Novel parathyroid hormone (PTH) antagonists that bind to the juxtamembrane portion of the PTH/PTH-related protein receptor.

Current antagonists for the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor (PTHR) are N-terminally truncated or N-terminally modified analogs of PTH(1-34) or PTHrP(1-34) and are thought to bind predominantly to the N-terminal extracellular (N) domain of the receptor. We hypothesized that ligands that bind only to PTHR region comprised of the extracellular loops and seven transmembrane helices (the juxtamembrane or J domain) could also antagonize the PTHR. To test this, we started with the J domain-selective agonists [Gln(10),Ala(12),Har(11),Trp(14),Arg(19) (M)]PTH(1-21), [M]PTH(1-15), and [M]PTH(1-14), and introduced substitutions at positions 1-3 that were predicted to dissociate PTHR binding and cAMP signaling activities. Strong dissociation was observed with the tri-residue sequence diethylglycine (Deg)(1)-para-benzoyl-l-phenylalanine (Bpa)(2)-Deg(3). In HKRK-B7 cells, which express the cloned human PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21), [Deg(1,3),Bpa(2),M]PTH(1-15), and [Deg(1,3),Bpa(2),M]PTH(1-14) fully inhibited (IC(50)s = 100-700 nm) the binding of (125)I-[alpha-aminoisobutyric acid(1,3),M]PTH(1-15) and were severely defective for stimulating cAMP accumulation. In ROS 17/2.8 cells, which express the native rat PTHR, [Deg(1,3),Bpa(2),M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) antagonized the cAMP-agonist action of PTH(1-34), as did PTHrP(5-36) (IC(50)s = 0.7 microm, 2.6 microm, and 36 nm, respectively). In COS-7 cells expressing PTHR-delNt, which lacks the N domain of the receptor, [Deg(1,3),Bpa(2), M]PTH(1-21) and [Deg(1,3),Bpa(2),M]PTH(1-15) inhibited the agonist actions of [alpha-aminoisobutyric acid(1,3)]PTH(1-34) and [M]PTH(1-14) (IC(50)s approximately 1 microm), whereas PTHrP(5-36) failed to inhibit. [Deg(1,3),Bpa(2),M]PTH(1-14) inhibited the constitutive cAMP-signaling activity of PTHR-tether-PTH(1-9), in which the PTH(1-9) sequence is covalently linked to the PTHR J domain, as well as that of PTHR(cam)H223R. Thus, the J-domain-selective N-terminal PTH fragment analogs can function as antagonists as well as inverse agonists for the PTHR. The new ligands described should be useful for further studies of the ligand binding and activation mechanisms that operate in the critical PTHR J domain.

Parathyroid hormone (PTH) 1 is a major regulator of ionized calcium and phosphate concentrations in the blood and extra-cellular fluids, and PTH-related protein (PTHrP) is a vital developmental morphogen (1,2). These two peptide ligands mediate their actions by binding to the same receptor, the PTH/PTHrP receptor or PTHR (or PTH receptor subtype 1). The PTHR is abundantly expressed in bone and kidney, the principal target organs of PTH, and in a variety of developing tissues (e.g. skeleton, heart, and mammary glands), where it mediates the actions of PTHrP. Excessive circulating levels of PTH, as occurs in cases of hyperparathyroidism, or PTHrP, as frequently occurs in cancer because of secretion by malignant tumors, produces a hypercalcemic state, which can be severely debilitating and potentially fatal (1,2). For both PTH and PTHrP, the first 34 amino acids encode all of the information needed for binding to and activating the PTHR.
The PTHR is a class 2 G protein-coupled receptor that activates the adenylyl cyclase/cAMP signaling pathway. Like all class 2 G protein-coupled receptors that bind peptide hormones, the PTHR has a relatively large (ϳ160 amino acid) N-terminal extracellular domain, herein termed the N domain (3), that plays a major role in hormone binding (4,5). The portion of the PTHR comprised of the extracellular loops and seven helical transmembrane domains (TMs), herein termed the juxtamembrane or J domain (3), is thought to mediate most, or all, of the ligand interactions that are involved in receptor activation and G protein coupling (5)(6)(7)(8). The molecular mechanism by which PTH (and PTHrP) binds to the PTHR has been extensively analyzed through the approaches of receptor mutagenesis (4 -6, 9 -11), photochemical crosslinking (12)(13)(14), and molecular modeling (15)(16)(17). These studies have led to a "two-site" model for the ligand-receptor interaction mechanism, according to which the C-terminal portion of PTH(1-34) (i.e. residues in the 23-34 region) binds to the N domain of the receptor to provide the majority of the binding energy to the complex, and the N-terminal portion of the ligand interacts with a lower affinity with the J domain of the receptor to induce activation (3,5,18). A similar two-site binding mechanism appears to be used by other class 2 G protein-coupled receptors that bind peptide hormones (19 -27).
