Prolonged signaling at the parathyroid hormone receptor by peptide ligands targeted to a specific receptor conformation

Abstract

The parathyroid hormone receptor (PTHR) is a class B G protein-coupled receptor that plays critical roles in bone and mineral ion metabolism. Ligand binding to the PTHR involves interactions to both the amino-terminal extracellular (N) domain, and transmembrane/extracellular loop, or juxtamembrane (J) regions of the receptor. Recently, we found that PTH(1–34), but not PTH-related protein, PTHrP(1–36), or M-PTH(1–14) (M = Ala/Aib¹,Aib³,Gln¹⁰,Har¹¹,Ala¹²,Trp¹⁴,Arg¹⁹), binds to the PTHR in a largely GTPγS-resistant fashion, suggesting selective binding to a novel, high-affinity conformation (R⁰), distinct from the GTPγS-sensitive conformation (RG). We examined the effects in vitro and in vivo of introducing the M substitutions, which enhance interaction to the J domain, into PTH analogs extended C-terminally to incorporate residues involved in the N domain interaction. As compared with PTH(1–34), M-PTH(1–28) and M-PTH(1–34) bound to R⁰ with higher affinity, produced more sustained cAMP responses in cells, formed more stable complexes with the PTHR in FRET and subcellular localization assays, and induced more prolonged calcemic and phosphate responses in mice. Moreover, after 2 weeks of daily injection in mice, M-PTH(1–34) induced larger increases in trabecular bone volume and greater increases in cortical bone turnover, than did PTH(1–34). Thus, the putative R⁰ PTHR conformation can form highly stable complexes with certain PTH ligand analogs and thereby mediate surprisingly prolonged signaling responses in bone and/or kidney PTH target cells. Controlling, via ligand analog design, the selectivity with which a PTH ligand binds to R⁰, versus RG, may be a strategy for optimizing signaling duration time, and hence therapeutic efficacy, of PTHR agonist ligands.

The parathyroid hormone (PTH) receptor (PTHR) mediates the biological actions of two ligands, PTH and PTH-related protein (PTHrP), and thereby plays critical roles in calcium and phosphate homeostasis, via PTH, and in bone growth and development, via PTHrP (1). As a class B G protein-coupled receptor, the PTHR uses two somewhat autonomous domains to engage its peptide ligands: the amino-terminal extracellular (N) domain, and the transmembrane/extracellular loop or juxtamembrane (J) region (2–6). Additionally, studies have revealed that the N domain of the PTHR mediates key binding interactions involving residues in the (residues 20–34) region of the ligand, whereas the J domain region forms the core binding pocket used by the N-terminal, signaling portion of the ligand.

We have shown previously that despite the absence of the C-terminal binding residues, N-terminal PTH fragments, such as PTH(1–14) and PTH(1–20), modified with certain “M” substitutions (M = Ala/Aib¹,Aib³,Gln¹⁰,Har¹¹,Ala¹²,Trp¹⁴,Arg¹⁹) bind to the PTHR J domain and activate signal transduction with much higher affinities and potencies than do the corresponding native PTH fragments (4, 5). Moreover, in PTHR binding studies performed in membranes and in the presence of GTPγS, or in membranes prepared from cells genetically lacking G_αS, the M-PTH(1–14) fragment analog binds poorly, if at all to the PTHR, whereas unmodified PTH(1–34) binds under such conditions with nearly the same high affinity that it does in the absence of GTPγS or presence of G_αS (7–9). These and related studies (10) have led us to the hypothesis that the PTHR has the capacity to form at least two pharmacologically distinguishable, high-affinity, conformational states. One high-affinity state, called RG, depends on G protein coupling, as demonstrated by the effects of GTPγS or absence of G_αS in the membrane binding studies. A second high-affinity state, called R⁰, does not depend on G protein coupling (7–9); furthermore, our studies predict that certain PTH ligands will bind selectively to one high-affinity conformation versus the other and that such selectivity of receptor binding can be a modulating factor of that ligand's biological activity. Indeed, PTHrP(1–36) appears to bind less selectivity to R⁰ than does PTH(1–34), in that it forms relatively unstable complexes with the receptor in membranes in the presence of GTPγS. Moreover, PTHrP(1–36) produces a relatively short-lived cAMP response in cells after ligand wash-out, whereas PTH(1–34) induces a longer cAMP response that persists for up to 1 h after wash-out (9). The cellular mechanisms underlying the more prolonged signaling response mediated by PTH(1–34) are uncertain at present, but the effect appeared to correlate with the capacity of the ligand to bind to the R⁰ PTHR conformation. These effects may have physiological relevance in that PTH(1–34) and PTHrP(1–36) have been noted to cause differential effects on calcium and vitamin D metabolism in humans (11, 12). We thus hypothesized that the selectivity with which a PTH ligand binds to the R⁰, versus RG, conformation of the PTHR would determine the duration time of signaling for that ligand, and hence its biological activity profile. As duration time of signaling likely plays an important role in the physiologic and therapeutic actions of PTH and PTHrP ligands (13–15), we sought to test our hypothesis further with additional PTH ligand analogs and by extending the studies to in vivo conditions.

