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Endocrinology Vol. 146, No. 4 1863-1870
Copyright © 2005 by The Endocrine Society

Receptors Specific for the Carboxyl-Terminal Region of Parathyroid Hormone on Bone-Derived Cells: Determinants of Ligand Binding and Bioactivity

P. Divieti, A. I. Geller, G. Suliman, H. Jüppner and F. R. Bringhurst

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

Address all correspondence and requests for reprints to: Paola Divieti M.D., Ph.D, Endocrine Unit, Wellman 5, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: divieti{at}helix.mgh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTH comprises 84 amino acids of which the first 34 are sufficient for full activation of the classical PTH/PTHrP receptor, the type 1 PTH receptor. It is known that multiple carboxyl (C)-terminal fragments of PTH are present in the blood and that they comprise the majority of circulating PTH. C-PTH fragments, previously regarded as by-products of PTH metabolism, are directly secreted by the parathyroid glands or arise from the peripheral cleavage of the intact hormone. Compelling evidence now strongly suggests that these C-PTH fragments mediate biological effects via activation of a receptor that specifically recognizes the C-terminal portion of intact PTH, and this receptor is therefore named the carboxyl-terminal PTH receptor (CPTHR). We have previously reported that osteocytes abundantly express this novel receptor and that its activation is involved in cell survival and communication. Here we report the characterization of determinants of PTH that are required for high-affinity binding to the CPTHR. Using synthetic PTH peptides harboring alanine substitution or truncations, we showed the existence of discrete binding domains and critical residues within the intact hormone. We have furthermore identified eight amino acids within the PTH sequence that play key roles in optimizing the binding affinity of C-PTH fragments to CPTHRs. These include the tripeptide sequence Arg25-Lys26-Lys27, the dibasic sequence Lys53-Lys54, and three additional residues within the PTH (55–84) sequence, Asn57, Lys65, and Lys72. Functional analysis of these residues demonstrated a strong correlation between binding affinity and biological effect and points to a potential role of CPTHR activation in regulating bone cell survival.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTH REGULATES BLOOD calcium concentration via activation of its classical receptor, the type 1 PTH/PTHrP receptor (PTH1R). This receptor recognizes the amino (N)-terminal portion of PTH (and the homologous N-terminal portion of PTHrP) and is activated with indistinguishable efficacy and efficiency by PTH (1–34), by PTHrP (1–36), and by intact PTH (1–84). There is no evidence to suggest that regions of PTH (1–84) located carboxyl (C)-terminal to residue 34 contribute to ligand binding or activation of the PTH1R. On the other hand, large portions of the C terminus of PTH (1–84) are highly conserved across species. For example, the sequences PTH (53–61) and PTH (65–75) are 80% identical and otherwise differ only by conservative substitutions across mammalian species. This high degree of evolutionary conservation strongly suggests the possibility of additional, independent biological function(s) for this region of the PTH molecule.

Evidence of cellular receptors with specificity for the C-terminal portion of PTH (1–84) (CPTHRs) has accumulated steadily over the past 25 yr, as recently reviewed (1). Initial observations suggestive of more than a single class of receptors for intact PTH on bone and kidney cells, including a class with apparent specificity for C-terminal PTH (CPTH) peptides (2, 3, 4, 5, 6) were followed by demonstration of unique biological effects of CPTH peptides such as PTH (53–84) on bone-derived cells in vitro (6, 7, 8, 9, 10). Inomata et al. (11) subsequently demonstrated specific binding and chemical cross-linking of a CPTHR-specific radioligand [125I-labeled [Tyr34] human (h)PTH (19–84)] to proteins on the surface of rat osteosarcoma and parathyroid-derived cells. We then reported abundant expression of CPTHRs (2–3 x 106/cell), detected using the same 125I-labeled [Tyr34] hPTH (19–84) radioligand, on the surface of clonal osteocytic cells isolated from calvarial bone of fetal PTH1R-null mice, thus providing the first conclusive evidence that CPTHRs exist independently of PTH1Rs (12). That study also reported that PTH (1–84) and certain CPTH ligands, such as PTH (24–84) and PTH (39–84), increased the rate of apoptosis in PTH1R-null osteocytes.

