International Union of Basic and Clinical Pharmacology. XCIII. The parathyroid hormone receptors--family B G protein-coupled receptors.

The type-1 parathyroid hormone receptor (PTHR1) is a family B G protein-coupled receptor (GPCR) that mediates the actions of two polypeptide ligands; parathyroid hormone (PTH), an endocrine hormone that regulates the levels of calcium and inorganic phosphate in the blood by acting on bone and kidney, and PTH-related protein (PTHrP), a paracrine-factor that regulates cell differentiation and proliferation programs in developing bone and other tissues. The type-2 parathyroid hormone receptor (PTHR2) binds a peptide ligand, called tuberoinfundibular peptide-39 (TIP39), and while the biologic role of the PTHR2/TIP39 system is not as defined as that of the PTHR1, it likely plays a role in the central nervous system as well as in spermatogenesis. Mechanisms of action at these receptors have been explored through a variety of pharmacological and biochemical approaches, and the data obtained support a basic "two-site" mode of ligand binding now thought to be used by each of the family B peptide hormone GPCRs. Recent crystallographic studies on the family B GPCRs are providing new insights that help to further refine the specifics of the overall receptor architecture and modes of ligand docking. One intriguing pharmacological finding for the PTHR1 is that it can form surprisingly stable complexes with certain PTH/PTHrP ligand analogs and thereby mediate markedly prolonged cell signaling responses that persist even when the bulk of the complexes are found in internalized vesicles. The PTHR1 thus appears to be able to activate the Gα(s)/cAMP pathway not only from the plasma membrane but also from the endosomal domain. The cumulative findings could have an impact on efforts to develop new drug therapies for the PTH receptors.

Altered selectivity of parathyroid hormone (PTH) and PTH-related protein (PTHrP) for distinct conformations of the PTH/PTHrP receptor.

PTH and PTHrP use the same G protein-coupled receptor, the PTH/PTHrP receptor (PTHR), to mediate their distinct biological actions. The extent to which the mechanisms by which the two ligands bind to the PTHR differ is unclear. We examined this question using several pharmacological and biophysical approaches. Kinetic dissociation and equilibrium binding assays revealed that the binding of [(125)I]PTHrP(1-36) to the PTHR was more sensitive to GTPgammaS (added to functionally uncouple PTHR-G protein complexes) than was the binding of [(125)I]PTH(1-34) ( approximately 75% maximal inhibition vs. approximately 20%). Fluorescence resonance energy transfer-based kinetic analyses revealed that PTHrP(1-36) bound to the PTHR more slowly and dissociated from it more rapidly than did PTH(1-34). The cAMP signaling response capacity of PTHrP(1-36) in cells decayed more rapidly than did that of PTH(1-34) (t(1/2) = approximately 1 vs. approximately 2 h). Divergent residue 5 in the ligand, Ile in PTH and His in PTHrP, was identified as a key determinant of the altered receptor-interaction responses exhibited by the two peptides. We conclude that whereas PTH and PTHrP bind similarly to the G protein-coupled PTHR conformation (RG), PTH has a greater capacity to bind to the G protein-uncoupled conformation (R(0)) and, hence, can produce cumulatively greater signaling responses (via R(0)-->RG isomerization) than can PTHrP. Such conformational selectivity may relate to the distinct modes by which PTH and PTHrP act biologically, endocrine vs. paracrine, and may help explain reported differences in the effects that the ligands have on calcium and bone metabolism when administered to humans.

Ligand-receptor interactions at the parathyroid hormone receptors: subtype binding selectivity is mediated via an interaction between residue 23 on the ligand and residue 41 on the receptor.

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind and activate the PTH/PTHrP receptor (PTH-1R). However, while the related receptor PTH-2R responds potently to PTH, it is not activated by PTHrP. Two hormone sites are known to be responsible for these different potencies. First, the absence of efficacy for PTHrP at PTH-2R is due to the presence of His-5 in PTHrP (Ile-5 in PTH), which interacts with the receptor's juxtamembrane domain. Second, PTHrP has lower affinity than PTH for PTH-2R because of the presence of Phe-23 (Trp-23 in PTH), which interacts with the receptor's N-terminal extracellular domain. We used these different receptor subtype properties to demonstrate that residue 41 in PTH-1R, when either the native Leu or substituted by Ile or Met, can accommodate either Phe or Trp at position 23 of the ligand. However, when Leu-41 is substituted by a smaller side chain, either Ala or Val (its equivalent residue in PTH-2R), the receptor becomes highly selective for those peptide ligands with Trp-23. Hence, despite the conservative nature of the substitutions found in the native ligands (Phe for Trp) and receptors (Leu for Val), they nevertheless enable a significant degree of selectivity to be achieved. Analysis of this functionally important ligand-receptor contact, within the context of the recent X-ray structure of the peptide-bound PTH-1R N domain, reveals the nature of the selectivity filter and how it is by-passed in PTH-1R.

