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Originally published In Press as doi:10.1074/jbc.M503272200 on June 1, 2005

J. Biol. Chem., Vol. 280, Issue 31, 28610-28622, August 5, 2005
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Insights into Interactions between the {alpha}-Helical Region of the Salmon Calcitonin Antagonists and the Human Calcitonin Receptor using Photoaffinity Labeling*

Vi Pham{ddagger}, Maoqing Dong§, John D. Wade{ddagger}, Laurence J. Miller§, Craig J. Morton||, Hooi-ling Ng||, Michael W. Parker||, and Patrick M. Sexton{ddagger}**

From the {ddagger}Howard Florey Institute, The University of Melbourne, Victoria 3010, Australia, §Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, Arizona 85259, and ||St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia

Received for publication, March 24, 2005 , and in revised form, June 1, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Fish-like calcitonins (CTs), such as salmon CT (sCT), are widely used clinically in the treatment of bone-related disorders; however, the molecular basis for CT binding to its receptor, a class II G protein-coupled receptor, is not well defined. In this study we have used photoaffinity labeling to identify proximity sites between CT and its receptor. Two analogues of the antagonist sCT(8-32) containing a single photolabile p-benzoyl-L-phenylalanine (Bpa) residue in position 8 or 19 were used. Both analogues retained high affinity for the CT receptor and potently inhibited agonist-induced cAMP production. The [Bpa19]sCT(8-32) analogue cross-linked to the receptor at or near the equivalent cross-linking site of the full-length peptide, within the fragment Cys134-Lys141 (within the amino terminus of the receptor, adjacent to transmembrane 1) (Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. (2004) J. Biol. Chem. 279, 6720-6729). In contrast, proteolytic mapping and mutational analysis identified Met49 as the cross-linking site for [Bpa8]sCT(8-32). This site differed from the previously identified cross-linking site of the agonist [Bpa8]human CT (Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 31177-31182) and may provide evidence for conformational differences between interaction with active and inactive state receptors. Molecular modeling suggests that the difference in cross-linking between the two Bpa8 analogues can be accounted for by a relatively small change in peptide orientation. The model was also consistent with cooperative interaction between the receptor amino terminus and the receptor core.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcitonins (CTs)1 are 32-amino acid peptide hormones with a wide spectrum of biological activity. The most recognized action is the inhibition of osteoclast-mediated bone resorption, which forms the basis for its primary clinical use in the treatment of bone-related disorders such as Paget disease, osteoporosis, and hypercalcemia of malignancy (1-3). CT, however, also has activity that includes modulation of renal ion excretion (4-7), analgesia (8), inhibition of appetite (9), and gastric acid secretion (10-12), as well as effects on reproduction via effects on embryological implantation and sperm function (13-15).

Calcitonin receptors (CTRs) belong to the class II subfamily of G protein-coupled receptors, which also includes receptors for other peptides such as parathyroid hormone (PTH) and PTH-related peptide, secretin, vasoactive intestinal peptide, glucagon, glucagon-like peptide-1, growth hormone-releasing hormone, calcitonin gene-related peptide, and corticotropin-releasing factor. These peptide hormone class II G protein-coupled receptors share 30-50% amino acid identity as well as a number of conserved structural motifs and are thought to interact with their ligands in a similar manner (16-18).

Alternative RNA splicing yields multiple CTR mRNA isoforms. In man, at least six potential variants exist (19-26); however, the most common hCTR isoforms differ by the presence (hCTRb) or absence (hCTRa) of a 16-amino acid insert between amino acids 174 and 175, within the first intracellular loop of the receptor (23). Of these, the hCTRa is the major human receptor isoform and is expressed in essentially all tissues known to express the CTR.

CTs from different species can be subdivided into three major classes: human/rodent, artiodactyl, and teleost/avian. Of these, the members of the teleost/avian group are generally the most potent, although relative potency varies in a species- and isoform-specific manner (19, 27-31). The higher potency combined with a longer in vivo half-life has led to fish-like CTs, exemplified by salmon CT (sCT), being the principle form of CT used for the clinical treatment of bone disorders (32, 33).

However, the usefulness of CT is limited by the development of clinical resistance. This can be due to the development of circulating antibodies against non-human CT (34-38), but it also occurs from loss of responsiveness to CT, presumably via receptor down-regulation and inhibition of new receptor synthesis (39-41). The optimal use of CTs remains unresolved, a situation that stems in part from lack of understanding of the bimolecular interaction between CT and its receptor and how this leads to receptor activation.



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  		FIG. 1.
Design of photoactive sCT analogues. Amino acid sequences of sCT and the two photoactive sCT(8-32) analogues used throughout the study are shown. For synthesis of the analogues, all the Lys residues at position 11 and 18 in sCT were replaced with Arg (Arg11,18, represented in gray) to derive fully active sCT analogues and render the ligands resistant to enzymatic cleavage by endoproteinase Lys-C, as required by the mapping scheme. This was followed by replacement of individual residue at position 8 and 19 (numbering refers to the full sequence) with a photolabile Bpa moiety (shown in black circles) across the sCT sequence. All peptides were oxidatively radioiodinated with 125I via Tyr22.

 
Like other class II receptor ligands, CT has a diffuse pharmacophore with residues throughout the peptide sequence contributing to binding affinity and/or agonist potency. Whereas it is clear that much of the binding energy for high affinity interaction of the peptide and its receptor derives from interaction of the peptide with the amino-terminal extracellular domain of the receptor (42-44), detailed information on how receptor and ligand interact is scant.