The specific structural determinants of CPTHR binding and biological activity have not yet been well defined. The reports by Inomata et al. (11) and by Divieti et al. (12) indicated that hPTH (1–84), [Tyr34] hPTH (19–84), and [Tyr34] hPTH (24–84) bind with similar affinity (IC50 = 10–30 nM), whereas hPTH (39–84) and hPTH (53–84) exhibited at least 10- to 50-fold lower binding affinities. This finding was consistent with the presence of important binding determinants within the region hPTH (24–38). More recently, hPTH (28–84) was shown to displace 125I-labeled [Tyr34] hPTH (19–84) with 10-fold lower binding affinity than hPTH (24–84), indicating that the region hPTH (24–27) may contain important binding determinants (1). Biological responses in various bone-derived cells have been reported using hPTH (39–84), hPTH (53–84), and hPTH (69–84) (6, 7, 8, 9, 13, 14), and cytosolic calcium responses have been seen in fetal chondrocytes using hPTH (52–84), hPTH (57–76), hPTH (61–80), and hPTH (64–84) but not hPTH (53–72) (10). In one report, removal of the C-terminal Gln84 residue abrogated binding and biological activity of hPTH (53–84) (6).

In this study, we have employed previously isolated clonal, PTH1R-null osteocytes with abundant CPTHR expression, together with the 125I-labeled [Tyr34] hPTH (19–84) CPTHR-specific radioligand and a series of CPTH peptide analogs, to probe the key structural determinants within the PTH sequence required for binding and activation of skeletal CPTHRs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Culture media and other tissue culture reagents were purchased from Invitrogen Life Technologies (Grand Island, NY), and other reagents and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) or Fisher Co. (Pittsburgh, PA). Radioactive Na[125I] was purchased from NEN Life Science Products (Boston, MA).

Human PTH peptides
Recombinant hPTH (1–84), [Tyr34] hPTH (19–84), [Tyr34] hPTH (24–84), hPTH (28–84), hPTH (34–84), and hPTH (37–84) were gifts of Chugai Pharmaceutical Co. (Shizuoka, Japan); [Asp76] hPTH (39–84) was purchased from Peninsula Laboratories (Belmont, CA); hPTH (7–84) and hPTH (53–84) was purchased from Bachem California Inc. (Torrance, CA). All other PTH fragments were synthesized in the Peptide and Oligonucleotide Core Laboratory of the Endocrine Unit (Massachusetts General Hospital, Boston, MA).

Cell culture
OC59 cells, isolated as previously described by enzymatic digestion from calvarial bones derived from an 18.5-d-old tsA58(+)/PTH1R(–/–) fetus (15) were cultured at 33 C in a humidified atmosphere (95% air/5% CO2) using growth medium [{alpha}-MEM containing 10% fetal bovine serum (lot 1011961, Invitrogen Life Technologies) and 1% penicillin-streptomycin].

Radioreceptor assay
[Tyr34] hPTH (19–84) was radioiodinated with Na[125I] (2000 Ci/mmol) by the chloramine-T method and purified by HPLC, as previously described (11). For binding experiments, cells (50,000 cells per well) were plated in 24-well dishes and cultured at 33 C for 10–14 d. Confluent monolayers then were rinsed once with 0.5 ml binding buffer [100 mM NaCl, 5 mM KCl, 2 mM CaCl2, 50 mM Tris-HCl (pH 7.8) plus 5% heat-inactivated horse serum] before incubation with 125I-labeled [Tyr34] hPTH (19–84) (100,000–200,000 cpm/well) and increasing concentration of different peptides in a final volume of 0.5 ml binding buffer for 4 h at 15 C, before washing, solubilization in NaOH, and determination of cell-associated radioactivity, as previously described (12). Nonspecific binding was ascertained in the presence of 1 x 10–6 M hPTH (1–84) or 10 x 10–6 M hPTH (53–84).