Results

Determinants of R⁰ Binding Affinity.

To further define the determinants of R⁰ binding affinity, we extended the M-PTH(1–14) scaffold, which binds weakly to R⁰ despite strong binding to RG and the PTHR J domain, C-terminally so as to incorporate residues involved in the N domain interaction, and assessed the capacity of the ligands to bind to the R⁰ PTHR conformation, as well as to RG.

Each of the ligands bound to the RG conformation within a narrow range of affinities: IC₅₀s ≈0.5–12 nM (Fig. 1A and Table 1). A much broader range of affinities was observed in the R⁰ binding assays as M-PTH(1–14) bound with an affinity that was ≈600-fold weaker than that of PTH(1–34), whereas M-PTH(1–34) and M-PTH(1–28) each bound with an affinity that was ≈24-fold stronger than that of PTH(1–34) (Fig. 1B and Table 1). Thus, R⁰ binding affinity improved progressively with C-terminal chain-length extension, indicating that the N domain interaction contributes importantly to the stability of the R⁰–ligand complex. Furthermore, comparing the binding affinities of the M-substituted ligands, M-PTH(1–28) and M-PTH(1–34), with that of their native ligand counterparts, reveals that the optimized J-domain interaction, in combination with the C-terminally stabilized N-domain interaction, results in considerably higher R⁰ complex stability than occurs with PTH(1–34).

Fig. 1.

Binding and signaling properties of PTH analogs at the PTHR. (A) Ligand binding to the RG PTHR conformation was assessed in the presence of a high-affinity Gαs mutant using ¹²⁵I-M-PTH(1–15) tracer radioligand; (B) binding to the R⁰ conformation was assessed in the presence of GTPγS (10⁻⁵ M) using ¹²⁵I-PTH(1-34) as a tracer radioligand. (C) Ligand-induced cAMP signaling was assessed in MC3T3-E1 mouse osteoblastic cells by dose-response and (D) ligand wash-out protocols. In the wash-out assays, IBMX was added for 5 min at times after ligand wash-out; cAMP levels are expressed as a percentage of the maximum cAMP observed in cells treated with ligand [10⁻⁷ M, or 10⁻⁶ M for M-PTH(1–14) analog] for 10 min in the presence of IBMX (range of maximum cAMP values 52-55 pmol per well; basal cAMP = 1.6 ± 0.5 pmol per well). In separate, more extended wash-out experiments, cAMP levels returned to or near basal levels by 24 h (data not shown). (C Inset) Correlation plot of the cAMP EC₅₀ values and corresponding RG binding IC₅₀ values. (D Inset) Correlation plot of the area-under-the-curve (AUC) values obtained from the cAMP wash-out assays and the corresponding R⁰ binding IC₅₀ values. Binding and cAMP parameter values are reported in Table 1. Data are means (± SEM) of three to five experiments, each performed in duplicate.

Table 1.