Structure and functional expression of a complementary DNA for porcine parathyroid hormone/parathyroid hormone-related peptide receptor.

A complementary DNA (cDNA) encoding the receptor for porcine parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) was isolated from a porcine kidney cDNA library. The porcine PTH/PTHrP receptor is a 585 amino acid protein containing seven putative membrane-spanning domains. The porcine PTH/PTHrP receptor has amino acid identity of 95.6%, 80.4%, and 88.7% with human, opossum, and rat PTH/PTHrP receptors, respectively and 53.4% identity to the recently cloned human PTH2 receptor. The receptor cDNA was subsequently cloned into a mammalian cell expression vector (pRC/CMV) which contains a human cytomegalovirus promoter. A human kidney cell line (293), stably transfected with this vector, expressed the receptor at a high level and, when challenged with human PTH(1-34), increased cytoplasmic cAMP and inositol triphosphate production. Radioligand binding studies revealed that the receptor bound both human PTH(1-34), and PTHrP(1-36). Scatchard analyses of three clones showed that the cells harbor a single class of high affinity receptor (Kd = 1-4 nM for human PTH(1-34)) but had varying receptor numbers (10(5)-10(6) receptors/cell). In contrast to PTH(1-34), the [Arg2]PTH(1-34) analog bound to the porcine PTH/PTHrP receptor with low affinity and was a weak agonist for cAMP stimulation with the cloned receptor. These response characteristics differentiate the porcine receptor from the previously cloned rat and opossum PTH/PTHrP receptors.

Interaction of the parathyroid hormone receptor with the 14-3-3 protein.

The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) regulates calcium homeostasis, bone remodeling and skeletal development. 14-3-3 proteins bind to signaling proteins and act as molecular scaffolds and regulators of subcellular localization. We show that the parathyroid hormone receptor (PTHR) interacts with 14-3-3 and the proteins colocalize within the cell. 14-3-3 interacts with the C-terminal tail of the receptor containing a consensus 14-3-3 binding motif, but additional binding sites are also used. Protein kinase-A treatment of the receptor and especially the C-terminal tail reduces 14-3-3 binding. The expressed C-terminal tail is primarily localized in the nucleus, supporting the function of a putative nuclear localization signal that could be involved in the previously described nuclear localization of PTHR. The observed interaction between PTHR and the 14-3-3 protein implies that 14-3-3 could contribute to regulation of PTHR signaling.

Molecular properties of the PTH/PTHrP receptor.

The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) is a G protein-coupled receptor (GPCR) that plays a key role in controlling blood Ca(2+) concentration and endochondral bone formation. This review focuses on the molecular mechanisms by which the receptor recognizes the PTH and PTHrP peptide ligands and transmits their signal across the cell membrane. The available data suggest that there are two principal components to the ligand-receptor interaction. First, a docking interaction between the C-terminal portion of PTH(1-34) and the N-terminal extracellular domain of the receptor; and second, a weaker interaction between the N-terminal portion of the ligand and the juxtamembrane region of the receptor, which induces signal transduction. A full understanding of these processes could lead to new PTH/PTHrP receptor ligands that are effective in controlling diseases of bone and mineral metabolism, such as osteoporosis.

Multiple regions of ligand discrimination revealed by analysis of chimeric parathyroid hormone 2 (PTH2) and PTH/PTH-related peptide (PTHrP) receptors.