Cell survival assays
For analysis of apoptosis or cell death, cells were plated at 50,000 cells per well (25,000 cells/cm2) in 24-well dishes and maintained in growth medium at 33 C for 2 d before shifting them to nonpermissive conditions (39 C) that inactivate the transforming SV40 ts-A58 T-antigen expressed by these cells (12). Cells were maintained at 39 C for an additional 4–6 d in {alpha}-MEM supplemented with 2.5% fetal bovine serum and 1% penicillin-streptomycin before incubation with different peptides for the indicated times, after which cells were suspended with trypsin-EDTA (including nonadherent cells), centrifuged, and resuspended in solutions appropriate for subsequent analysis.

Apoptosis was assessed by flow-cytometric detection of exposed phosphatidylserine using phycoerythrin-tagged annexin V (Guava Nexin method; Guava Technologies, Hayward, CA). For these measurements, cells were resuspended in Nexin assay buffer (Guava Technologies), protected from light, and incubated on ice for 20 min with Nexin V, following the manufacturer’s instructions. Cell-associated fluorescence then was analyzed by the Guava PCS personal cell analyzer system and results expressed as the percentage of 2000 counted cells that were annexin V positive.

In some experiments, apoptotic cells were detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) reaction, using the Apotag Plus kit (Chemicon International, Temecula, CA). For this purpose, cells were plated and cultured as above in four-chamber slide flasks (Lab-Tek glass slide, Nalge Nunc International, Naperville, IL) and incubated for 6 h with the appropriate hormone before the monolayers were fixed in 1% paraformaldehyde/PBS and stained according to the manufacturer’s instructions. Apoptosis was quantified by scoring the percentage of TUNEL-positive cells present in two adjacent fields (x20 magnification).

For assessment of cell death, cells were resuspended in 0.1% trypan blue solution in PBS (Bio Whittaker, Walkersville, MD), and the percentage of cells exhibiting both nuclear and cytoplasmic staining then was determined using a hemocytometer to count at least 500 cells.

Statistical analysis
Results were expressed as the mean ± SD or SE. Each experiment was repeated three to five times. Significance of differences between treatment and control groups was assessed by one-way ANOVA using Bonferroni correction and Prism 3 software (GraphPad, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ligand recognition by the CPTHR
To identify region(s) of the PTH molecule important for binding to the CPTHR, competitive displacement analysis was performed using OC59 cells, the CPTH radioligand 125I-labeled [Tyr34] hPTH (19–84), which does not bind to the PTH1R (11), and various recombinant or synthetic hPTH fragments. This clonal, osteocytic cell line (OC59) lacks PTH1Rs but expresses abundant CPTHRs (12). As shown in Fig. 1Go and Table 1Go, N-terminally truncated hPTH peptides hPTH (7–84), [Tyr34] hPTH (11–84), [Tyr34] hPTH (13–84), [Tyr34] hPTH (19–84), and [Tyr34] hPTH (24–84) displaced the radioligand as effectively as hPTH (1–84) (IC50, 10–40 nM), whereas a group of shorter peptides, including hPTH (28–84), hPTH (34–84), hPTH (37–84), [Asn76] hPTH (39–84), and hPTH (53–84), bound with lower apparent affinity (IC50, 200–600 nM). Further minimal N-terminal truncation beyond position 53, as in hPTH (55–84), hPTH (57–84), and hPTH (60–84), effectively abolished measurable binding affinity for CPTHRs (IC50 >> 10,000 nM) (Fig. 1Go and Table 1Go). This initial structural analysis of intact PTH highlighted the presence of at least two domains required for maximal binding affinity, one within the sequence hPTH (24–27), binding domain 1 (BD1), and another represented by the dibasic sequence (Lys53-Lys54), termed binding domain 2 (BD2).



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  		FIG. 1. Binding of N-terminally truncated human PTH fragments to CPTHRs on OC59 cells. The hPTH peptides shown (see Materials and Methods) were tested for their ability to competitively displace the 125I-labeled [Tyr34] hPTH (19–84) radioligand from sites on OC59 cells. Cells were plated at 25,000 cells/cm2 in 24-well plates and maintained in culture at 33 C for 10–14 d before use. Results are expressed as the mean ± SE (n = 3) of the percentage of maximal specific binding observed in the absence of competing ligand.