Binding and signaling properties of PTH analogs

	Binding				cAMP			IP3
	R0		RG		EC50 Log M	nM	Emax pmol/well (basal = 0.8 ± 0.1 pmol/well)	EC50 Log M	nM	Emax cpm/well (basal = 82 ± 41 cpm/well)
	IC50 Log M	nM	IC50 Log M	nM
PTH(1–34)	−7.37 ± 0.09	55	−9.37 ± 0.05	0.45	−9.74 ± 0.03	0.18	88 ± 1	−7.84 ± 0.07	14	772 ± 50
PTH(1–28)	−5.50 ± 0.14	3,900	−8.53 ± 0.06	3.0	−9.01 ± 0.03	0.99	94 ± 1	−7.44 ± 0.13	36	653 ± 53
M-PTH(1–34)	−8.70 ± 0.11	2.3	−9.16 ± 0.08	0.75	−9.06 ± 0.05	0.87	93 ± 2	−7.62 ± 0.08	24	600 ± 30
M-PTH(1–28)	−8.56 ± 0.09	3.0	−9.28 ± 0.07	0.56	−9.01 ± 0.05	0.97	94 ± 2	−7.39 ± 0.08	41	702 ± 44
M-PTH(1–21)	−7.07 ± 0.09	92	−9.34 ± 0.07	0.49	−8.93 ± 0.002	1.2	91 ± 1	−7.82 ± 0.07	15	820 ± 33
M-PTH(1–14)	−4.48 ± 0.05	34,000	−7.93 ± 0.04	12	−7.53 ± 0.05	30	88 ± 2	−7.28 ± 0.04	52	635 ± 16

Data (means ± SEM) are from five (binding) or three (IP₃ and cAMP) experiments, each performed in duplicate.

Potency and Duration of cAMP Signaling.

Despite having enhanced apparent affinity for the R⁰ PTHR conformation, M-PTH(1–28) and M-PTH(1–34) exhibited slightly weaker potencies for stimulating cAMP responses in MC3T3-E1 cells than did PTH(1–34) (Fig. 1C and Table 1). Overall, the potencies (EC₅₀s) with which the ligands stimulated cAMP production, as assessed by dose–response assays, correlated better with the apparent affinities (IC₅₀s) with which they bound to the RG PTHR conformation, than that with which they bound to the R⁰ conformation (R² = 0.72 and 0.43, respectively; Fig. 1C Inset and Table 1). Thus, RG binding affinity was a better predictor of the potency with which a given PTH ligand induced a cAMP response than was R⁰ binding affinity.

Our previous studies on PTH(1–34) and PTHrP(1–36) analogs suggested a possible correlation between the affinity with which a PTH or PTHrP ligand bound to R⁰ and its capacity to produce a cAMP response at times after ligand wash-out (9). We thus assessed the current series of PTH ligands by using such a cAMP wash-out procedure in MC3T3-E1 cells. As shown in Fig. 1D, the cAMP signaling capacity of cells treated briefly (10 min) with a concentration of M-PTH(1–14) (1 × 10⁻⁶) or PTH(1–28) (1 × 10⁻⁷) returned to near baseline levels by 15 min after ligand wash-out. In contrast, the cAMP signaling capacity of cells treated with M-PTH(1–34) or M-PTH(1–28) was markedly prolonged and persisted at nearly 50% of the initial maximum level for at least 2 h after wash-out. The cumulative cAMP response capacity of each ligand after wash-out was quantified as the area under curve (AUC) for the 0- to 120-min time interval; the resulting values correlated better with the corresponding affinities with which the ligands bound to the R⁰ conformation than they did with those for the RG conformation (R² = 0.88 and 0.42, respectively; Fig. 1D). Thus, R⁰ binding affinity was a better predictor of the duration of the cAMP response induced by a given PTH ligand after initial binding to the receptor than was RG binding affinity.

FRET Recordings of Ligand–Receptor Interaction.