PTH and PTH-related peptide (PTHrP) bind to the PTH/PTHrP receptor and stimulate cAMP accumulation with similar efficacy. Only PTH activates the PTH2 receptor. To examine the structural basis for this selectivity, we analyzed receptor chimeras in which the amino terminus and third extracellular domains of the two receptors were interchanged. All chimeric receptors bound radiolabeled PTH with high affinity. Transfer of the PTH2 receptor amino terminus to the PTH/PTHrP receptor eliminated high-affinity PTHrP binding and significantly decreased activation by PTHrP. A PTH/PTHrP receptor N terminus modified by deletion of the nonhomologous E2 domain transferred weak PTHrP interaction to the PTH2 receptor. Introduction of the PTH2 receptor third extracellular loop into the PTH/PTHrP receptor increased the EC50 for PTH and PTHrP, while preserving high-affinity PTH binding and eliminating high-affinity PTHrP binding. Similarly, transfer of the PTH/PTHrP receptor third extracellular loop preserved high-affinity PTH binding by the PTH2 receptor but decreased its activation. Return of Gln440 and Arg394, corresponding residues in the PTH/PTHrP and PTH2 receptor third extracellular loops, to the parent residue restored function of these receptors. Simultaneous interchange of wild-type amino termini and third extracellular loops eliminated agonist activation but not binding for both receptors. Function was restored by elimination of the E2 domain in the receptor with a PTH/PTHrP receptor N terminus and return of Gln440/Arg394 to the parent sequence in both receptors. These data suggest that the amino terminus and third extracellular loop of the PTH2 and PTH/PTHrP receptors interact similarly with PTH, and that both domains contribute to differential interaction with PTHrP.

Arginine 186 in the extracellular N-terminal region of the human parathyroid hormone 1 receptor is essential for contact with position 13 of the hormone.

PTH maintains blood calcium concentrations within the physiological range by acting on a G protein-coupled heptahelical receptor (PTH1 Rc) located primarily in cells in bone and kidney. We have undertaken a photoaffinity cross-linking approach to elucidate the nature of the bimolecular interaction of PTH with the human (h) PTH1 Rc. Specifically, we have studied the region of the receptor that interacts with the midregion of PTH-(1-34), position 13, using a benzophenone-containing photoaffinity ligand, 125I-[Nle(8,18),Lys13(epsilon-pBz2),L-2-NaI23,Arg(26,2 7),Tyr34]bPTH-(1-34)NH2 (125I-K13). Using site-directed mutagenesis in combination with biochemical analysis, we have reduced our previously identified contact domain, 17 residues in the extracellular region of the receptor (173-189), to an 8-amino acid domain (182-189). Furthermore, we have found arginine 186 to be of critical importance to the interaction of the hPTH1 Rc with 125I-K13: modification of Arg186 to either lysine or alanine does not modify receptor avidity or signal transduction by the receptor, but eliminates cross-linking to 125I-K13.

Identification of a contact site for residue 19 of parathyroid hormone (PTH) and PTH-related protein analogs in transmembrane domain two of the type 1 PTH receptor.

Recent functional studies have suggested that position 19 in PTH interacts with the portion of the PTH-1 receptor (P1R) that contains the extracellular loops and seven transmembrance helices (TMs) (the J domain). We tested this hypothesis using the photoaffinity cross-linking approach. A PTHrP(1-36) analog and a conformationally constrained PTH(1-21) analog, each containing para-benzoyl-l-phenylalanine (Bpa) at position 19, each cross-linked efficiently to the P1R expressed in COS-7 cells, and digestive mapping analysis localized the cross-linked site to the interval (Leu232-Lys240) at the extracellular end of TM2. Point mutation analysis identified Ala234, Val235, and Lys240 as determinants of cross-linking efficiency, and the Lys240-->Ala mutation selectively impaired the binding of PTH(1-21) and PTH(1-19) analogs, relative to that of PTH(1-15) analogs. The findings support the hypothesis that residue 19 of the receptor-bound ligand contacts, or is close to, the P1R J domain-specifically, Lys240 at the extracellular end of TM2. The findings also support a molecular model in which the 1-21 region of PTH binds to the extracellular face of the P1R J domain as an alpha-helix.

Analysis of parathyroid hormone (PTH)/secretin receptor chimeras differentiates the role of functional domains in the pth/ pth-related peptide (PTHrP) receptor on hormone binding and receptor activation.