 

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  		TABLE 1. Apparent binding affinity of hPTH peptides to CPTHRs

 
To further analyze these two domains, a series of short synthetic peptides spanning either one or both binding regions, including hPTH (20–30), hPTH (50–60), and hPTH (24–54), were synthesized and tested (Table 2Go). The fragments hPTH (20–30) and hPTH (50–60), either alone or in combination, failed to substantially displace 125I-labeled [Tyr34] hPTH (19–84), even at concentrations as high as 100 µM each (Table 2Go). The peptide hPTH (24–54), incorporating both BD1 and BD2, did exhibit concentration-dependent radioligand displacement (Fig. 1Go) that was parallel to that of hPTH (1–84) but showed a much higher IC50 (16.7 ± 1.3 µM vs. 30.9 ± 15.1 nM) (Table 2Go). These results suggest that the domains PTH (24–27) and PTH (53–54) both must be present within the same linear sequence to permit effective interaction with the CPTHR. Importantly, the approximately 1000-fold disparity between the IC50 of hPTH (24–84) (13.5 ± 7.4 nM; Fig. 1Go and Table 1Go) and that of hPTH (24–54) (16.7 ± 1.3 µM; Table 2Go) points to the presence of additional major determinants of binding affinity within the region hPTH (55–84), thereby defining a third binding domain (BD3). It is likely that the BD3 region must be tethered to more N-terminal regions of the PTH sequence to facilitate high-affinity binding, as equimolar mixtures of hPTH (24–54) and hPTH (53–84) (from 50–50,000 nM of each peptide) displaced the 125I-labeled [Tyr34] hPTH (19–84) radioligand no more effectively (640 ± 100 nM; Table 2Go) than did hPTH (53–84) alone (648 ± 132 nM; Table 1Go).


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  		TABLE 2. Apparent CPTHR binding affinity of domain-spanning hPTH (1–84) fragments and analogs

 
Alanine-scanning analysis of major CPTHR binding domains
The importance for CPTHR binding affinity of specific amino acids within the BD1 and BD2 regions of the ligand was further examined using analogs of the readily synthesized model peptide, hPTH (24–54). Progressive N-truncation of hPTH (24–54) indicated that the Leu24 residue contributed only modestly to binding affinity but that further removal of the adjacent Arg25, as represented in hPTH (26–54), led to virtually complete loss of hPTH (24–54) binding (Fig. 2AGo and Table 2Go). Similar results were obtained in experiments in which these amino acids were substituted with single alanine residues (Fig. 2BGo and Table 2Go). Thus, [Ala24] hPTH (24–54) exhibited the same modest (2- to 4-fold) reduction in binding affinity vs. hPTH (24–54) seen with hPTH (25–54), whereas individual alanine substitution for Arg25,Lys26, or Lys27 each severely impaired binding affinity relative to hPTH (24–54), perhaps most so for [Ala25] hPTH (24–54). In the case of BD2 (Lys53-Lys54), single-Ala substitutions induced at least 10-fold loss in apparent affinity, indicating that both of these basic residues are critical for preservation of binding affinity attributable to this domain (Fig. 2CGo and Table 2Go).



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  		FIG. 2. Effects of truncation and alanine scanning on binding of hPTH (24–54). Analogs of the short synthetic peptide hPTH (24–54) (see Fig. 1Go) spanning BD1 and BD2, either truncated at the amino terminus (A) or incorporating single-Ala substitutions at the amino-terminal (B) or carboxyl-terminal (C) region, were tested for their ability to displace the 125I-labeled [Tyr34] hPTH (19–84) radioligand from sites on OC59 cells. A, Serial amino-terminal truncation of hPTH (24–54): {blacksquare}, hPTH (24–54); {square}, hPTH (25–54); {circ}, hPTH (26–54). B, N-terminal (BD1) single-alanine mutations: {blacksquare}, hPTH (24–54); {square}, [Ala24] hPTH (24–54); {triangleup}, [Ala25] hPTH (24–54); {circ}, [Ala26] hPTH (24–54); {diamond}, [Ala27] hPTH (24–54). C, C-terminal (BD2) single-alanine mutations: {blacksquare}, hPTH (24–54); {triangleup}, [Ala53] hPTH (24–54); {square}, [Ala54] hPTH (24–54).