Photoaffinity labeling, which relies on the spatially restricted (within 3.1 Å) cross-linking of photolabile amino acids within peptide ligands and their receptors, presents a mechanism for establishing proximity between individual amino acids of a peptide ligand and small fragments (or even individual amino acids) of the receptor. This provides essential information about nature of the interaction. We have previously shown that position 19 of sCT is in close contact with the receptor region Cys134-Lys141 (45). In human CT (hCT), amino acid 16, located one helical turn apart from residue 19, cross-linked to Phe137, consistent with orientation of the {alpha}-helix of agonist peptides with the membrane-proximal region of the receptor amino terminus (46). We have also demonstrated that hCT amino acid 26 interacts with Thr30 in the distal amino terminus (46), whereas hCT amino acid 8 interacts with Leu368 in the third extracellular loop (47).

Amino-terminal truncation of the disulfide-bridged loop (amino acids 1-7) of sCT leads to the generation of potent antagonists (48, 49) and allows for comparative analysis of the site of cross-linking of equivalent amino acids of agonist and antagonist peptides. In this study we have generated antagonist peptides substituted with the photolabile amino acid p-benzoyl-L-phenylalanine (Bpa) at residue 8 ([Bpa8]sCT(8-32)) and 19 ([Bpa19]sCT(8-32)) and compared their interaction with the published sites of interaction for full-length agonist peptides. The data show that the [Bpa19]sCT(8-32) analogue cross-links to the equivalent domain of the receptor labeled by the full-length peptide. In contrast, [Bpa8]sCT(8-32) cross-linked to Met49 in the distal amino terminus, a region quite distinct from the site labeled by [Bpa8]hCT.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—125I-Na (specific activity, 2200 Ci/mmol) was purchased from Amersham Biosciences. sCT, sCT(8-32), hCT, and all amino acid derivatives were synthesized by Auspep (Parkville, Australia). CNBr, protein G-agarose beads, 3-isobutyl-1-methylxanthine, and iodoacetamide were obtained from Sigma. Sequencing grade endoproteinases Lys-C and Asp-N, N-glycosidase F, and Complete protease inhibitor mixture tablets were purchased from Roche Applied Science. Endoglycosidase F was prepared as previously described (50). Bovine serum albumin (BSA) was from ICN. Penicillin G/streptomycin was from Multicell Technologies (Warwick, RI). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, HEPES, fungizone, Lipofectamine transfection reagent, and protein molecular mass markers were obtained from Invitrogen. Tissue culture disposables and plastic ware were purchased from Falcon (Bedford, MA).

Synthesis of the sCT(8-32) Photoactive Analogues—The two sCT(8-32) antagonist analogues were synthesized with a Bpa moiety incorporated into either position 8 (designated as [Bpa8]sCT(8-32)) or position 19 (designated as [Bpa19]sCT(8-32)), where 8 is the most amino-terminal amino acid (Fig. 1). In addition, for each of the photoactive analogues, lysine residues at positions 11 and 18 in the native sCT sequence were replaced with arginines to render the ligand resistant to enzymatic cleavage by endoproteinase Lys-C; this substitution does not alter activity of the peptide (51). Both photoactive peptide analogues were prepared by solid phase peptide synthesis as described previously (45). Analysis of the synthetic sCT analogues, [Bpa19]sCT(8-32) and [Bpa8]sCT(8-32), by matrix-assisted laser desorption ionization time-of-flight mass spectrometry showed the principal products with the expected molecular masses (Bpa8 = 2937.6 Da and Bpa19 = 2925 Da), and amino acid analysis further validated the authenticity of the analogues. The synthetic peptides were purified to homogeneity by analytical reversed-phase high performance liquid chromatography (HPLC) in good overall yield.

Iodination of Peptides—sCT, [Bpa8]sCT(8-32), and [Bpa19]sCT(8-32) were iodinated using chloramine-T method, as described previously, with specific activity of about 700 Ci/mmol (29, 45). In some experiments, the [Bpa8]sCT(8-32) peptide analogue was also radiolabeled using IODO-BEAD (Pierce) as a solid-phase oxidant of a chloramine-T analogue followed by reversed-phase HPLC (52). Briefly, 15 µg of the [Bpa8]sCT(8-32) peptide was solubilized in 100 µl of 40% acetonitrile followed by 100 µl of 0.2 M borate buffer, pH 9.0, and 10 µl of Na125I (1 mCi). This reaction mixture was exposed to IODO-BEAD for 15 s and subsequently diluted with 0.5 ml of 0.1% trifluoroacetic acid before purification by reversed-phase HPLC on a C18 column. Fractions containing the radioactive peaks (specific activity, ~ 2000 Ci/mmol) from the HPLC were collected and stored in 50-µl aliquots at -20 °C until use.

Receptor Mutagenesis—The WT HA-hCTRa (Leu447 polymorphic variant) was generated as described previously (45). The CTR mutants M48I, M59L, and M376L were generated using QuikChange site-directed mutagenesis (Stratagene), using the WT HA-hCTRa as template. All other receptor mutants were generated as previously described (45). Oligonucleotide primer pairs (sense and antisense) were synthesized by GeneWorks (Hindmarsh, Australia). Selected clones were chosen for plasmid isolation (Qiagen), and the fidelity of mutations was confirmed by nucleotide sequencing (Australian Genome Research Facility, Parkville, Australia). Once the authenticity of mutagenesis was confirmed by sequencing, large-scale plasmid preparations were purified using a Qiagen kit (Qiagen).

Cell Culture and DNA Transfection—Monkey kidney epithelium (COS-7 and COS-1) or human embryonic kidney HEK-293 cells stably expressing the hCTRa were maintained in complete DMEM supplemented with either 5% heat-inactivated fetal bovine serum or fetal clone-2 (Hyclone Laboratories, Logan, UT), 100 units/ml penicillin G, 100 µg/ml streptomycin, 16 mM HEPES, and 50 µg/ml fungizone at 37 °C in a humidified atmosphere of 95% air/5% CO2. For radioligand receptor binding assay, COS-7 cells were seeded into 24-well plates. For cAMP assay and cross-linking studies, COS-7 cells were grown in 60-cm2 dishes and 140-cm2 dishes, respectively. Once the cell monolayers were at 95% confluence, cells were transfected in serum- and antibiotic-free DMEM using 0.1, 3, or 7 µg of plasmid DNA for 24-well plates, 60-cm2 dishes, and 140-cm2 dishes, respectively, as instructed by the manufacturer. Before addition to cells, DNA was complexed with the transfection lipid reagent Lipofectamine at a ratio of 1 µl/0.1 µg DNA. Following transfection, cells were incubated for at least 4 h at 37 °C in a CO2 incubator, and the culture medium was replaced with complete DMEM as described above. All transient transfections were performed with plasmids encoding either the WT HA-hCTRa or the required mutant constructs. Transfected cells were grown for at least 48 h before radioligand binding studies, cAMP assay, or photoaffinity labeling.