The use of antagonist ligands contributes importantly to the analysis of ligand binding and signal transduction mechanisms used by G protein-coupled receptors. For the PTHR, all current antagonists are PTH or PTHrP analogs that extend C-terminally to at least residue 34 and are thought to depend strongly, although probably not completely (11), on interactions with the N domain of the receptor to achieve high binding affinity and, hence, inhibitory potency. The lack of signaling activity in these antagonist can be attributed to either the deletion, or modification, of N-terminal residues of the ligand, which are well known to play a critical role in PTHR activation (34 -36). The most potent PTHR antagonists to date are based on the PTH(7-34) scaffold peptide (36); these "classical" PTHR antagonist include [Leu 11 ,D-Trp 12 ]PTHrP(7-34) (37) and [Ile 5 ,Trp 23 , Tyr 36 ]PTHrP(5-36) (38). The N-and C-terminally intact antagonists include PTH  or PTHrP  analogs that have either valine 2 substituted by arginine (39), tryptophan or para-benzoyl-L-phenylalanine (Bpa) (38,40), or serine 3 substituted by Tyr or Phe (41). Several of the previous PTHR antagonists, including [Leu 11 ,D-Trp 12 ]PTHrP , [Leu 11 ,D-Trp 12 ]PTHrP , and [Bpa 2 ]PTHrP(1-36), function as inverse agonists on the constitutively active mutant PTHRs, PTHR cam T410P, and/or PTHR cam H223R (42,43).
Cell Culture and DNA Transfection-The cell lines HKRK-B28 and HKRK-B7 are clonal derivatives of the porcine kidney cell line, LLC-PK 1 , and were transfected with pCDNA1-based plasmid DNA to stably express recombinant PTH receptors at surface densities of ϳ280,000 and ϳ950,000 receptors/cell, respectively (44). HKRK-B28 cells express a fully functional PTHR chimera comprised of the opossum PTHR from the N terminus to the middle of TM3 and the rat PTHR from the middle of TM3 to the C terminus (11); HKRK-B7 cells express the wild-type human PTHR (44). COS-7 cells were transfected to transiently express the PTHR using the FuGENE 6 (Roche Applied Science) reagent and CsCl-purified, pCDNA1-based plasmid DNA (200 ng/well of a 24-well plate) encoding either the wild-type human PTHR (45) or a human PTHR derivative, PTHR-delNt (6, 7), PTHR cam H223R (46), or PTHRtether-PTH(1-9). The PTHR-tether-PTH(1-9) construct is similar to constructs described by us previously (31) and consists of the following contiguous (N to C terminus) segments: the prepro sequence of native human PTH, residues 1-9 of native rat PTH (Ala-Val-Ser-Glu-Ile-Gln-Leu-Met-His), a tetraglycine linker, and the hPTHR sequence Glu 182 -Met 593 , which extends from the extracellular terminus of TM1 to the C terminus of the receptor. Upon expression and processing in COS-7 cells, the prepro-PTH sequence is removed so as to leave alanine 1 of the PTH sequence as the free N terminus of the protein (confirmed by N-terminal sequence analysis of the purified protein). 2 Competition Binding Assays-Binding studies were performed using 125 I-[Aib 1,3 ,M]PTH(1-15) as the tracer radioligand and varying concentrations of unlabeled test peptides as competitors, as described previously (29,30). In brief, cells in 24-well plates were rinsed in binding buffer (50 mM Tris-HCl, 100 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 5% heat-inactivated horse serum, 0.5% fetal bovine serum, adjusted to pH 7.7 with HCl),and the following three components were added to the well successively, 100 l of binding buffer, 100 l of binding buffer containing an unlabeled test peptide, and 100 l binding buffer containing 125 I-[Aib 1,3 ,M]PTH(1-15) (ϳ100,000 cpm/well). All solutions were ice-cold, and the plates were embedded in a tray of ice during the addition phase. The plates were then incubated in a 15°C water bath for 4 h, after which the well contents were removed by aspiration, and the cell monolayers were rinsed three times by adding and rapidly removing 0.5 ml of binding buffer. Then, 0.5 ml of 5 N NaOH was added to lyse the monolayers followed by the addition of 2.0 ml of H 2 0, and the entire lysate was counted for radioactivity. Nonspecific binding was determined in wells containing an excess (1 ϫ 10 Ϫ5 M) of an unlabeled high affinity ligand, and the total specific binding at each competing ligand concentration was calculated as a percentile of the total specific binding observed in the absence of competitor. The data were then analyzed by non-linear regression analysis to obtain fitted curves and IC 50 values (see below).