 
As noted above, the striking difference in apparent binding affinities of hPTH (24–84) and hPTH (24–54) indicated that the C-terminal region PTH (55–84) (BD3) must play a major role in supporting the CPTHR binding affinity of longer PTH peptides, even though the hPTH (55–84) peptide alone does not measurably displace the 125I-labeled [Tyr34] hPTH (19–84) radioligand (Fig. 1Go and Table 1Go). To identify key residues involved in the contribution of BD3 to overall CPTHR ligand binding affinity, clustered triple-alanine substitutions were introduced across the sequence of hPTH (53–84) (the shortest BD3-containing peptide with measurable binding affinity) to produce nine mutant hPTH (53–84) peptides (Fig. 3Go). Five of these peptides (M1, M2, M3, M5, and M7) exhibited apparent binding affinities similar to that of the unsubstituted hPTH (53–84) (data not shown), whereas three of them, M4, M6, and M9, with substitutions at positions 71–74, 64–66, and 55–57, respectively, showed dramatic (roughly 100-fold) reductions in apparent affinity (Fig. 4Go). Interestingly, in one mutant, M8, the replacement of the hydrophobic sequence Val58-Leu59-Val60 with three alanines actually slightly enhanced CPTHR binding affinity (by 2-fold; Fig. 4Go). To further identify, for each of these mutant peptides, the amino acid(s) important for high-affinity binding, peptides incorporating corresponding single-Ala substitutions were synthesized and tested. As shown in Fig. 5Go, this approach identified three key residues, Asn57, Lys65, and Lys72, that appear to be critical for high-affinity binding to CPTHRs, in that single-alanine substitutions of each resulted in the same 50- to 100-fold reduction in hPTH (53–84) binding affinity previously observed with the corresponding triple-Ala mutants. Single-alanine substitutions of those neighboring residues within M4, M6, and M9 previously converted to alanines in the triple-Ala strategy, led to minimal or no independent effects on binding affinity (data not shown). Interestingly, the enhanced binding of the M8 mutant peptide could not be reproduced by any single-Ala substitution within the Val-Leu-Val sequence (data not shown).



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  		FIG. 3. Triple-alanine mutation strategy for hPTH (53–84), with a schematic representation of the mutant hPTH (53–84) peptides containing three consecutive alanines. The top line depicts the native sequence of hPTH (53–84). For each of the nine mutant synthetic peptides shown (M1–M9), the alanine mutations are indicated in bold. Naturally occurring alanines in M4 and M5 were not altered.

 


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  		FIG. 4. CPTHR binding of triple-alanine hPTH (53–84) mutants. Triple-alanine hPTH (53–84) mutants (see Fig. 3Go) were tested for competitive displacement of the 125I-labeled [Tyr34] hPTH (19–84) radioligand in OC59 cells. Results are shown only for those mutants for which apparent binding affinities differed from that of hPTH (53–84). Results are expressed as described in Fig. 1Go.

 


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  		FIG. 5. CPTHR binding of single-alanine hPTH (53–84) mutants. Displacement of 125I-labeled [Tyr34] hPTH (19–84) is shown for native hPTH (53–84) ({blacksquare}) and for single-alanine-substituted analogs of M4, M6, and M9 for which apparent binding affinities differed from that of hPTH (53–84). Results are expressed as in Fig. 1Go.