Receptor Binding Assays—The binding of the radiolabeled analogues to HA-hCTRa was assessed in 24-well plates as previously described (45). Briefly, the cells were incubated with ~90 pM 125I-sCT, 125I-sCT(8-32), or [125I-Bpa]sCT(8-32) analogue in binding buffer (DMEM and 0.1% BSA) in the absence (total binding) or presence of varying concentrations of unlabeled peptides. Cells were incubated for 1 h at 37 °C and subsequently washed with phosphate-buffered saline and lysed with 0.5 M NaOH. Nonspecific binding was determined in wells containing 1 µM unlabeled sCT(8-32), and maximal specific binding at each competing ligand concentration was calculated as a percentile of the total specific binding observed in the absence of competitor. The entire cell lysate was counted in a PerkinElmer Life Sciences {gamma}-irradiation counter to determine the bound radioactivity.

cAMP Assays—Intracellular cAMP assay was performed in 384-well plates using an Alpha Screen cAMP kit (PerkinElmer Life Sciences). In brief, transiently transfected COS-7 cells in 60-cm2 dishes were harvested, counted, and resuspended in stimulation buffer (phenol red-free DMEM, 0.1% BSA, and 1 mM 3-isobutyl-1-methylxanthine) and preincubated at 37 °C for ~20 min. Required concentrations of peptides and cAMP were prepared in stimulation buffer. To generate a cAMP standard curve, increasing concentrations of cAMP (10-11 to 10-6 M) were added to wells. To generate agonist dose-response curves, cells (10,000 cells/well) were then incubated at 37 °C for 30 min in the presence of increasing concentrations of agonists (10-13 to 10-8 M), either sCT or sCT analogues. To generate antagonist dose-response curves, cells (10,000 cells/well) were incubated at 37 °C for 30 min with increasing concentrations of hCT (10-12 to 10-5 M) in the presence of 0, 10-6, 10-7, or 10-8 M of the antagonist sCT(8-32) or its analogues.

Following incubation, cells were lysed with lysis buffer (5 mM HEPES, 0.3% Tween 20, and 0.1% BSA). Anti-cAMP acceptor beads (7.5 µg/ml), which were prepared in lysis buffer, were added to all wells and incubated at room temperature for 30 min in the dark. The detection mix of biotinylated cAMP (5 mM)/streptavidin donor beads (10 µg/ml) in lysis buffer, which was preincubated in the dark for 30 min at room temperature, was then added to all wells. The assay plate was incubated overnight at room temperature before reading on a Fusion plate reader (PerkinElmer Life Sciences). For each experiment, forskolin and cAMP dose-response curves were performed in parallel to allow translation of the {alpha}-screen signal to either cAMP or a percentage of the maximum forskolin response.

Data were analyzed using Graphpad Prism 4.02. (San Diego, CA). In each assay, the quantity of cAMP generated was back-calculated from the raw data using a cAMP standard curve. For agonist responses, concentration-effect curves were fitted to a four-parameter logistic equation (53).

For calculation of antagonist potency, agonist concentration-response curves in the absence and presence of antagonist were globally fitted to the following equation using Prism (53):

(Eq.1)

where Emax represents the maximal asymptote of the concentration-response curves, Emin represents the lowest asymptote of the concentration-response curves, pEC50 represents the negative logarithm of the agonist EC50 in the absence of antagonist, [A] represents the concentration of the agonist, [B] represents the concentration of the antagonist, nH represents the Hill slope of the agonist curve, s represents the Schild slope for the antagonist, and pA2 represents the negative logarithm of the concentration of antagonist that shifts the agonist EC50 by a factor of 2.

Photoaffinity Labeling of Analogues to the Receptors and Mapping of the Binding Domain—The radiolabeled photoactive peptide [Bpa19]sCT(8-32) or [Bpa8]sCT(8-32) was cross-linked to hCTRs expressed in intact COS-7 cells followed by multiple enzymatic/chemical cleavage as previously described (45). Briefly, WT or mutated receptor HA-hCTRa transiently transfected into COS-7 cells was incubated for 1 h in darkness with binding buffer (DMEM and 0.1% BSA) containing ~900 pM 125I-Bpa-substituted sCT(8-32). Labeled cells were washed with phosphate-buffered saline and immediately irradiated for 30 min with a 365-nm UV lamp on ice. Cells were collected in 0.1 M Tris and 10 mM EDTA, pH 7.3, and subsequently subjected to immunoprecipitation according to the methodology of Sengstag et al. (54). The precipitated receptors were then subjected to enzymatic or chemical cleavage using methods described previously (45). For the [Bpa8]sCT(8-32) probe, a modified approach was also used because it yielded more complete digestion of the receptor. Briefly, plasma membranes from COS-1 cells transiently expressing WT or mutant receptor (M59L, M376L, or M48I) containing 50 µg of protein were incubated with 0.1 nM 125I-[Bpa8]sCT(8-32) in Krebs-Ringer-HEPES buffer (0.25 M HEPES, pH 7.4, 1.04 M NaCl, 50 mM KCl, 10 mM KH2PO4, and 12 mM MgSO4)in the dark for 1 h. The reaction was immediately photolysed in a Rayonet photochemical reactor (Southern New England Ultraviolet, Hamden, CT) at 4 °C for 30 min and then washed twice with Krebs-Ringer-HEPES buffer. Photolabeled membranes were then subjected to reduction/alkylation (45) or solubilized in 1x Tris-glycine SDS sample buffer for SDS-PAGE followed by elution, lyophilization, ethanol/acetone precipitation, and endoglycosidase F/CNBr cleavage (55).