A bimolecular fluorescent FRET approach (2) was used to examine more directly the mechanisms by which these ligands interact with the receptor. The approach uses HEK-293 transiently transfected with ^GFPPTHR (GFP inserted into the N domain), and PTH ligands tagged at position with 13 with tetramethylrhodamine (TMR). In these experiments, as the TMR ligand binds to the ^GFPPTHR, the GFP fluorescence diminishes, because of FRET-based quenching by the TMR group, and then, upon ligand dissociation, the GFP fluorescence recovers. For the studies, we used PTH (1–28)^TMR and M-PTH (1–28)^TMR representing strongly RG- and R⁰-selective ligands, respectively (Fig. 1). Fig. 2A shows a kinetic trace of the FRET experiment performed with PTH(1–28)^TMR. Addition of ligand induced a rapid reduction in GFP fluorescence, and subsequent wash-out resulted in a rapid and nearly complete recovery of fluorescence. Thus, nearly all (≈90%) of the complexes formed with native PTH(1–28) were in an unstable state and sensitive to wash-out. The modified analog, M-PTH (1–28)^TMR, induced a similar rapid decrease in FRET upon ligand addition, but in this case, little or no recovery of fluorescence occurred after wash-out (Fig. 2B). Thus, nearly all (≈90%) of the complexes formed with M-PTH(1–28) were in a stable state and resistant to wash-out. The marked difference in complex stability observed for PTH(1–28) and M-PTH(1–28) in these FRET assays parallels the large (1,300-fold) difference in affinities observed for these equal-length ligands in the R⁰ binding assays.

Fig. 2.

FRET and confocal microscopy analysis of conformationally selective PTH ligands in HEK-293 cells. Single-cell, intermolecular FRET experiments were performed to monitor the binding of (A) PTH(1–28)^TMR and (B) M-PTH(1–28)^TMR to the ^GFPratPTHR transiently expressed in HEK-293 cells. The cells were super-fused with buffer containing ligand (1 × 10⁻⁷ M) for ≈30 s and then with buffer alone for 30 min (ligand application phase indicated by black bar). Spinning-disk confocal microscopy was used to assess binding and subcellular localization events induced by (C) PTH(128)^TMR and (D) M-PTH(1–28)^TMR in HEK-293 cells expressing ^GFPhumanPTHR. Images were recorded for both green (Top) and red (Middle) fluorescence, starting several seconds prior to ligand application and extending for 30 min thereafter; cells were superfused with ligand for ≈30 s and then with buffer alone for the remainder of the experiment (Magnification: 600×.)

Subcellular Localization of Ligand–Receptor Complexes.

The markedly prolonged activity observed for M-PTH(1–28) and M-PTH(1–34) in the cAMP wash-out assays (Fig. 1D) prompted us to investigate whether or not the ligands could induce receptor internalization responses. Internalization and desensitization responses have been well documented for the PTHR and unmodified PTH or PTHrP ligands (16–18). In the experiments of Fig. 2 C and D and supporting information (SI) Fig S1, we used spinning disk confocal microscopy to examine the internalization responses induced by PTH (1–28)^TMR and M-PTH (1–28)^TMR, respectively, in HEK-293 cells expressing ^GFPPTHR. Before ligand addition, ^GFPPTHR was predominantly located at the cell surface. Immediately upon addition, each of the TMR-labeled ligands appeared at the cell periphery and colocalized with ^GFPPTHR. No TMR fluorescence was detected in the presumably untransfected cells that lacked ^GFPPTHR fluorescence (not seen in fields shown), indicating that the observed TMR cell-surface fluorescence was PTHR-dependent. In addition, most, if not all, of the ^GFPPTHR appeared to colocalize with TMR ligand, which is consistent with the use of a high concentration of ligand (1 × 10⁻⁷ M). The cells were washed with buffer at ≈30 s after initial ligand application so as to remove unbound ligand. By 30 min after wash-out, only a very low level of PTH(1–28)^TMR fluorescence signal remained, which appeared to be localized to small internalized punctae. Thus, most of the PTH(1–28)^TMR had dissociated from the cell, leaving most of the ^GFPPTHRs on the surface in an unoccupied state. In contrast, at the same 30-min time point intense red fluorescence could be detected for M-PTH(1–28)^TMR, which appeared as larger-sized punctae both internally and at or near the plasma membrane, and in a colocalized state with the ^GFPPTHR. These data confirm that PTH(1–28), and M-PTH(1–28) form complexes of differential stability with the PTHR and indeed show that M-PTH(1–28) is not deficient for inducing receptor internalization.

Altered Actions in Vivo.