 
Functional analysis of the CPTHR binding domains
We previously reported that CPTHR activation by PTH (1–84), PTH (24–84), and PTH (39–84) in osteocytes exerts a proapoptotic effect (12). To analyze the relation between apparent binding affinity to CPTHRs and biological activity of CPTH fragments and mutant ligands, selected peptides were tested for their ability to induce programmed cell death in OC59 cells. Increased apoptosis, as determined by TUNEL staining, was observed after 6 h of treatment with the intact hormone, 100 nM PTH (1–84), and with the shorter fragment PTH (53–84) (1000 nM) (Fig. 6Go). Treatment with both the intact hormone and the CPTH fragment induced, respectively, a 4.4- and 4.1-fold increase in apoptosis [basal, 2 ± 0.5%; PTH (1–84), 8.4 ± 3.4%; and PTH (53–84), 8 ± 1.9% positive cells] that was similar to the effect induced by dexamethasone (1000 nM) (3.6-fold increase and 7.1 ± 1.2% positive cells). We previously showed that measures of apoptosis, such as TUNEL staining and nuclear pyknosis, observed after 6 h of treatment of OC59 cells with hPTH (1–84), correlated with loss of trypan blue exclusion, a measure of cell death, at 16 h (12). As shown in Fig. 7AGo, treatment of OC59 cells with 100 nM hPTH (53–84) for 16 h induced a 1.8-fold increase in trypan blue-stained cells that was comparable to the effect of 10 nM hPTH (1–84) (1.8-fold). The lower potency of hPTH (53–84) vs. hPTH (1–84) is consistent with the higher binding IC50 of this shorter peptide (Table 1Go). This increase in cell death at 16 h induced by PTH (1–84) and hPTH (53–84) was mirrored by increased apoptosis at 6 h, as assessed by increased amounts of exposed phosphatidylserine residues on the outer plasma membrane leaflet, detected by flow cytometry after binding of fluorescent annexin V (16, 17, 18). Thus, as shown in Fig. 7BGo, treatment with PTH (53–84) (1 µM) for 6 h produced a 70% increase in annexin V staining [basal, 9.25 ± 0.9%; PTH (53–84), 15.65 ± 1.4% of cells] that was comparable to the effect of the intact hormone (100 nM) [PTH (1–84), 15.40 ± 1.1% of cells]. Moreover, the relative increase over untreated control in the annexin V assay (1.8-fold) was similar to that observed in the trypan blue assay (1.8- to 2-fold; Fig. 7AGo). A comparable effect also was observed with 100 nM hPTH (7–84) (data not shown).



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  		FIG. 6. Induction of apoptosis by intact PTH and CPTH fragment in OC59 cells. OC59 cells were plated in four-chamber slides, cultured at 33 C for 3 d, and then incubated at 39 C for 5 d before addition of PTH peptides for another 6 h. At the end of the incubation, cells were fixed and stained by TUNEL. Arrows indicate apoptotic cells. Two adjacent x10 magnification fields were scored (>700 cells per field), and the results are expressed as the percentage of TUNEL-positive cells (D). Representative fields are shown for control (A), PTH (1–84) (B), and PTH (53–84) (C). *, P < 0.05 vs. control.

 


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  		FIG. 7. Cell death induced by PTH fragments and synthetic peptides in OC59 cells. OC59 cells were plated, cultured at 33 C for 3 d, and then incubated at 39 C for 5 d before addition of PTH peptides for another 16 h for trypan blue staining (A, C, and D) or 6 h for annexin V staining (B) (see Materials and Methods). A, hPTH (1–84) (white bars) and hPTH (53–84) (black bars) were added at the indicated concentrations 16 h before assessment of the percentage of nonviable trypan blue-stained cells (see Materials and Methods). Values are expressed as percentage of controls and shown as mean ± SD of quadruplicate determinations. B, OC59 cells were treated with vehicle alone (white bar),10 nM hPTH (1–84) (black bar), or 100 nM hPTH (53–84) (gray bar) for 6 h before assessment of annexin V binding (see Materials and Methods). C, Response to 100 nM hPTH (1–84) and 1000 nM hPTH (24–54). D, Response to 100 nM PTH (1–84) vs. 10,000 nM hPTH (20–30) or hPTH (50–60). *, Significantly different from controls (P < 0.05).

 
As shown in Fig. 7CGo, treatment of OC59 cells for 16 h with 1 µM hPTH (24–54) also increased cell death (1.6-fold over basal), whereas the short peptides hPTH (20–30) and hPTH (50–60), which cannot bind to CPTHRs, did not increase cell death, even at concentrations as high as 10 µM (Fig. 7DGo).