cAMP Stimulation Assays-Assays were performed in whole cells in 24-well plates, in which the cells had formed confluent monolayers 2-4 days prior to assay and had attained densities of 200,000 -300,000 cells/well, as described previously (29,30). The assays utilized binding buffer containing 2 mM IBMX (3-isobutyl-1-methylxanthine). For cAMP response assays involving the addition of a single peptide, cells were rinsed in IBMX-binding buffer, and the following two components were added to the wells successively, 200 l of IBMX-binding buffer and 100 l of IBMX-binding buffer containing the test peptide. For assays involving the addition of two peptides (agonist plus antagonist), cells were rinsed with IBMX-binding buffer and the following three components were added to the wells successively, 100 l of IBMX-binding buffer, 100 l of IBMX-binding buffer containing an antagonist peptide, and 100 l of IBMX-binding buffer containing an agonist peptide. Following these additions, the plates were incubated at room temperature for 30 min. The buffer was then rapidly removed, the plates were embedded in powdered dry ice to freeze the monolayer, and 0.5 ml of 50 mM HCl was added. After freezing and thawing the contents, 2.0 ml of H 2 0 was added to each well and an aliquot (0.5-20 l) was withdrawn and was processed by a radioimmunoassay to quantify the concentration of (intracellular) cAMP.
Inositol Phosphate (IP) Stimulation Assays-COS-7 cells in 24-well plates were transiently transfected as above with the human PTHR and 3-4 days later were treated with serum-free, inositol-free Dulbecco's modified Eagle's medium containing 0.1% bovine serum albumin and [ 3 H]myo-inositol (PerkinElmer Life Sciences) (2 Ci/ml) for 16 h. The cells were then rinsed with binding buffer containing LiCl (30 mM), and the following three components were added to the wells successively, 200 l of LiCl-binding buffer, 40 l of LiCl-binding buffer containing or lacking a test antagonist peptide, and 10 l of LiCl-binding buffer containing or lacking an agonist peptide. The cells were then incubated at 37°C for 40 min, after which the buffer was removed and replaced by 0.5 ml of ice-cold 5% trichloroacetic acid solution. After 2 h on ice, the lysate was collected, extracted twice with diethyl ether, applied to an ion exchange column (Dowex AG1-X8 resin, 0.5-ml bed volume), and total inositol phosphates were eluted as described previously (47) and counted in liquid scintillation mixture for radioactivity.
Data Calculations-All calculations were performed using the Excel software package (Microsoft Corp., Redmond, WA). Curves were fit to data points using a four parameter logistic equation: where y is the observed response (specific binding of radioligand or cAMP value), min and max are the minimum and maximum response values, respectively, x is ligand concentration (nM), IC 50 (or EC 50 in cAMP assays) is the ligand concentration (nM) at which 50% of the maximum response occurs, and n is the slope of the plot of y versus x and the Excel Solver function to optimize the parameters by nonlinear regression analysis (48). Paired data sets were statistically compared using the Student's t test (two-tailed) assuming unequal variances for the two sets.