The prolonged cAMP responses and stable receptor complex formation observed for some of the modified analogs prompted us to consider whether such effects in vitro would translate into altered physiological effects in vivo. It is well documented that a single injection of PTH(1–34) into animals and humans results in acute rises in blood calcium and decreases in blood phosphate concentrations. We thus compared the two ligands with high R⁰ binding affinity: M-PTH(1–34) and M-PTH(1–28), to control PTH(1–34) for effects on acute changes in blood Ca²⁺ and Pi levels in mice after single-dose injection. Upon injection of PTH(1–34) (20 nmol/kg, i.v.), blood Ca²⁺ levels increased to a maximum value at 1 h that was 10% above the preinjection (t = 0) level, and the levels returned to baseline by 4 h (Fig. 3A). At the same dose, M-PTH(1–34) and M-PTH(1–28) increased Ca²⁺ to a similar level at 1 h, but levels continued to rise to maximum values at 2 h that were 17% and 20%, respectively, above preinjection levels, and these elevated levels were sustained for at least 8 h after injection (Fig. 3A). In another similar experiment, blood calcium levels were still elevated in M-PTH(1–34)-treated mice at 21-h postinjection (data not shown). Similarly, blood phosphate levels were suppressed to greater extents and for longer durations in M-PTH(1–28)- and M-PTH(1–34)-treated mice, as compared with PTH(1–34)-treated mice (Fig. 3B). We tested whether these prolonged physiological effects of M-PTH(1–28) and M-PTH(1–34) could be caused by the persistence of the peptides in the circulation by using a cAMP-based bioactivity assay for analyzing PTH peptide content in the plasma [Fig. S2 shows validation of this assay using a PTH(1–34)-directed ELISA kit]. The results of the bioactivity PK assay (Fig. 3C) indicated that M-PTH(1–28) and M-PTH(1–34) attained lower peak concentrations and disappeared more rapidly from the blood than did PTH(1–34).

Fig. 3.

Actions of PTH ligands in vivo. Peptide ligands or vehicle were injected i.v. into C57/BL6 mice for (A–C) acute time course analyses or (D) two-week bone-metabolic studies. Acute studies were performed with hPTH(1–34), M-PTH(1–28), and M-PTH(1–34) (20 nmol/kg), and vehicle control. Blood withdrawn immediately prior to injection (t = 0) and at times thereafter was assessed for (A) ionized calcium, (B) inorganic phosphate, or (C) PTH analog content. Analog content was determined by treating HEK-293 cells transiently transfected with the hPTHR with 1 μl of plasma for 30 min in the presence of IBMX and then measuring intracellular cAMP. C Inset shows that the peptides, when measured directly, are approximately equipotent in the HEK-293/hPTHR cell assay; no cAMP response was detected for the plasma samples in mock-transfected HEK-293 cells (not shown). The data show means (± SEM) of values from a single experiment in which (A) five, (B) six, or (C) three mice were used per group. For each analysis, similar results were obtained in at least two other experiments. Two-week daily injection studies were performed with rPTH(1–34) and M-PTH(1–34) (5 nmol/kg per day; n = 7 per group) in D. At the two-week end-point, femurs and tibiae were isolated and imaged by μCT (panes 1–6) or histology with von Kossa staining (panes 7–9). Shown are transverse views of the distal, metaphyseal (panes 1–3) and mid-diaphyseal (panes 4–6) regions of the femurs, and sagittal views of proximal tibiae (panes 7–9). (Magnification: D, 7–9, 40×.)

Exogenous administration of PTH ligands over extended periods is well known to stimulate processes of both bone formation and bone resorption, with intermittent (once daily) injection generally promoting net bone formation and continuous infusion promoting net bone resorption (13–15, 19–22). The mechanisms that govern the balance between the anabolic and catabolic effects of PTH are complex and not well understood, but one key determinant appears to be the duration and phasing of ligand exposure. The prolonged actions of M-PTH(1–34) observed in the above in vitro and acute in vivo assays prompted us to evaluate this ligand for effects on bone formation and bone resorption processes in mice. Mice were thus injected once daily via the tail vein for 2 weeks with either vehicle, PTH(1–34) or M-PTH(1–34) at equivalent doses (5 nmol/kg per day).