As shown in Fig. 8Go, neither the triple-alanine-substituted peptide M9 (see Fig. 3Go) nor the single-alanine-substituted analog [Ala57] hPTH (53–84) (N57A), at 1000 nM, increased the rate of cell death at 16 h in OC59 cells. Both of these peptides exhibit greatly reduced CPTHR binding affinity relative to hPTH (53–84) (Figs. 4Go and 5Go), In contrast, the single-alanine-substituted analogs [Ala56] hPTH (53–84) (D56A) and [Ala58] hPTH (53–84) (V58A), with apparent binding affinities similar to that of native hPTH (53–84) (data not shown), were as effective as hPTH (53–84) in inducing OC59 cell death (Fig. 8Go).



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  		FIG. 8. Effect of alanine mutations in hPTH (53–84) on bioactivity. Cell death in response to treatment for 16 h with the indicated peptides was assessed by trypan blue exclusion, as described in Fig. 7Go. Results are expressed as mean ± SD of the control percentage of trypan blue-stained cells in five independent experiments for each peptide [except hPTH (53–84), where the experiment was repeated three times]. All peptides were added at 1000 nM except hPTH (1–84) (100 nM). C, Control; 1–84, hPTH (1–84); 53–84, hPTH (53–84); M9, [Ala55–57] hPTH (53–84); D56A, [Ala56] hPTH (53–84); N57A, [Ala57] hPTH (53–84); V58A, [Ala58] hPTH (53–84). Significantly different from controls: *, P < 0.05; **, P < 0.01. NS, P > 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we have employed a unique clonal osteocytic cell line (OC59) that expresses abundant CPTHRs but no PTH1Rs (12), together with the CPTHR-specific radioligand 125I-labeled [Tyr34] hPTH (19–84) (11) and various recombinant and synthetic CPTH peptide analogs to map ligand determinants of CPTHR binding and bioactivity. Building on preliminary observations previously reported by us and others (1, 11, 12), we now have identified eight specific amino acids within the PTH sequence that play key roles in optimizing affinity of PTH binding to CPTHRs. These include the tripeptide sequence Arg25-Lys26-Lys27, the dibasic sequence Lys53-Lys54, and three additional residues, Asn57, Lys65, and Lys72, that are roughly evenly spaced within the N-terminal portion of the hPTH (53–84) sequence. Remarkably, each of these residues is highly conserved among known mammalian PTH sequences. The preponderance of basic residues is notable as well. This might reflect important intra- or intermolecular ionic interactions necessary to promote optimal conformations for CPTH/CPTHR binding or could indicate sites of pre- or postsecretory proteolysis that could serve to modulate the overall activity of circulating PTH species at skeletal CPTHRs.

With regard to the latter possibility, it is of interest that CPTH fragments with N termini at Leu24 and Leu28 have been identified as products of parathyroid cells (19, 20, 21, 22), and various PTH peptides with N termini located at positions within the region PTH (34–43) are produced both by the parathyroid glands and by postsecretory hepatic proteolysis of circulating PTH (1–84) (23, 24). Our data indicate that CPTH peptides shorter than PTH (25–84) would be expected to have less CPTHR binding and activity than would longer PTH peptides, including intact PTH (1–84) and the as-yet-unidentified extended CPTH fragments found in both normal and uremic human serum that coelute on HPLC systems with synthetic hPTH (7–84) (25, 26, 27, 28, 29). Our results also suggest that CPTH peptides shorter than PTH (54–84) would not be expected to bind to skeletal CPTHRs at all. The molecular sequence and structure of CPTHRs remain unknown, and it is possible that distinct CPTHRs in different tissues possess different ligand selectivities. Analysis of the binding properties of CPTHRs on clonal osteocytes, osteoblasts, chondrocytes, and marrow stromal cells, however, indicates that the overall pattern of CPTH ligand selectivity reported here is conserved, at least among these skeletally derived cell lines (1).