PTHR Binding and cAMP Signaling Properties of PTH Analogs in LLC-PK1
Cells-Starting with the potent agonist [Ac 5 c 1 ,Aib 3 ,M]PTH(1-14) (30) as a parent scaffold peptide, we first introduced at positions 1 or 2 single-residue modifications that were predicted to dissociate PTHR binding affinity and cAMP-signaling capacity. These modifications included the removal of the N-terminal amino function (35), as provided by des-NH 2 -Ac 5 c 1 and des-NH 2 -Aib 1 (Aib is structurally related to Ac 5 c and when substituted at position 1 results in activity profiles that are nearly indistinguishable from those seen with Ac 5 c 1 (30)) and the substitution of valine 2 by arginine (39), tryptophan, or Bpa (38, 40) ( Table  I). The resulting analogs were tested for PTHR binding affinity and cAMP-stimulating potency using HKRK-B28 cells, an LLC-PK1-derived cell line in which a rat/opossum chimeric PTHR is expressed at a density of ϳ280,000 receptors/ cell (44). As summarized in Table II, each of the singleresidue modifications resulted in some loss of binding affinity, as assessed in competition experiments performed with 125 I-[Aib 1,3 ,M]PTH(1-15) tracer radioligand, with the strongest (830-fold) reduction occurring with the Arg 2 substitution and the mildest (25-fold) reduction occurring with the Trp 2 substitution. In the cAMP formation assays we used a peptide concentration of 10 M, at which most, if not all, of the analogs could be predicted to attain at least half-maximum receptor occupancy. Each of the substituted analogs stimulated no more than 25% of the maximum cAMP response (E max ) observed with the parental peptide [Ac 5 c 1 , Aib 3 ,M]PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), and the Arg 2 -, Bpa 2 -, and Trp 2 -substituted analogs stimulated no more than 10% of the parental response.
Inhibition of Constitutively Active Mutant PTH Receptors in COS-7 Cells-We then investigated the capacity of the antagonist analogs to inhibit the basal cAMP-stimulating activity of two constitutively active mutant PTH receptor derivatives. The first of these derivatives was a "tethered" ligand-receptor construct in which the N-terminal domain of the PTH receptor is replaced by the (1-9) segment of native PTH, representing the minimum agonist pharmacophore of the ligand (31). In this construct, the ligand sequence is covalently joined to the body of the PTHR via a tetra-glycine linker between His 9 of the ligand sequence and Glu 182 of the receptor, which is located approximately nine residues N-terminal of the predicted extracellular end of TM1. When transiently transfected into COS-7 cells, this construct, in the absence of exogenously added ligand, results in basal cAMP levels that are comparable to those seen in COS-7 cells transfected with the wild-type PTHR and stimulated maximally with PTH(1-34). As shown in Fig. 5A, the addition of [Deg 1,3 ,Bpa 2 ,M]PTH(1-14) (10 M) to such transfected COS-7 cells caused an ϳ40% diminishment in the basal cAMP levels. The analog PTHrP  resulted in a small (16%) yet significant (p ϭ 0.02) reduction in basal signaling activity (Fig. 5A).
The second constitutively active mutant PTHR utilized was PTHR cam H223R. This intact mutant PTHR contains an activating point mutation (His 223 3 Arg) at or near the cytoplasmic terminus of TM2 (46). We included in these studies well as two C-terminally extended PTHrP ligands known to function as inverse agonists on this constitutively active PTHR, [Leu 11 ,D-Trp 12 ]PTHrP(5-36) and [Bpa 2 ]PTHrP(1-36) (43). Each of the tested analogs, applied at a concentration of 10 M, reduced to at least some degree the intracellular cAMP levels in COS-7 cells expressing PTHR cam H223R (Fig. 5B). The strongest reduction (72%) occurred with [Ac 5 c 1 ,Bpa 2 ,Aib 3 ,M]-PTH(1-14) 4  Inhibition of Ligand-induced Inositol Phosphate Signaling in COS-7 Cells Expressing the Wild-type PTHR-Because the wild-type PTHR can also couple to the PLC/IP signaling pathway, we analyzed the capacity of the antagonist ligands to inhibit this mode of PTHR signaling. As shown in the experiments of Fig. 6A  These IP antagonism results are fully consistent with the cAMP-antagonism data described above and are also consistent with the notion that mechanism of ligand-induced IP signaling in the PTHR is similar to the mechanism of ligand-induced cAMP signaling, in that each involves interactions between the N-terminal residues of the ligand and the juxtamembrane region of the receptor (28,30,51). DISCUSSION In this study we explored whether a PTHR ligand that binds exclusively to the portion of the receptor comprised of the extracellular loops and seven transmembrane helices (the J region) could function as a PTHR antagonist. Our results showed that this is indeed possible, as PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), and PTH(1-21) agonist analogs, previously shown to interact mainly, if not exclusively, with the PTHR J domain (7,11,29,30), when modified with activity-lowering substitutions at positions 1-3, exhibit antagonist properties on both the intact PTHR, as well as on PTHR-delNt, which lacks most of the N-terminal extracellular