is hPTH1-Rc-(409-437) (mass = 3273.9 Da) which is represented in the ~7-kDa Ib/IIb band (Fig. 3, A and B). Therefore, hPTH1-Rc-(409-437) is the minimal contact domain that interacts with position 1 in the PTH analog, Bpa1-PTH-(1-34). This region contains two Met residues (positions 414 and 425).

Exhaustive cyanogen bromide digestion of the intact 125I-Bpa1-PTH-(1-34)-PTH1-Rc conjugate (87 kDa) (III) (Fig. 4) and of the Lys-C-generated fragment Ia (11 kDa) yields a similar band IIIa with a low apparent molecular mass (~4 kDa) (Fig. 3C, lane 2). The electrophoretic mobility of this band is similar to that of the ligand itself (mass = 4,487 Da) and distinctly lower than any potential covalent ligand-receptor complex produced by cyanogen bromide cleavage.

Photocross-linking of a benzophenone-containing ligand through insertion into a C-H bond of the S-CH3 group in Met residues will generate upon cyanogen bromide treatment a ligand-CH3SCN adduct, which increases its molecular mass by only 73 Da (19). Electrophoretically, the CH3SCN-125I-Bpa1-PTH-(1-34) adduct will be indistinguishable from the non-modified photoreactive radioligand (Fig. 3C). This adduct could be generated by cross-linking to either one of the two methionine residues present in the minimal contact domain hPTH1-Rc-(409-437) and included in the conjugate fragment (Ib/IIb) (Figs. 4, 3A, lane 3, and 3B, lane 3). Both methionines, Met414 and Met 425, are located in TM6 and are therefore potential contact points between the N-terminal residue of PTH and the receptor.

Using site-directed mutagenesis to produce both M414L and M425L mutated Rcs, the Met residue involved in the cross-linking can be assigned to Met425. The M414L mutant is fully active and like the native PTH1-Rc it photocross-links to 125I-Bpa1-PTH-(1-34). In contrast, M425L mutant, although fully active, does not cross-link to 125I-Bpa1-PTH-(1-34). Therefore, this mutation perturbs the close spatial proximity required for effective cross-linking with an N-terminal benzophenone-containing ligand. The identification of Met425 as the contact point for position 1 of PTH-(1-34) provides an additional, and important constraint in defining the topology of the PTH-(1-34)-hPTH1-Rc complex.

The structure of the hPTH1-Rc obtained by homology modeling and MD using a two-phase solvent cell (33) suggests that the segment Arg179-Arg189 consists of an amphipathic alpha -helix whose axis is parallel to the membrane surface and directed away from the helical bundle of the receptor. The structure of hPTH-(1-34) used in the molecular modeling was determined by NMR in a zwitterionic, micellar environment (37) as a mimetic of the cellular membrane. Throughout the MD simulations, deviations from the experimentally determined structure of hPTH-(1-34), consisting of alpha -helices for residues 4-10 and 20-32, were not allowed. A number of starting structures with Lys13 of hPTH placed at different locations along the 17-amino acid cross-linking domain (hPTH1-Rc-(173-189)) (17) were used for MD simulations. During these simulations, utilizing the biphasic solvent mixture to mimic the membrane environment (33), the interactions between the amphipathic helix of the receptor, segment 179-186, just exterior to TM1, and the C-terminal helix (residues 20-32) of hPTH were optimized. Throughout these simulations, the N-terminal residue of hPTH could be easily placed in close proximity to Met425, which is on the surface of the membrane at the C-terminal end of TM6 (Fig. 6). In contrast, all attempts to place position 1 of hPTH in close proximity to Met414, while maintaining the experimentally determined conformation of hPTH and Lys13 of hPTH close to its cross-linking domain, failed. In our model, Met414 is on the intracellular half of TM6, projecting toward the membrane, a full three helical turns removed from Met425. Thus, the biochemical analysis of the 125I-Bpa1-PTH-(1-34)-PTH1-Rc conjugate, site-directed mutagenesis of the hPTH1-Rc, and molecular modeling simulation strongly suggest that Met425 is the "contact point" of the hPTH1-Rc and the cross-linking site for 125I-Bpa1-PTH-(1-34).

Previous mutagenesis studies have implied that TM6 and the third extracellular loop are important for hormone binding and signal transduction. Homologous substitution of these regions in the rat Rc with the corresponding portions of either the opossum PTH1-Rc (45) or the secretin Rc (46) identified several residues (i.e. Leu427, Trp437) which affect hormone binding and/or signaling. Interestingly, mutation of Thr410 in TM6 generated a constitutively active receptor associated with the clinical skeletal disorder, Jansen's metaphyseal chondrodysplasia (47). TM6 seems to be directly involved in signaling in other G protein-coupled receptors. Mutations in this region of the m5 muscarinic (48) and alpha -factor (49) receptors result in constitutive receptor activation. In addition, TM6 is contiguous with the third intracellular loop, which has been implicated in the interaction of G proteins in several seven TM-domain-containing receptors, including the PTH1-Rc (50).