Polyacrylamide Electrophoresis and Autoradiography—Radioactive protein samples were warmed to 70 °C for 10 min and then analyzed using a combination of 10% SDS-glycine, 16.5% SDS-Tricine, or 10% NuPAGE bis-Tris pre-cast gels (Invitrogen), depending on the molecular weight of the protein of interest. Following electrophoresis, if necessary, gels were stained with Coomassie Blue G, destained, dried, and exposed to phosphorimaging screens (Fuji-Bas, Tokyo, Japan) for 1-5 days. The screens were then scanned and developed using Bio-Imaging Analyzer Bas 2000 Software (Fuji-Bas) and AIS Analytical Imaging (Imaging Research Inc., Ontario, Canada) to visualize radiolabeled fragments. The relative molecular weight of the radiolabeled bands was determined, by interpolation, on a plot of the mobility of protein standards versus the log values of their apparent molecular mass.

Molecular Modeling—The amino-terminal domain and the seven transmembrane (7TM) helical bundle of hCTRa were modeled separately and then combined to form a complete hCTRa structure. Homology modeling of the amino-terminal domain was carried out with COMPOSER as contained in Sybyl7.0 (Tripos, St. Louis, MO) using the NMR structure of the amino-terminal domain of corticotropin releasing factor receptor 2 (Protein Data Bank code 1U34) (56) as a template. An extensive amino-terminal tag sequence was removed from the template structure before modeling commenced. The relatively unstructured carboxyl-terminal end of the model was removed prior to docking of the amino terminus with the seven transmembrane helix (7TMH) receptor core as described below.

The 7TM helical bundle of hCTRa was built using the G protein-coupled receptor mode of SwissModel (57-59). The positions of the TM helices of the protein were identified through comparison of the results of three separate prediction algorithms: TMHMM (60), TMpred (61), and Toppred (62). For all seven helices, there was good agreement between the predicted position, with any helix varying by at most three residues. Once the helices had been assembled by SwissModel, the extracellular and cytoplasmic loops were added using Biopolymer as contained in Sybyl7.0 using the Loop Search method. The conserved disulfide bond between Cys219 and Cys289 was added during the loop building process, and the completed 7TM bundle was minimized using the Tripos forcefield.

Assembly of the completed models of the amino-terminal and 7TM domains into putative hCTRa structures was carried out using Hex (63) (www.csd.abdn.ac.uk/hex/). Docking was carried out with the Full Rotation search and Shape and Electrostatics fitting, with post-processing MM minimization as described in the Hex manual. The Hex centroid of the 7TM domain was moved toward the extracellular loops to help reduce selection of docking orientations where the amino-terminal domain was interacting with the cytoplasmic or membrane-inserted sections of the protein. The top five solutions from Hex were selected, and the two regions of the protein were joined using the Loop Tweak algorithm in Biopolymer. The carboxyl-terminal tail of the amino-terminal domain was rebuilt into each Hex solution in an optimal conformation to complete the structure, and then the entire model was minimized using the Tripos forcefield. The five final models were inspected using Sybyl.

A model of sCT was obtained from the NMR structure of eel CT (Protein Data Bank code 1BKU [PDB] ) (64). The model was constructed because the published NMR structure of sCT is S-sulfonated and unsuitable for our purposes. The model peptide was manually docked into each of the hCTRa models using Sybyl.



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  		FIG. 2.
Pharmacological characterization of the photoactive analogues on COS-7 cells transiently expressing HA-hCTRa. A, binding assays showed that the amino-terminally truncated analogues [Bpa8]sCT(8-32) and [Bpa19]sCT(8-32) competed with radiolabeled 125I-sCT for binding to the receptor with similar affinity to the parent antagonist sCT(8-32). Antagonist activity of sCT(8-32) (B), [Bpa8]sCT(8-32) (C), and [Bpa19]sCT(8-32) (D) of hCT-stimulated cAMP activation. All values are the mean ± S.E. of triplicate data from at least three independent experiments.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Pharmacological Characterization of the Antagonist Peptide Analogues—To explore the ability of photoactive sCT(8-32) analogues to bind to the HA-hCTRa transiently transfected into COS-7 cells, competition for 125I-sCT binding by the unlabeled cold analogue peptides were assessed. IC50 values of the antagonist analogues, [Bpa8]sCT(8-32) and [Bpa19]sCT(8-32), were 2.32 ± 0.14 and 3.77 ± 0.69 nM, respectively, similar to that observed for unmodified sCT(8-32) peptide (5.51 ± 0.13 nM) (Fig. 2A).

The major signaling pathway activated by the CTR is the production of cAMP, which occurs following activation of the enzyme adenylate cyclase by the G protein, G{alpha}s. Antagonism of hCT-induced cAMP production by sCT(8-32) analogues was assessed in COS-7 cells transiently transfected with the HA-hCTRa. [Bpa8]sCT(8-32) and [Bpa19]sCT(8-32) were each potent antagonists of hCT-induced cAMP accumulation with pA2 values of 8.91 ± 0.12 and 9.01 ± 0.30, respectively; the native sCT(8-32) had a pA2 value of 8.69 ± 0.21 (Fig. 2, B-D).

Following iodination, the unlabeled analogues competed with their corresponding radiolabeled analogues (125I-[Bpa8]sCT(8-32) and 125I-[Bpa19]sCT(8-32)) with high affinity, similar to that seen for unmodified sCT (Fig. 3, A and B). Therefore, the two analogues were suitable for photoaffinity labeling of the receptor and assessment of sites of proximity between specific individual amino acids of the peptide and the receptor.