domain (N domain) of the receptor. These new antagonist ligands differ structurally and mechanistically from all previous PTHR antagonists as the latter analogs are C-terminally extended to at least position 34 and depend strongly on interactions with the N domain of the receptor to achieve their binding affinity and, hence, inhibitory potency (4,5,52). The modifications that conferred antagonist properties to the new N-terminal PTH analogs included the removal of the N-terminal amino group, the substitution of valine 2 by arginine, tryptophan, or Bpa, and the substitution of residues 1 (normally serine or alanine) and 3 (normally serine) by Deg. The activity-diminishing effects observed for these substitutions in the context of the modified N-terminal PTH fragment analogs are fully consistent with the known importance of the first few residues of native PTH  in inducing PTHR activation (34 -36,41), particularly that of valine 2 (9,38,40). In the current analogs, the strongest dissociation of binding and signaling activities occurred with the valine 2 3 Bpa substitution; combining Bpa 2 with the Deg 1,3 substitutions led to analogs with little or no cAMP or IP agonist activity, even when present at concentrations that would nearly saturate the receptor binding sites (Table II, Fig. 6). The tri-residue sequence, Deg 1 -Bpa 2 -Deg 3 , thus constitutes a strong antagonist pharmacophore for the PTHR and, as such, was incorporated into the affinity-enhanced   analog scaffolds for subsequent functional studies. The functional properties of these three analogs were found to be very similar; the slightly (6-fold) higher binding affinity of the longer PTH(1-21) analog, compared with the PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and PTH(1-15) analogs (Table III) resulted in slightly higher inhibitory potencies, as seen in some of the antagonism assays (e.g. Fig. 2A), reflecting mostly the contribution of residue 19 to binding affinity (11).
One goal of these studies was to obtain new antagonist ligands that could be used to further explore the ligand binding and activation mechanisms that operate in the PTHR. In this regard, the results of our studies are generally consistent with and extend the two-site model that has been proposed for the PTH/PTHR interaction mechanism (3)(4)(5)(6). The principal postulates of this model are depicted in Fig. 7    maintained approximately the same inhibitory potency on PTHR-delNt as they did on the intact PTHR, whereas in contrast, PTHrP(5-36), a highly effective antagonist on the intact PTHR, exhibited little or no inhibitory capacity on the Ntruncated receptor. These divergent functional activities observed for the N-and C-truncated antagonists on PTHR-delNt provided the principal support for our conclusion that the new N-terminal antagonist analogs bind purely to the J domain of the receptor, and thus differ mechanistically from the previous C-terminally extended antagonists, including the classical Nterminally truncated antagonists (36 -38), which derive the major portion of their binding energy from interactions with the N domain of the receptor (4, 5, 52). Our findings with PTHR-delNt also demonstrate that interactions with the N 4 In the experiments of Fig. 5A, this analog reduced cAMP signaling by PTHR-tether-PTH(1-9) by 30%, p Ͻ 0.0001 (data not shown). domain of the receptor are not necessary for antagonizing the PTHR, at least in the case where the antagonist binds with high affinity to the J domain, as do the new N-terminal PTH antagonist analogs described here.
The overall results so far on the PTH/PTHR interaction mechanism achieved with multiple PTH ligand analogues, mutant PTH receptors, and photo-affinity cross-linking studies, provide an initial definition of the regions of interaction between the ligand and the receptor. Although the new antagonists of the current study bind exclusively to the J domain, we also found evidence to suggest that the classical, N-terminally truncated antagonists, represented here by one of the strongest antagonists, PTHrP(5-36) (38, 43), do not bind purely to the N domain of the receptor, a possibility that has been raised previously (3,6). The new evidence for this includes the capacity of PTHrP  to fully inhibit the cAMP-stimulating activity of [Ac 5 c 1 ,Aib 3 ,M]PTH(1-14) (a J domain-specific agonist) on the PTHR (Fig. 2B), as well the capacity of PTHrP  to fully inhibit the binding of the 125 I-[Aib 1,3 ,M]PTH(1-15) radioligand to the intact receptor (Fig. 1A). These results thus suggest that some portion of PTHrP(5-36) directly interacts with the PTHR J domain. Consistent with this possibility, recent mutational and photo-affinity cross-linking data provide evidence for interactions or proximities between residues in the 5-27 region of the ligand and the PTHR J domain. Thus, the extension of PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) peptides to position 20/21 and inclusion of the Glu 19 3 Arg substitution enhances binding affinity and/or signaling potency on PTHR-delNt (the isolated J domain) by as much as 100-fold (11). In cross-linking studies, a PTH(1-34) analog modified