The identification of Met425 in the extracellular end of TM6 as the contact point for the N terminus of PTH, together with the emerging model of ligand-receptor interaction, offers new insights into the nature of hormone-receptor interactions and signal transduction in this system. The contact of the principal activation domain of PTH-(1-34) with residues in the extracellular end of TM6, which in turn is connected to the third intracellular loop (considered to contain a G protein contact domain) suggests a possible relay mechanism that communicates an extracellular stimulus (i.e. agonist binding) into an intracellular signaling event (i.e. activation of the G protein).

Our modeling of the bimolecular PTH-hPTH1-Rc interaction potentially can be greatly enhanced by identification of the specific amino acid in hPTH1-Rc involved in cross-linking with residue 13 of hPTH. Narrowing the 17-amino acid contact domain to a smaller fragment, plus identification of additional contact domains in the Rc with other amino acids of the hormone, will refine our experimentally based model and provide greater detail regarding the hPTH-hPTH1-Rc bimolecular interface.

    ACKNOWLEDGEMENT

We thank Alex Kelly (Clark University) for assistance in the initial stages of the molecular modeling studies.

    FOOTNOTES

* This work was supported, in part, by Grant RO1-DK47940 (to M. R.) and GM54082 (to D. F. M.) 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.

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

The abbreviations used are: PTH, parathyroid hormone; b, bovine; BNPS-skatole, 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine; Bpa, p-benzoylphenylalanineBpan-PTH-(1-34), [Bpan,Nle8,18,Arg13,26,27,L-2-NaI23,Tyr34]bPTH-(1-34)NH2Endo-F, endoglycosidase F/N-glycosidase FFura-2, fura-2/acetomethyl esterG protein, guanyl nucleotide-binding proteinh, humanHEK, human embryonic kidneyLys-C, lysyl endopeptidaseMD, molecular dynamicsNal, naphthylalanineNle, norleucinePTH-(1-34), [Nle8,18,Tyr34]bPTH-(1-34)NH2125I-PTH-(1-34), 125I-[Nle8,18,Tyr34]bPTH-(1-34)NH2PTH-(7-34), [Nle8,18,D-Trp12,Tyr34]bPTH-(7-34)NH2Rc, receptorRP-HPLC, reverse phase-high performance liquid chromatographyTM, transmembraneTricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineD-MEM, Dulbecco's modified Eagle's mediumPBS, phosphate-buffered salineBoc, t-butoxycarbonylPAGE, polyacrylamide gel electrophoresis.
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Top
Abstract
Introduction
Procedures
Results
Discussion
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Determining the Environment of the Ligand Binding Pocket of the Human Angiotensin II Type I (hAT1) Receptor Using the Methionine Proximity Assay
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Insights into the Structure and Molecular Basis of Ligand Docking to the G Protein-Coupled Secretin Receptor Using Charge-Modified Amino-Terminal Agonist Probes
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Parathyroid Hormone Secretion and Action: Evidence for Discrete Receptors for the Carboxyl-Terminal Region and Related Biological Actions of Carboxyl- Terminal Ligands
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Novel Parathyroid Hormone (PTH) Antagonists That Bind to the Juxtamembrane Portion of the PTH/PTH-related Protein Receptor
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Molecular Approximation between a Residue in the Amino-terminal Region of Calcitonin and the Third Extracellular Loop of the Class B G Protein-coupled Calcitonin Receptor
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Spatial Proximity between a Photolabile Residue in Position 19 of Salmon Calcitonin and the Amino Terminus of the Human Calcitonin Receptor
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Spatial Approximation between the Amino Terminus of a Peptide Agonist and the Top of the Sixth Transmembrane Segment of the Secretin Receptor
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Identification of a Contact Site for Residue 19 of Parathyroid Hormone (PTH) and PTH-Related Protein Analogs in Transmembrane Domain Two of the Type 1 PTH Receptor
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Spatial Approximation between Two Residues in the Mid-region of Secretin and the Amino Terminus of Its Receptor: INCORPORATION OF SEVEN SETS OF SUCH CONSTRAINTS INTO A THREE-DIMENSIONAL MODEL OF THE AGONIST-BOUND SECRETIN RECEPTOR
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Sauvagine Cross-links to the Second Extracellular Loop of the Corticotropin-releasing Factor Type 1 Receptor
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Identification of Determinants of Inverse Agonism in a Constitutively Active Parathyroid Hormone/Parathyroid Hormone-related Peptide Receptor by Photoaffinity Cross-linking and Mutational Analysis
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Regions in Rat and Human Parathyroid Hormone (PTH) 2 Receptors Controlling Receptor Interaction with PTH and with Antagonist Ligands
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Analysis of Parathyroid Hormone (PTH)/Secretin Receptor Chimeras Differentiates the Role of Functional Domains in the PTH/ PTH-Related Peptide (PTHrP) Receptor on Hormone Binding and Receptor Activation
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K. B. Jonsson, M. R. John, R. C. Gensure, T. J. Gardella, and H. Juppner