Micro-computed tomography (μCT) views of the distal femurs and histological images of the proximal tibiae revealed marked increases in trabecular bone in the M-PTH(1–34)-treated mice (Fig. 3D, panels 1–3 and 7–9). Calculations of volumetric bone parameters in the distal femur metaphyses confirmed significant increases in trabecular bone in the M-PTH(1–34)-treated mice, as compared with the vehicle controls, as well as to PTH(1–34)-treated mice (Table S1). Additionally, areal bone-density scans of total femur and lumbar spine showed that M-PTH(1–34) increased BMD to a greater extent than did PTH(1–34), which had only small effects on bone parameters (Fig. 3D and Table S1), although comparable to those seen in a previous study, in which a two-fold higher dose of PTH(1–34) was used (23). The dose of ligand used in our study (5 nmol/Kg) was chosen in order to avoid possible harmful calcemic effects that might occur from daily treatment with higher doses of M-PTH(1–34) (Fig. 3A).

At the cortical bone site of the mid-femur, M-PTH(1–34) produced areas of apparent erosion on the endosteal bone surface not seen in vehicle controls or with PTH(1–34) treatment (Fig 3D, panels 4–6). Calculations of the cortical wall thickness in the mid-femur diaphysis revealed a significant reduction in the M-PTH(1–34)-treated mice, relative to vehicle controls (172 ± 4 μm vs. 151 ± 3 μm, P = 0.003) (Table S1). The greater impact that M-PTH(1–34) had on trabecular and cortical bone structural parameters, relative to those of PTH(1–34), was paralleled by greater increases in serum markers of both bone formation (PINP and osteocalcin) and bone resorption (CTX) (Table S1). We also noted that the higher magnification views of the proximal tibiae revealed an apparent expansion of the integrated stromal cell population in the M-PTH(1–34)-treated animals, relative to the control and PTH(1–34)-treated groups (Fig. S3).

Discussion

Our in vitro studies on the prolonged signaling properties of the high-affinity PTHR/PTH conformation that we term R⁰ (8–10), and our continuing structure/activity studies with PTHrP, PTH, and modified fragments of PTH, led us to explore the hypotheses that we could use our cumulative body of data to: (i) design analogs of even greater affinity for the R⁰ conformation than PTH(1–34), and (ii) demonstrate that the relative propensity of such ligands to bind to this R⁰ conformation would determine the duration and extent of action of these ligands on mineral ion and bone metabolism in vivo. Our hypotheses have been confirmed by the data summarized herein, thereby pointing to a hitherto underappreciated role of receptor occupancy per se in biological responses in vivo for this G protein-coupled receptor/ligand system, well known to play critical roles in mineral ion and bone metabolism processes. The structure/activity approach involved extending the RG-selective ligand, M-PTH(1–14), C-terminally to incorporate residues concerned in the N-domain interaction (6). Combining the activity-enhancing features of the M series of N-terminal substitutions (24–26) with addition of significant portions of the carboxyl-terminal domain of PTH(1–34) produced ligands, M-PTH(1–28) and M-PTH(1–34), that bound more strongly to the R⁰ conformation, and produced more prolonged calcemic and hypophosphatemic responses in mice than did unmodified PTH(1–34) when each peptide was administered at equivalent doses. The prolonged effects in vivo were shown not to be attributable to prolonged pharmacokinetics and are thus consistent with a protracted mode of action at the receptor. The striking effects on trabecular bone mass in mice observed with M-PTH(1–34), which considerably exceeded those seen with PTH(1–34) (again at an equivalent dose) over a 2-week period further attests to the prolonged biological action of ligands favoring the R⁰ conformation. Overall, the bone phenotype observed with intermittent daily injection of M-PTH(1–34), increased trabecular bone with stromal cell expansion, and increased cortical resorption, parallels that seen in rodents treated continuously with unmodified PTH(1–34) (13, 14, 20, 21) and in transgenic mice overexpressing a constitutively active PTHR mutant in osteoblasts (27). These parallels are consistent with our view that the biological effects observed for M-PTH(1–34) in vivo involve selective binding of the ligand to a stable PTHR conformation, R⁰, that results in persistent signaling.