The type 1 parathyroid hormore receptor (PTH1r) belongs to the class II family of G protein-coupled receptors. To delineate the sites in the PTH1r's N-terminal region, and the carboxy-core domain (transmembrane segments + extracellular loops) involved in PTH binding, we have evaluated the functional properties of 27 PTH1-secretin chimeras receptors stably expressed in HEK-293 cells. The wild type and chimeric receptors were analyzed for cell surface expression, binding for PTH and secretin, and functional responsiveness (cAMP induction) toward secretin and PTH. The expression levels of the chimeric receptors were comparable to that of the PTH1r (60-100%). The N-terminal region of PTH1r was divided into three segments that were replaced either singly or in various combinations with the homologous region of the secretin receptor (SECr). Substitution of the carboxy-terminal half (residues 105-186) of the N-terminal region of PTH1r for a SECr homologous segment did not reduced affinity for PTH but abolished signaling in response to PTH. This data indicate that receptor activation is dissociable from high affinity hormone binding in the PTH1r, and that the N-terminal region might play a critical role in the activation process. Further segment replacements in the N-termini focus on residues 105-186 and particularly residues 146-186 of PTH1r as providing critical segments for receptor activation. The data obtained suggest the existence of two distinct PTH binding sites in the PTH1r's N-terminal region: one site in the amino-terminal half (residues 1-62) (site 1) that participates in high-affinity PTH binding; and a second site of lower affinity constituted by amino acid residues scattered throughout the carboxy-terminal half (residues 105-186) (site 2). In the absence of PTH binding to site 1, higher concentrations of hormone are required to promote receptor activation. In addition, elimination of the interaction of PTH with site 2 results in a loss of signal transduction without loss of high-affinity PTH binding. Divers substitutions of the extracellular loops of the PTH1r highlight the differential role of the first- and third extracellular loop in the process of PTH1r activation after hormone binding. A chimera containing the entire extracellular domains of the PTH1r and the transmembrane + cytoplasmic domains of SECr had very low PTH binding affinity and did not signal in response to PTH. Further substitution of helix 5 of PTH1r in this chimera increased affinity for PTH that is close to the PTH affinity for the wild-type PTH1r but surprisingly, did not mediate signaling response. Additional substitutions of PTH1r's helices in various combinations emphasize the fundamental role of helix 3 and helix 6 on the activation process of the PTH1r. Overall, our studies demonstrated that several PTH1r domains contribute differentially to PTH binding affinity and signal transduction mechanism and highlight the role of the N-terminal domain and helix 3 and helix 6 on receptor activation.

Identification of phosphorylation sites in the G protein-coupled receptor for parathyroid hormone. Receptor phosphorylation is not required for agonist-induced internalization.

In some G protein-coupled receptors (GPCRs), agonist-dependent phosphorylation by specific GPCR kinases (GRKs) is an important mediator of receptor desensitization and endocytosis. Phosphorylation and the subsequent events that it triggers, such as arrestin binding, have been suggested to be regulatory mechanisms for a wide variety of GPCRs. In the present study, we investigated whether agonist-induced phosphorylation of the PTH receptor, a class II GPCR, also regulates receptor internalization. Upon agonist stimulation, the PTH receptor was exclusively phosphorylated on serine residues. Phosphoamino acid analysis of a number of receptor mutants in which individual serine residues had been replaced by threonine identified serine residues in positions 485, 486, and 489 of the cytoplasmic tail as sites of phosphorylation after agonist treatment. When serine residues at positions 483, 485, 486, 489, 495, and 498 were simultaneously replaced by alanine residues, the PTH receptor was no longer phosphorylated either basally or in response to PTH. The substitution of these serine residues by alanine affected neither the number of receptors expressed on the cell surface nor the ability of the receptor to signal via Gs. Overexpression of GRK2, but not GRK3, enhanced PTH-stimulated receptor phosphorylation, and this phosphorylation was abolished by alanine mutagenesis of residues 483, 485, 486, 489, 495, and 498. Thus, phosphorylation of the PTH receptor by the endogenous kinase in HEK-293 cells occurs on the same residues targeted by overexpressed GRK2. Strikingly, the rate and extent of PTH-stimulated internalization of mutated PTH receptors lacking phosphorylation sites were identical to that observed for the wild-type PTH receptor. Moreover, overexpressed GRK2, while enhancing the phosphorylation of the wild-type PTH receptor, had no affect on the rate or extent of receptor internalization in response to PTH. Thus, the agonist-occupied PTH receptor is phosphorylated by a kinase similar or identical to GRK2 in HEK-293 cells, but this phosphorylation is not requisite for efficient receptor endocytosis.

Parathyroid hormone receptor recycling: role of receptor dephosphorylation and beta-arrestin.