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  		FIG. 3.
Binding characterization of radiolabeled probes. Competition for binding of the labeled probes 125I-[Bpa8]sCT(8-32) (A) and 125I-[Bpa19]sCT(8-32) (B) by either unmodified sCT(8-32) or homologous peptide analogue revealed that each radioligand behaved similarly to the corresponding unlabeled analogue.

 
Identification of the Labeling Domain of [Bpa8]sCT(8-32)—Photocross-linking of 125I-[Bpa8]sCT(8-32) to HEK-293 cells stably expressing the hCTRa revealed a radiolabeled band of Mr ~97,000 following resolution on SDS-glycine gels (Fig. 4A). The receptor labeling was decreased in a dose-dependent manner by competition with unlabeled [Bpa8]sCT(8-32) with a complete inhibition of binding seen at 10-6 M. Deglycosylation of the [Bpa8]sCT(8-32)·hCTRa complex shifted the Mr ~97,000 band to Mr ~52,000, the core size of the receptor protein (Fig. 4A).

Following elution from SDS-glycine gels, the radiolabeled native receptors or deglycosylated receptors were submitted to CNBr digestion. Fig. 4B illustrates that the cleavage product migrated with a Mr of ~3800 for both glycosylated and deglycosylated receptors, indicating an absence of N-glycosylation on the Bpa8-labeled fragment. Given the molecular mass of the probe (3060.6 Da), two candidate receptor fragments, Asp50-Met59 (1291 Da) and Leu368-Met376 (1100 Da), were investigated as potential sites for the interaction of Bpa8 (Fig. 4C); the latter fragment is the region of cross-linking of the agonist probe [Bpa8]hCT (47).



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  		FIG. 4.
Photoaffinity labeling and CNBr cleavage of [Bpa8]sCT(8-32)·hCTRa conjugates. A, photocross-linking of 125I-[Bpa8]sCT(8-32) to hCTRa stably expressed in HEK-293 cells revealed a protein band of Mr ~97,000, which shifted to Mr ~52,000 after endoglycosidase F deglycosylation following resolution on a 10% SDS-glycine gel. B, CNBr cleavage of the purified native receptor generated a radioactive band of Mr ~3800, which did not alter upon deglycosylation. Shown is a typical of autoradiograph of a 10% NuPAGE gel. The positions of MultiMark protein standards (Invitrogen) are indicated on the left. The autoradiograph is a representative of five distinct experiments. C, schematic diagram of the amino acid sequence of HA-hCTRa, including the predicted sites for CNBr digestion. Theoretically, CNBr digestion of the intact receptor, which cleaves at the carboxyl terminus of methionine residues (black circles), generates 16 fragments, 2 of which contain consensus sites for N-linked glycosylation (Y). The molecular mass of each CNBr-digested fragment is shown. The site of insertion of the double HA epitope tag is also illustrated. The two potential candidate fragments for cross-linking to [Bpa8]sCT(8-32) are highlighted in gray circles.

 
To resolve the site of cross-linking, Met residues at either amino acid 59 or 376 were replaced with Leu by site-directed mutagenesis, leading to abolition of the predicted CNBr cleavage sites flanking the candidate receptor fragments identified above. Both mutant receptors, when transiently transfected into COS-7 cells, bound sCT with high affinity (Fig. 5A; Table I) and stimulated the accumulation of cAMP in response to sCT with similar potency to the WT receptor (Fig. 5B; Table I). Both constructs were efficiently and specifically affinity labeled by 125I-[Bpa8]sCT(8-32) (data not shown). However, CNBr cleavage of either the photolabeled receptor mutant M59L or mutant M376L produced a Mr ~3800 band similar to that observed with the WT receptor and the radioiodinated free probe, indicating that neither receptor fragment Asp50-Met59 nor Leu368-Met376 was a contact site labeled by the probe (Fig. 5C). Further examination of the receptor sequence suggested that the probe may cross-link directly to Met49, with release of the Met49-[Bpa8]sCT(8-32) "fragment" via cleavage of both Met49 and Met48.


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  		TABLE I
Effects of point mutations on receptor function

WT HA-hCTRa and mutated receptors were transiently transfected into COS-7 cells and assessed for apparent sCT binding affinity and sCT-induced cAMP response. All values are expressed as the mean ± S.E.

 



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  		FIG. 5.
Assessment of the effect of M59L and M376L point mutations on receptor function. WT HA-hCTRa or mutated receptors (M59L and M376L) were transiently transfected into COS-7 cells and assessed for apparent sCT binding affinity (A) and sCT-induced cAMP response (B). The data are the mean ± S.E. of two to three pooled independent experiments conducted in triplicate. Basal cAMP levels for individual experiments ranged from 4 to 8 nM, and Emax values ranged from 25 to 35 nM. C, CNBr cleavage of the photoaffinity-labeled WT HA-hCTRa or HA-hCTRa mutants M59L and M376L transiently expressed in COS-1 cells. Shown is a typical of autoradiograph of a 10% NuPAGE gel. CNBr cleavage of either the purified native receptor or mutated receptors generated a similar radioactive band of Mr ~3800, migrating similarly to the radioiodinated free probe. The positions of MultiMark protein standards are indicated on the left. The autoradiograph is a representative of two distinct experiments.

 
To test this hypothesis, the mutant receptor M48I was generated. The M48I receptor mutant retained both high affinity sCT binding and functional response to sCT (Fig. 6; Table I). Photolabeling of the mutant receptor M48I revealed the expected band of Mr ~97,000, with a similar labeling intensity as the WT receptor (Fig. 7A). However, CNBr cleavage of the photolabeled mutant receptor M48I generated a fragment of Mr ~19,000, in contrast to the Mr 3800 photoligand band observed with the WT receptor (Fig. 7B). This fragment was further resolved to Mr ~6000 for the deglycosylated M48I receptor (Fig. 7B). These data are consistent with photocross-linking to the amino terminus of the mutant receptor with cleavage at Met49 and confirm Met49 as the site of cross-linking for [Bpa8]sCT(8-32) (Fig. 7C).