with benzophenone at position 17 cross-linked to Leu 261 in the first extracellular loop (53), a benzophenone 13-modified PTH(1-34) peptide cross-linked to Arg 186 near the N domain/ TM1 boundary (13), and [Bpa 19 ]-PTHrP(1-36), as well as [Bpa 19 ,M]PTH(1-21), each cross-linked to the extracellular terminus of TM2 (32). At least some such proximities in the PTHR J domain must contribute to the overall binding energy with which PTHrP(5-36) interacts with the receptor. In further support of this hypothesis we found that although PTHrP  failed to inhibit the binding of 125 I-[Aib 1,3 ,M]PTH(1-15) to PTHR-delNt or the cAMP-signaling activity of [Ac 5 c 1 ,Aib 3 ,M]-PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and [Aib 1,3 ]PTH(1-34) on this truncated receptor (emphasizing the critical dependence of the ligand on the presence of the N domain of the receptor) it did inhibit marginally, yet significantly, signaling by PTHR-tethered-PTH(1-9), which like PTHR-delNt lacks nearly all of the N domain of the receptor. Any interactions that occur between PTHrP(5-36) and the PTHR J domain would therefore be too weak by themselves to enable the inhibition of binding of N-terminal PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) analogs modified with affinity-enhancing substitutions yet be strong enough to enable at least partial inhibition of the binding of the unmodified N-terminal PTH (1-9) sequence to the PTHR J domain.
Another possibility not entirely excluded by the present data is that PTHrP(5-36) inhibits the modified PTH(1-14) agonist actions on the intact PTHR via an allosteric mechanism, for example, by binding to the PTHR N domain in a way that interferes with movements in the J domain that are required for the formation of a PTHR active state (54) that is specifically recognized by the PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) agonist. In this regard, it is worth noting that several non-peptidic antagonist compounds have been identified for other class 2 G protein-coupled receptors, and some of these appear to bind outside of the receptor region occupied by the agonist pharmacophore of the native ligand. Thus, a key binding determinant for a non-peptide antagonist for the calcitonin gene-related protein receptor maps to the receptor-associated-modifying protein that modulates the li-gand-binding specificity of that receptor (55), and a binding determinant for a non-peptide antagonist of the glucagon-likepeptide-1 receptor was identified as a tryptophan residue located at position 23 near the N terminus of that receptor (56). On the other hand, Hoare and colleagues (27) have shown that several small molecule antagonists of the corticotropin-releasing factor (CRF) receptor bind directly to the isolated CRF receptor J domain, and residues required for the binding of such antagonists have been identified in the midregions of TM3 and TM6 of the intact CRF receptor and within ϳ10 amino acids of residues at the extracellular ends of the same helices that contribute to CRF binding (57). It would thus seem that these CRF receptor antagonists could inhibit CRF action via direct, competitive mechanisms. A small molecule antagonist for the glucagon receptor also binds within the J domain of that receptor and requires for this binding residues within the midregions of TM2 and TM3; in this case, however, the data point to a non-competitive mechanism of inhibition (58).
The mechanism by which PTHrP(5-36) inhibits the J domain-selective agonists on the PTHR is not at present clear. It is clear, however, that the PTH(1-14)-based antagonists bind to the J domain of the receptor and appear to function via a direct competitive mechanism (Fig. 4B). This mode of inhibition renders these new antagonists potentially useful probes of the region of the PTHR J domain that is involved in binding and responding to the PTH agonist pharmacophore. That the Bpa 2 substitution in these antagonists produces approximately the same profile of antagonism and inverse agonism as that seen with Bpa 2 -modified PTH  or PTHrP(1-36) further suggests that the modified, N-terminal PTH antagonists interact with the PTHR in a fashion similar to that used by the N-terminal portion of the relatively unmodified, longer length ligands and thus do not mediate antagonism via long range, allosteric effects.
The overall results of our study show that it is possible to antagonize the PTH receptor by targeting ligands specifically to the PTHR J domain. The data provide support for and extend the current two-domain model of the PTH/PTHR binding and activation mechanisms. The principal determinants of the antagonist pharmacophore of the new analogs are the photolabile Bpa 2 group and the conformationally constraining Deg 1,3 substitutions. The ligands should therefore be useful for further mapping sites of interaction in the PTH/PTH receptor interface, specifically those that occur within the J domain of the receptor and are involved in activation (16), and potentially for the further design of ligands (peptidic or non-peptidic) that exhibit potent, agonist or antagonist properties on the PTH/ PTHrP receptor.