The recovery of PTH receptor (PTHR) function after acute homologous receptor desensitization and down-regulation in bone and kidney cells has been attributed to receptor recycling. To determine the role of receptor dephosphorylation in PTHR recycling, we performed morphological and functional assays on human embryonic kidney 293 cells stably expressing wild-type (wt) or mutant PTHRs. Confocal microscopy and ligand binding assays revealed that the wt PTHR is rapidly recycled back to the plasma membrane after removal of the agonist. Receptors that were engineered to either lack the sites of phosphorylation or to resemble constitutively phosphorylated receptors were able to recycle back to the plasma membrane with the same kinetics as the wt PTHR. The PTHR was found to be dephosphorylated by an enzyme apparently distinct from protein phosphatases 1 or 2A. The PTHR and beta-arrestin-2-green fluorescent protein (GFP) were found to stably colocalize during PTHR internalization, whereas after agonist removal and during receptor recycling, the colocalization slowly disappeared. Experiments using phosphorylation-deficient PTHRs and a dominant-negative form of beta-arrestin showed that beta-arrestin does not regulate the efficiency of PTHR recycling. These studies indicate that, unlike many G protein-coupled receptors, PTHR recycling does not require receptor dephosphorylation or its dissociation from beta-arrestin.

Direct identification of two contact sites for parathyroid hormone (PTH) in the novel PTH-2 receptor using photoaffinity cross-linking.

Direct examination of the interacting sites between PTH and the human PTH2 receptor (PTH2R) was conducted by photoaffinity cross-linking followed by protein digestion and mapping of the radiolabeled photoconjugated receptor. Photoreactive analogs of PTH, individually substituted with an L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in cells stably expressing recombinant PTH2R. One highly bioactive analog, [Bpa1,Nle8,18,Arg13,26,27,L-2-Nal23,Tyr34]PTH-(1-34)NH 2 (Bpa1-PTH), was chosen for cross-linking studies. In addition, a PTH analog in which the photoreacive moiety is at the mid-region position 13 (K13) was demonstrated to be bioactive, then cross-linked to PTH2R. The minimal digestion-restricted domain containing the contact site ("contact domain") for 125I-Bpa1-PTH is in the sixth transmembrane domain and part of the third extracellular loop, spanning residues Ser364-Met395 of the receptor. This domain was further confirmed and refined by cross-linking 125I-Bpa1-PTH to two receptor mutants, PTH2R[V380M]- and PTH2R[V380M,M395L]-receptors. Treatment of the cross-linked conjugates with cyanogen bromide identified a single amino acid (position 380) as the putative contact point. The contact domain for 125I-K13 is located in the N-terminal extracellular tail of the receptor (in the C-terminal portion) and spans Gln138-Met147. Further validation of this contact domain was accomplished by photocross-linking to point-mutated PTH2R[K137R] receptor. Previous studies in which PTH analogs were cross-linked to human PTH/PTHrP receptor (PTH1R) identified Met425 and Phe173-Met189 as the contact sites for Bpa1-PTH and K13, respectively. These studies demonstrate that both receptor subtypes, PTH1- and PTH2-receptors, use analogous sites for interaction with positions 1 and 13 in PTH.

Selective and nonselective inverse agonists for constitutively active type-1 parathyroid hormone receptors: evidence for altered receptor conformations.

The spontaneous signaling activity of some G protein-coupled receptors and the capacity of certain ligands (inverse agonists) to inhibit such constitutive activity are poorly understood phenomena. We investigated these processes for several analogs of PTH-related peptide (PTHrP) and the constitutively active human PTH/PTHrP receptors (hP1Rcs) hP1Rc-H223R and hP1Rc-T410P. The N-terminally truncated antagonist PTHrP(5-36) functioned as a weak partial/neutral agonist with both mutant receptors but was converted to an inverse agonist for both receptors by the combined substitution of Leu(11) and D-Trp(12). The N-terminally intact analog [Bpa(2)]PTHrP(1-36)-a partial agonist with the wild-type hP1Rc-was a selective inverse agonist, in that it depressed basal cAMP signaling by hP1Rc-H223R but enhanced signaling by hP1Rc-T410P. The ability of [Bpa(2)]PTHrP(1-36) to discriminate between the two receptor mutants suggested that H223R and T410P confer constitutive receptor activity by inducing distinct conformational changes. This hypothesis was confirmed by the observations that: 1) the double mutant receptor hP1Rc-H223R/T410P exhibited basal cAMP levels that were 2-fold higher than those of either single mutant; and 2) hP1Rc-H223R and hP1Rc-T410P internalized (125)I-PTHrP(5-36) to markedly different extents. The overall results thus reveal that two different types of inverse agonists are possible for PTHrP ligands (nonselective and selective) and that constitutively active PTH-1 receptors can access different conformational states.

Dual regulation of the parathyroid hormone (PTH)/PTH-related peptide receptor signaling by protein kinase C and beta-arrestins.