Our observations concerning the binding and bioactivity of CPTH peptides are generally consistent with limited data available from previous reports using other systems (25, 26, 27, 28, 29). One difference relates to the role of the C-terminal glutamine residue. Takasu et al. (6) previously reported, using [35S]hPTH (1–84) radioligand and ROS 17/2.8 osteosarcoma cells (which express both PTH1Rs and CPTHRs), that removal of Gln84 led to loss of binding of hPTH (53–83) and of its ability to up-regulate alkaline phosphatase activity. We found that removal of Gln84 decreased CPTHR binding affinity only modestly (3-fold), whereas its mutation to alanine (as in peptide M1, [Ala82–84] hPTH (53–84) had no effect upon binding affinity. The explanation for this discrepancy is not clear but presumably is attributable in part to differences in cell lines, culture conditions, and reagents employed.

Our results indicate that the intact PTH ligand contains at least three critical domains, all of which must be present within the same peptide to achieve maximal binding affinity. The 10- to 20-fold greater apparent affinity of hPTH (1–84) than of C-PTH fragments such as hPTH (39–84) is of interest, given that CPTH fragments circulate in blood at concentrations almost 10-fold higher than intact PTH (1–84). Finally, despite its reduced binding affinity, hPTH (53–84), at an appropriately relatively higher concentration vs. the intact hormone, can exert full agonism via the CPTHR. This suggests that sequences important for bioactivity must be located C-terminal to Lys53, a prediction compatible with the reported bioactivity in other systems of fragments such as hPTH (57–76), hPTH (61–80), and hPTH (69–84) (10, 30), for which binding affinity is too low even to be detected by available techniques. Indeed, the potencies of active CPTHR agonists [i.e. PTH (1–84), PTH (53–84), and hPTH (7–84)] in promoting apoptosis, as observed here, are as much as two orders of magnitude greater than predicted by the IC50 of these peptides in the radioligand binding assay. This could indicate the presence of spare receptors in these cells, which express over 1 million CPTHR sites per cell (12), relative to the levels of occupied receptors required to maximally activate biological responses via more distal effectors engaged by these receptors.

Functional studies revealed that the CPTHR activation may be involved in cell survival and cell-to-cell communication (12), at least in the osteocytic cells studied here. Our previous data demonstrated that activation of the receptor induces osteocytic cell death in vitro, as demonstrated by increased nuclear pyknosis, chromatin condensation, and TUNEL staining (12). This proapoptotic effect of the C fragment does not require the presence of the first binding domain hPTH (24–27), or the sequence hPTH (28–52), because similar effects were obtained with hPTH (39–84) (12) and hPTH (53–84). Our data are most consistent with a model in which the basic structural determinants of PTH required for productive CPTHR interaction (both binding and bioactivity) are located in the PTH (53–84) region, wherein several strictly conserved (mostly basic) residues (Arg53, Arg54, Asn57, Lys65, and Lys72) play especially critical roles. The three subdomains we have identified seem incapable of functioning independently of one another [i.e. PTH (20–30), PTH (50–60), and PTH (55–84) each are inactive in both binding and functional assays], which implies that they may work in concert to direct the CPTH ligand’s conformations that are optimal for CPTHR interaction. Additional work is needed to determine whether structural determinants of bioactivity can be dissociated from those required for binding, but the present results provide a basic conceptual framework for future research in this area.

It is of interest to note that Jilka et al. (31) have reported an antiapoptotic action of hPTH (1–34), via PTH1R activation, in dexamethasone-stimulated osteocytic cells. In our cells, which lack endogenous PTH1Rs, the intact hormone exerts only a proapoptotic effect (via CPTHRs). This suggests the possibility that PTH1Rs and CPTHRs may exert opposite effects on osteocytes and osteoblasts and that the two receptors might be preferentially activated in response to changes in circulating intact hormone and CPTH fragments. Additional studies in clonal cells expressing both CPTHRs and PTH1Rs are needed to assess this hypothesis.


   Acknowledgments
 
We thank Dr. H. M. Kronenberg for critical review of the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grants DK-02889 and DK-65032 (to P.D.) and AR-47062 (to F.R.B.) and an educational grant from N.P.S. Pharmaceuticals, Inc.

First Published Online December 29, 2004

Abbreviations: BD1, Binding domain 1; CPTHR, carboxyl-terminal PTH receptor; h, human; PTH1R, type 1 PTH/PTHrP receptor; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Received September 23, 2004.

Accepted for publication December 21, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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