Identification of the Binding Domain of the Antagonist [Bpa19]sCT(8-32)—As previously identified (45), position 19 of the full-length photoactive agonist [Bpa19]sCT is in close proximity to the receptor region delimited by residues Cys134 and Lys141. To further address the molecular nature of the interaction between agonist and antagonist with the receptor, we synthesized the antagonist sCT analogue [Bpa19]sCT(8-32). Like the photoactive agonist, the novel antagonist of [Bpa19]sCT(8-32) efficiently labeled the HA-hCTRa receptor transiently expressed in COS-7 cells with a single radioactive band of Mr ~97,000, which shifted to Mr ~52,000 after N-glycosidase F deglycosylation (Fig. 8A). CNBr digestion of the 125I-[Bpa19]sCT(8-32)-labeled receptor generated a band of Mr ~9300, which was unaltered upon N-glycosidase F treatment (Fig. 8B). Given the molecular mass of the 125I-[Bpa19]sCT(8-32) of ~3.3 kDa and based on the previous study of the full-length [Bpa19]sCT(8-32), the region Cys134-Met187 (Fig. 8C) was the most probable candidate receptor fragment matching the labeled band. As expected, the M133A/L mutation completely abolished the generation of the Mr 9300 band, whereas it gave rise to a higher band of Mr 20,000 (Fig. 8D), corresponding to two accumulated receptor fragments, Gln60-Met133 (Mr 8800) and Cys134-Met187 (Mr 6100). In addition, Asp-N digestion (Fig. 9A) and Lys-C cleavage (Fig. 9B) of the antagonist 125I-[Bpa19]sCT(8-32)-receptor conjugates generated similar protein patterns to those described for the full-length agonist peptide (45), indicative of cross-linking to the receptor fragments Asp113-Arg214 (Fig. 9C) and His121-Lys141 (Fig. 9D), respectively. These data led to a conclusion that the antagonist [Bpa19]sCT(8-32) labeled the same receptor fragment Cys134-Lys141 located near the first TMH. Also, like the full-length peptide, additional analysis of the efficiency of cross-linking of the 125I-[Bpa19]sCT(8-32) analogue to alanine scanning receptor mutants of the region Cys134-Lys141 did not identify a specific residue as the site of interaction (Fig. 10), consistent with flexibility of this region around the site of cross-linking.



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  		FIG. 6.
Assessment of the effect of the M48I mutation on receptor function. WT HA-hCTRa or mutated receptor M48I was transiently transfected into COS-7 cells and assessed for apparent sCT binding affinity (A) and sCT-induced cAMP response (B). The data are the mean ± S.E. of three to four pooled independent experiments conducted in triplicate. Basal cAMP levels for individual experiments ranged from 40 to 120 nM, and Emax values ranged from 110 to 550 nM.

 



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  		FIG. 7.
CNBr cleavage of the photoaffinity labeled WT HA-hCTRa and HA-hCTRa mutant M48I. A, photocross-linking of [Bpa8]sCT(8-32) to WT hCTRa or M48I mutant transiently expressed in COS-1 cells revealed a protein band of Mr ~97,000 following resolution on a 10% SDS-glycine gel. B, CNBr cleavage of the photolabeled M48I mutated receptor generated a radioactive band of Mr ~19,000, which, after endoglycosidase F deglycosylation, shifted to Mr ~6000. This is distinct from the cleavage pattern of the WT receptor. The right panel shows a typical of autoradiograph of a 10% NuPAGE gel. The positions of MultiMark protein standards are indicated on the left. The autoradiograph is a representative of two distinct experiments. C, a schematic presentation of the theoretical CNBr digestion mapping for the WT HA-hCTRa and mutated construct M48I.

 



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  		FIG. 8.
Photoaffinity labeling and CNBr cleavage of [Bpa19]sCT(8-32)·hCTRa/mutant conjugates. A, photocross-linking of 125I-[Bpa19]sCT(8-32) to hCTRa transiently expressed in COS-7 cells revealed a protein band of Mr ~97,000, which shifted to Mr ~52,000 after N-glycosidase F deglycosylation following resolution on a 10% SDS-glycine gel. The low molecular weight standards (Bio-Rad) are shown at the left. B, conjugates were immunoprecipitated and then treated with N-glycosidase F followed by CNBr. The radiolabeled protein was electrophoresed on a 16.5% SDS-Tricine gel. CNBr digestion of the native receptor yielded a prominent fragment of Mr ~9300. Sequential digestion with N-glycosidase F followed by CNBr did not alter the mobility of the Mr ~9300 fragment. The SeeBlue protein standards (Invitrogen) are shown at the left. C, a schematic presentation of the theoretical CNBr digestion mapping for the WT HA-hCTRa, containing a potential candidate fragment for cross-linking to [Bpa19]sCT(8-32) highlighted in gray circles. D, the truncated peptide 125I-[Bpa19]sCT(8-32) was photocross-linked to the WT HA-hCTRa or M133A/M133L mutants transiently expressed in COS-7 cells. The radiolabeled protein fragments were electrophoresed on a 16.5% SDS-Tricine gel. CNBr cleavage of either mutated receptor M133A or M133L gave rise to a single band of Mr ~40,000. Receptor deglycosylation prior to CNBr cleavage shifted this band to Mr ~20,000. Mutation of Met133 to either leucine or alanine completely abolished the generation of the lower Mr 9300 band, which was observed with the WT receptor. The SeeBlue protein standards (Invitrogen) are shown at the left. The autoradiographs are representative of more than three independent experiments.