Tuberoinfundibular Peptide 39 Binds to the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor, but Functions as an Antagonist
Endocrinology, February 1, 2001; 142(2): 704 - 709.
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Structural Insights into the Amino-Terminus of the Secretin Receptor: I. Status of Cysteine and Cystine Residues
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Uncovering Molecular Mechanisms Involved in Activation of G Protein-Coupled Receptors
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Photoaffinity Cross-linking Identifies Differences in the Interactions of an Agonist and an Antagonist with the Parathyroid Hormone/Parathyroid Hormone-related Protein Receptor
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The Hydrophobic Residues Phenylalanine 184 and Leucine 187 in the Type-1 Parathyroid Hormone (PTH) Receptor Functionally Interact with the Amino-terminal Portion of PTH-(1-34)
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Studies of the N-Terminal Region of a Parathyroid Hormone-Related Peptide(1-36) Analog: Receptor Subtype-Selective Agonists, Antagonists, and Photochemical Cross-Linking Agents
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M. Mannstadt, H. Juppner, and T. J. Gardella
Receptors for PTH and PTHrP: their biological importance and functional properties
Am J Physiol Renal Physiol, November 1, 1999; 277(5): F665 - F675.
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Endocytosis of Ligand-Human Parathyroid Hormone Receptor 1 Complexes Is Protein Kinase C-dependent and Involves beta -Arrestin2. REAL-TIME MONITORING BY FLUORESCENCE MICROSCOPY
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Direct Identification of Two Contact Sites for Parathyroid Hormone (PTH) in the Novel PTH-2 Receptor using Photoaffinity Cross-Linking
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Identification of an Interaction between Residue 6 of the Natural Peptide Ligand and a Distinct Residue within the Amino-terminal Tail of the Secretin Receptor
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Measurement of Agonist and Antagonist Ligand-Binding Parameters at the Human Parathyroid Hormone Type 1 Receptor: Evaluation of Receptor States and Modulation by Guanine Nucleotide
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M. D. Luck, P. H. Carter, and T. J. Gardella
The (1-14) Fragment of Parathyroid Hormone (PTH) Activates Intact and Amino-Terminally Truncated PTH-1 Receptors
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Arginine 186 in the Extracellular N-Terminal Region of the Human Parathyroid Hormone 1 Receptor Is Essential for Contact with Position 13 of the Hormone
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M. Dong, Y. W. Asmann, M. Zang, D. I. Pinon, and L. J. Miller
Identification of Two Pairs of Spatially Approximated Residues within the Carboxyl Terminus of Secretin and Its Receptor
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L. Jin, S. L. Briggs, S. Chandrasekhar, N. Y. Chirgadze, D. K. Clawson, R. W. Schevitz, D. L. Smiley, A. H. Tashjian, and F. Zhang
Crystal Structure of Human Parathyroid Hormone 1-34 at 0.9-A Resolution
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M. Shimizu, P. H. Carter, and T. J. Gardella
Autoactivation of Type-1 Parathyroid Hormone Receptors Containing a Tethered Ligand
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A. Piserchio, T. Usdin, and D. F. Mierke
Structure of Tuberoinfundibular Peptide of 39 Residues
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S. R. J. Hoare, J. A. Clark, and T. B. Usdin
Molecular Determinants of Tuberoinfundibular Peptide of 39 Residues (TIP39) Selectivity for the Parathyroid Hormone-2 (PTH2) Receptor. N-TERMINAL TRUNCATION OF TIP39 REVERSES PTH2 RECEPTOR/PTH1 RECEPTOR BINDING SELECTIVITY
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L. Mouledous, C. M. Topham, H. Mazarguil, and J.-C. Meunier
Direct Identification of a Peptide Binding Region in the Opioid Receptor-like 1 Receptor by Photoaffinity Labeling with [Bpa10,Tyr14]Nociceptin
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T. Asano and M. Ashida
Transepithelially Transported Pro-phenoloxidase in the Cuticle of the Silkworm, Bombyx mori. IDENTIFICATION OF ITS METHIONYL RESIDUES OXIDIZED TO METHIONINE SULFOXIDES
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S. R. J. Hoare, T. J. Gardella, and T. B. Usdin
Evaluating the Signal Transduction Mechanism of the Parathyroid Hormone 1 Receptor. EFFECT OF RECEPTOR-G-PROTEIN INTERACTION ON THE LIGAND BINDING MECHANISM AND RECEPTOR CONFORMATION
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R. C. Gensure, T. J. Gardella, and H. Juppner
Multiple Sites of Contact between the Carboxyl-terminal Binding Domain of PTHrP-(1-36) Analogs and the Amino-terminal Extracellular Domain of the PTH/PTHrP Receptor Identified by Photoaffinity Cross-linking
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N. Shimizu, J. Guo, and T. J. Gardella
Parathyroid Hormone (PTH)-(1-14) and -(1-11) Analogs Conformationally Constrained by alpha -Aminoisobutyric Acid Mediate Full Agonist Responses via the Juxtamembrane Region of the PTH-1 Receptor
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M. Shimizu, J. T. Potts Jr., and T. J. Gardella
Minimization of Parathyroid Hormone. NOVEL AMINO-TERMINAL PARATHYROID HORMONE FRAGMENTS WITH ENHANCED POTENCY IN ACTIVATING THE TYPE-1 PARATHYROID HORMONE RECEPTOR
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