The prolonged and robust actions in vivo of M-PTH(1–34) and M-PTH(1–28) were not predicted by the potency of the ligands in our cell-based cAMP or IP₃ dose–response assays (Table 1); rather, the better in vitro predictors of actions in vivo were the results seen with the cAMP wash-out assay and R⁰ binding assays. The specific cellular and molecular mechanisms responsible for the prolonged signaling actions of these modified ligands both in vitro and in vivo are, as yet, unclear and require further investigation. Certain of our findings, as evident in the spinning disk confocal microscopy experiments (Fig. 2 and Fig. S1), demonstrate differences in internalization patterns for the structurally and functionally distinct ligands, PTH(1–28) and M-PTH(1–28), and clearly reveal that the long-acting ligand does induce some form of internalization/sequestration. The differential effects that these ligands have on cellular signaling events suggest a form of biased agonism, by which structurally distinct ligands for a given receptor activate different mechanisms of downstream signaling (28–30). How our observations on ligands with prolonged signaling capability relate to other G protein-coupled receptors, particular the class B receptors, remains to be determined.

Although the mechanism of the prolonged signaling is not yet determined, it is clear that ligands that strongly favor the R⁰ conformation have striking biological effects in vivo, which, in turn, suggests potential medical applications for our observations. Certain forms of hypoparathyroidism that are difficult to treat conventionally (vitamin D and calcium supplements), but have responded more favorably to PTH injections, although requiring twice-daily injections (31), might be more approachable with a long-acting PTH ligand that favors the R⁰ conformation, such as M-PTH(1–34) or derivative peptides (32). On the other hand, such a PTH analog might not prove superior to PTH(1–34) for treating osteoporosis, at least following current daily injection protocols (33, 34), in that bone-resorptive effects could predominate, particularly within the cortical bone, as typically seen with continuous PTH treatments in animals (19–22). In this regard, a high-potency PTH analog that favors the RG conformation, and hence produces a shorter-lived and more pulsatile action at the receptor, might prove to be more efficacious in this class of patients. Additional studies in vitro and in vivo of such conformationally selective PTH and PTHrP ligand analogs are required to test these hypotheses further.

Materials and Methods

Peptides and Reagents.

Peptides were synthesized by the Massachusetts General Hospital Biopolymer Core facility and were C-terminal amides. Human PTH derivatives used were: PTH(1–34), M-PTH(1–34) (M = Ala^1,12,Aib³,Gln¹⁰,Har¹¹,Trp¹⁴,Arg¹⁹); M-PTH(1–28), M-PTH(1–21), and M-PTH(1–14). Derivative peptides were modified with TMR at Lys-13. Rat PTH(1–34)NH₂ was used for 2-week in vivo studies. Radioligands ¹²⁵I-PTH(1–34) ([¹²⁵I-[Nle^8,21,Tyr³⁴]ratPTH(1–34)NH₂), and ¹²⁵I-M-PTH(1–15) (¹²⁵I-[Aib^1,3,Nle⁸,Gln¹⁰,Har¹¹,Ala¹²,Trp¹⁴,Tyr¹⁵]PTH(1–15)NH₂) were prepared as described (9).

Binding and cAMP Assays.

Binding to the RG and R⁰ conformations of the rat PTHR was assessed by competition reactions performed in 96-well plates by using transiently transfected COS-7 cell membranes as described (9). In brief, binding to R⁰ was assessed by using ¹²⁵I-PTH(1–34) as tracer radioligand and including GTPγS in the reaction (1 × 10⁻⁵ M). Binding to RG was assessed by using membranes containing a high-affinity, negative-dominant Gα_S subunit (Gα_S^ND) (35), and ¹²⁵I-M-PTH(1–15) as tracer radioligand. cAMP responses were assessed in MC3T3-E1 cells at room temperature (9). For dose–response assays, cells were incubated with ligand in buffer containing 3-isobutyl-1-methylxanthine (IBMX) (2 mM) for 30 min; for wash-out assays, cells were treated with ligand [1 × 10⁻⁷, or 1 × 10⁻⁶ M for M-PTH(1–14) analog] in buffer lacking IBMX for 10 min, rinsed, incubated in buffer for up to 120 min, then in buffer containing IBMX (2 mM) for a final 5 min. Incubations were terminated with 50 mM HCl, and intracellular cAMP was quantified by RIA. Assays of IP₃ stimulation were performed in COS-7 cells as described (9).