 
Molecular Modeling—The five final conformations of the hCTRa models are shown in Fig. 11. The position of the amino-terminal domain with respect to the 7TM helices is poorly constrained by both the linker between the two regions, which is of sufficient length as to provide no real restriction, and the shape correlations identified by Hex. This is strongly suggestive of a weakly associated amino-terminal domain that is free to reposition itself with respect to the rest of the protein. Interaction with CT would in effect bridge the two domains, forming a more stable arrangement in the activated or antagonized receptor.




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  		FIG. 9.
Photoaffinity labeling followed by endoproteinase Asp-N or Lys-C cleavage. The truncated peptide 125I-[Bpa19]sCT(8-32) was photocross-linked to the WT HA-hCTRa transiently expressed in COS-7 cells. Conjugates were immunoprecipitated and then treated with either Asp-N or Lys-C enzymes followed by N-glycosidase F. The radiolabeled protein was electrophoresed on a 16.5% SDS-Tricine gel for 125I-[Bpa19]sCT(8-32). A, Asp-N digestion yielded two radiolabeled bands; one migrating at Mr ~52,000, and the other migrating at Mr ~26,000. After treatment with N-glycosidase F, these bands were shifted to Mr ~33,000 and Mr ~12,000, respectively. The upper bands represent partial digest products. B, Lys-C digestion yielded two fragments; one migrating at Mr ~40,000, and the other migrating at Mr ~20,000. When treated withN-glycosidase F, these bands were shifted to Mr ~30,000 and Mr ~8,000, protein respectively. The upper bands represent partial digestion products. The SeeBlue standards are shown at the left. The results shown are representative of three different experiments. C and D, schematic diagram of the amino acid sequence of HA-hCTRa, including the predicted sites for Asp-N and Lys-C digestions (black circles). Theoretically, Asp-N and Lys-C digestions of the intact receptor, which cleave at the amino terminus of aspartic acid residues and carboxyl terminus of lysine residues, respectively, generate 14 and 21 fragments, 3 of which contain consensus sites for N-linked glycosylation (Y). The molecular mass of each Asp-N-digested and Lys-C-digested fragment is shown. A potential candidate fragment for cross-linking to [Bpa19]sCT(8-32) is highlighted in gray circles.

 
Within three of five models (Fig. 11, A, C, and E), Leu368 and Met49, the Bpa8 cross-linking site of agonist and antagonist peptides, respectively, are located in close proximity to each other. In one model (Fig. 11A), the residues are only 8.5 Å apart (Fig. 12), a distance easily covered by residues on an {alpha}-helix with less than a 60° rotation along the helical axis.

Using the receptor model represented in Figs. 11A and 12, the helix in the model structure of sCT exhibits a sensible fit within a groove in the predicted structure of the amino-terminal receptor protein, where Val8 of the peptide is in close proximity to residues Met49 and Leu368 of the receptor, and peptide residues Leu16 and Leu19 are positioned over the loop containing Cys134-Lys141 (Fig. 13). The carboxyl-terminal end of CT is currently very difficult to model in the context of the receptor because it is almost completely unstructured in the NMR models.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The comparative analysis of the agonist [Bpa19]sCT in our earlier paper (45) and the antagonist [Bpa19]sCT(8-32) in this study revealed that both probes cross-linked to the same receptor region, Cys134-Lys141, indicating that orientation of this segment of the receptor does not alter dramatically during agonist activation of the receptor. This is not surprising and, indeed, is consistent with a previous report for another class II G protein-coupled receptor, the PTH1 receptor. In addition to the photoactive agonist [Bpa13]PTH (1-34), Chorev and co-workers developed a photoactive antagonist, PTH (7-34), substituted at position 13 to directly study the nature of the bimolecular interface interaction of PTH antagonist with its receptor (65). In this case, equivalent sites of cross-linking occurred for both peptides (Arg186 in the extracellular domain).



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  		FIG. 10.
Photocross-linking to alanine scanning mutants. Photoaffinity cross-linking of [Bpa19]sCT(8-32) to WT or mutated receptors (N135A, A136V, F137A, T138A, P139A, E140A, or K141A) transiently transfected into COS-7 cells generated a protein band of Mr ~ 97,000. The low molecular weight standards (Bio-Rad) are shown at the left. The autoradiograph is a representative of three independent experiments.

 



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  		FIG. 11.
Diagrammatic representation of the final five models of hCTRa. Each model is shown in cartoon representation with the same orientation of the 7TM region, so that the difference in position of the amino-terminal domain is emphasized. The figure was created using Molscript (81).

 
Analysis of photoaffinity cross-linking of [Bpa8]sCT(8-32) to the hCTRa identified Met49 as the attachment site of the Bpa moiety. The identification of a methionine residue as a contact site in the receptor has been commonly described in photoaffinity cross-linking studies using the benzophenone moiety, present in Bpa or pBz2 (66-69). The mechanism underlying this selectivity is based on an intermediate charge-transfer complex (i.e. thiolactone intermediate) generated from the photoactivable benzophenone radical, which interacts with the sulfur atom in the Met residue (70).



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  		FIG. 12.
Diagrammatic representation of one hCTRa model. The model shows the distance between Leu368 and Met49, the residues that cross-link to position 8 of CT agonist and antagonist peptides, respectively. The visible section of the protein is shown as a cartoon in gray, with the two residues shown as rods and colored black (Met49) and gray (Leu368). The distance measurement between the two residues is indicated.

 



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  		FIG. 13.
Diagrammatic representation of a model of the sCT·hCTRa complex. The proteins are both shown as cartoon representations, with side chains of particular interest shown as rods. The CT is colored yellow, with residues Val8 (purple), Leu16 (orange), and Leu19 (black) indicated. The amino-terminal domain of the hCTRa model is shown in blue, with the transmembrane helical bundle in gray. On the hCTRa model, residues Met49 (green), Leu368 (cyan), and the stretch of residues Cys134 to Lys141 (gray) are shown. The ability of both ends of the CT to interact simultaneously with the points of known cross-linking is clear. The figure was produced with Molscript (81) and Raster3D (82).