FRET.

The binding of PTH ligands to and dissociation from the PTHR was analyzed by an intermolecular FRET approach that used ^GFPPTHR(rat)-transfected HEK-293 cells and TMR-labeled PTH ligands (2). By this approach, the binding of the PTH^TMR ligand to the ^GFPPTHR causes fluorescence of GFP (λ_εx = 480 nm; λ_εm = 520 nm) to diminish, because of intermolecular FRET-based quenching by TMR (λ_ε = 520 nm; λ_εm = 580 nm) (2). Single cells were monitored over a 30-min period, beginning just before ligand addition by using excitation and detection wavelengths of 480 ± 20 and 520 ± 20 nm, respectively. FRET instrumentation used was as described (2). Data were corrected for photo-bleaching decay, which limited the duration of the recordings to ≈30 min.

Cellular Localization.

HEK-293 cells transiently transfected with ^GFPPTHR(human) were monitored by using a PerkinElmer LCI RS spinning disk confocal microscope before and after treatment with TMR-labeled PTH ligands. Cells on glass coverslips were monitored at 37°C, and fluorescent images were taken for a 30-min period beginning just before the addition of ligand (3 × 10⁻⁷ M). Images were processed by using the associated PerkinElmer software package.

In Vivo Pharmacology.

Mice (C57BL/6, male, age 9–12 weeks) were treated in accordance with the ethical guidelines adopted by Massachusetts General Hospital. Mice were injected i.v. via the tail vein, as opposed to s.c., to minimize possible effects of differences in ligand bioavailability or pharmacokinetics. For single-injection studies, mice were injected with either PTH(1–34), M-PTH (1–28), M-PTH(1–34) (20 nmol/kg), or vehicle (10 mM citric acid/150 mM NaCl/0.05% Tween-80, pH 5.0). Tail vein blood was collected immediately before and at times after injection for analysis. Plasma phosphate was measured with a UV spectroscopic assay (Stanbio Laboratory); blood Ca²⁺ was measured with a Chiron Diagnostics model 634 Ca²⁺/pH analyzer. Blood PTH peptide content was assessed by applying 1 μl of plasma, prepared in the presence of protease inhibitors (aprotinin, leupeptin, EDTA), to HEK-293 cells transiently transfected with the hPTHR for 30 min in the presence of IBMX, and measuring the resulting intracellular cAMP; mock-transfected cells were used to establish that the observed cAMP increases were PTHR-dependent. Plasma PTH(1–34) concentration was independently assessed with an ELISA kit specific for PTH(1–34) (Peninsula Laboratories).

For 2-week studies, mice were injected with rPTH(1–34) or M-PTH(1–34) (5 nmol/kg), or with vehicle once daily. Peptide activity in vivo was confirmed by measuring plasma cAMP responses in each animal 10 min after the first injection; these measurements yielded the expected and comparable cAMP increases for both PTH(1–34) and M-PTH(1–34) (data not shown). Blood was withdrawn at day 13 and analyzed by ELISA for P1NP (Immunodiagnostic Systems), osteocalcin, (Biomedical Technologies), and CTX (Nordic Bioscience Diagnostics). Mice were killed at day 14; serum was analyzed for total calcium (Stanbio Laboratory), and femurs, tibia, and lumbar vertebrae (L2-L5) were isolated. Femurs and vertebrae were analyzed for areal BMD by dual-energy, x-ray absorptiometry (PIXImus2; GE Lunar), and for volumetric BMD and structural parameters by using microcomputer tomography (μCT40; Scanco Medical AG) (15). Tibia were processed for histological analysis by von Kossa or hemotoxylin and eosin staining.

Data Calculations.

Data were processed by using Microsoft Excel and GraphPad Prism 4.0 software packages. Data from binding cAMP and IP₃ dose–response assays were analyzed by using a sigmoidal dose–response equation with variable slope. Paired data sets were statistically compared by using Student's t test (two-tailed) assuming unequal variances for the two sets.