 
Interestingly, the site of covalent attachment on the hCTRa for the current antagonist analogue Bpa8-substituted sCT(8-32) is very distinct from that recently identified for the agonist analogue Bpa8-substituted hCT (47). [Bpa8]sCT(8-32) cross-linked to Met49 of the receptor amino terminus in the current study, whereas [Bpa8]hCT interacted with the residue Leu368 in the third extracellular loop of the hCTRa (47). Although significant differences exist in the primary amino acid sequences of hCT and sCT, these peptides are predicted to adopt similar secondary structure, particularly within the region of strongest {alpha}-helical propensity (amino acids 8-16) (71-75). Despite this, it is possible that the difference in the site of cross-linking for [Bpa8]sCT(8-32) and [Bpa8]hCT (47) is due to differences in the orientation of the helix between sCT versus hCT peptides. More intriguing is the possibility that the different patterns of cross-linking arise from differences in receptor conformation of the agonist-bound state ([Bpa8]hCT) and antagonist-bound state ([Bpa8]sCT(8-32)).

De novo modeling of the hCT receptor suggests that amino acids Met49 and Leu368 may exist in close proximity to each other and that the CT peptide helix can be accommodated within a groove in the structure of the receptor amino terminus in a manner that is consistent with the cross-linking data for positions 8, 16, and 19 of calcitonin (Fig. 13). Within this model, only a relatively small reorientation of the position of Bpa8 would be required to explain the alternate site of cross-linking of the agonist versus antagonist peptides.

The molecular mechanism governing transition of the class II receptors between its active and inactive states currently remains unclear. Nonetheless, this is the first study of CT showing a potential conformational change in the amino terminus of the CT receptor accompanying transition of inactive state to active state. A recent study (56) has proposed a two-step model of corticotropin-releasing factor receptor activation based on the NMR structure of the extracellular domain of the mouse corticotropin-releasing factor receptor 2{beta} and NMR chemical-shift perturbation experiments for ligand binding. In this model, the first step involves the carboxyl terminus of the ligand binding to the extracellular domain of the receptor. In the following step, the amino terminus of the agonist ligand penetrates into the receptor core to activate the receptor by movement of TM helices. In this model an antagonist ligand lacking the amino-terminal activation domain does not penetrate deep into the receptor core and consequently does not activate the receptor. In the PTH1 (76-78) and secretin (79) receptors the "activation module" of the peptide amino terminus contacts the receptor near the apex of TMH6, and agonist and antagonist analogues of PTH exhibit distinct modes of binding around this region (78). Our data are also consistent with a model of receptor activation in which amino acid 8 is proximal to the receptor extracellular loop 3 leading to orientation of the CT activation domain (the amino terminus 1-7 disulfide-bridged loop) into the top of TMH6, in a similar mode to that seen for the secretin and PTH receptors. Our current modeling suggests that the amino-terminal domain is only weakly associated with the receptor core and that the linker between the structured component of the amino terminus and the top of helix 1 is of sufficient length to allow the domain to reposition itself with respect to the rest of the protein. In the depiction of the sCT-hCTRa complex in Fig. 13, the CT amino terminus currently sits above TMH6. It is possible that this more closely represents binding to inactive state receptor. One interpretation of the data may be that, in the presence of agonist or agonist-bound state, conformational change associated with receptor activation may bring the amino-terminal domain in closer proximity to the receptor core, specifically in this case, with extracellular loop 3, leading to positioning of the peptide amino terminus within the groove at the top of TMH6. Such a conformational change may favor cross-linking between CT amino acid 8 and extracellular loop 3 as seen with [Bpa8]hCT (47). Cooperative interaction of the amino-terminal domain and receptor core in CT binding has been proposed previously based on binding differences of receptor splice variants and chimeric receptors (1). Similar cooperative binding of agonists to the amino-terminal (N) and receptor core (J) domains has been proposed for peptide binding to the PTH1 receptors (80).

In conclusion, the current identification of contact domains of amino acids 8 and 19 of CT antagonist peptides provides important information on the relative orientation of the receptor amino terminus and the core of the receptor. Analysis of the modeled structures at this time suggests that a relatively small shift in orientation of CT peptides in active and inactive state receptors can account for the difference in the site of cross-linking identified for Bpa8 but allow for movement of the receptor amino terminus relative to the receptor core as part of the activation process. The level of accuracy possible with the current hCTRa model is inadequate for more detailed conclusions, but the model forms a basis for suggesting new experimental directions and a framework upon which further results can be assembled.


   FOOTNOTES
 
* This work was funded by National Health and Medical Research Council Project Grant 145703, the Potter Foundation Neuropeptide laboratory, and National Institutes of Health Grant DK46577 (to L. J. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

A National Health and Medical Research Council Principal Research Fellow. Back

** A National Health and Medical Research Council Senior Research Fellow. To whom correspondence should be addressed: Howard Florey Institute, The University of Melbourne, Gate 11, Royal Parade, Parkville, Victoria 3010, Australia. Tel.: 61-3-8344-1954; Fax: 61-3-9347-0446; E-mail: p.sexton{at}hfi.unimelb.edu.au.

1 The abbreviations used are: CT, calcitonin; CTR, calcitonin receptor; hCT, human calcitonin; hCTR, human calcitonin receptor; Bpa, p-benzoyl-L-phenylalanine; [Bpa8]sCT(8-32), [Arg11,18, Bpa8]sCT(8-32); [Bpa19]sCT(8-32), [Arg11,18, Bpa19]sCT(8-32); BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; HA, hemagglutinin; HPLC, high performance liquid chromatography; PTH, parathyroid hormone; sCT, salmon calcitonin; TM, transmembrane; TMH, transmembrane helix; WT, wild-type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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