We examined here the role of second messenger-dependent kinases and beta-arrestins in short-term regulation of the PTH receptor (PTHR) signaling. The inhibition of protein kinase C (PKC) in COS-7 cells transiently expressing PTHR, led to an approximately 2-fold increase in PTH-stimulated inositol phosphate (IP) and cAMP production. The inhibition of protein kinase A increased cAMP production 1.5-fold without affecting IP signaling. The effects of PKC inhibition on PTHR-mediated G(q) signaling were strongly decreased for a carboxy-terminally truncated PTHR (T480) that is phosphorylation deficient. PKC inhibition was associated with a decrease in agonist-stimulated PTHR phosphorylation and internalization without blocking PTH-dependent mobilization of beta-arrestin2 to the plasma membrane. Overexpression of beta-arrestins strongly decreased the PTHR-mediated IP signal, whereas cAMP production was impaired to a much lower extent. The regulation of PTH-stimulated signals by beta-arrestins was impaired for the truncated T480 receptor. Our data reveal mechanisms at, and distal to, the receptor regulating PTHR-mediated signaling pathways by second messenger-dependent kinases. We conclude that regulation of PTHR-mediated signaling by PKC and beta-arrestins are separable phenomena that both involve the carboxy terminus of the receptor. A major role for PKC and beta-arrestins in preferential regulation of PTHR-mediated G(q) signaling by independent mechanisms at the receptor level was established.

Tuberoinfundibular peptide 39 binds to the parathyroid hormone (PTH)/PTH-related peptide receptor, but functions as an antagonist.

The tuberoinfundibular peptide TIP39 [TIP-(1-39)], which exhibits only limited amino acid sequence homology with PTH and PTH-related peptide (PTHrP), stimulates cAMP accumulation in cells expressing the PTH2 receptor (PTH2R), but it is inactive at the PTH/PTHrP receptor (PTH1R). However, when using either (125)I-labeled rat [Nle(8,21),Tyr(34)]PTH-(1-34)amide (rPTH) or (125)I-labeled human [Tyr(36)]PTHrP-(1-36)amide [PTHrP-(1-36)] for radioreceptor studies, TIP-(1-39) bound to LLCPK(1) cells stably expressing the PTH1R (HKrk-B7 cells), albeit with weak apparent affinity (243 +/- 52 and 210 +/- 64 nM, respectively). In comparison to the parent peptide, the apparent binding affinity of TIP-(3-39) was about 3-fold higher, and that of TIP-(9-39) was about 5.5-fold higher. However, despite their improved IC(50) values at the PTH1R, both truncated peptides failed to stimulate cAMP accumulation in HKrk-B7 cells. In contrast, the chimeric peptide PTHrP-(1-20)/TIP-(23-39) bound to HKrk-B7 cells with affinities of 31 +/- 8.2 and 11 +/- 4.0 nM when using radiolabeled rPTH and PTHrP-(1-36), respectively, and it stimulated cAMP accumulation in HKrk-B7 and SaOS-2 cells with potencies (EC(50), 1.40 +/- 0.3 and 0.38 +/- 0.12 nM, respectively) and efficacies (maximum levels, 39 +/- 8 and 31 +/- 3 pmol/well, respectively) similar to those of PTH-(1-34) and PTHrP-(1-36). In both cell lines, TIP(9-39) and, to a lesser extent, TIP-(1-39) inhibited the actions of the three agonists with efficiencies similar to those of [Leu(11),D-Trp(12),Trp(23),Tyr(36)]PTHrP-(7-36)amide, an established PTH1R antagonist. Taken together, the currently available data suggest that the carboxyl-terminal portion of TIP-(1-39) interacts efficiently with the PTH1R, at sites identical to or closely overlapping those used by PTH-(1-34) and PTHrP-(1-36). The amino-terminal residues of TIP-(1-39), however, are unable to interact productively with the PTH1R, thus enabling TIP-(1-39) and some of its truncated analogs to function as an antagonist at this receptor.

The extracellular amino-terminal region of the parathyroid hormone (PTH)/PTH-related peptide receptor determines the binding affinity for carboxyl-terminal fragments of PTH-(1-34).

The recombinant human PTH/PTH-related peptide (PTHrP) receptor, when transiently expressed in COS-7 cells, binds [Nle8,18,Tyr34] bovine PTH-(7-34)amide [PTH-(7-34)], human PTH-(10-34)amide [PTH-(10-34)], and bovine PTH-(15-34)amide [PTH-(15-34)] with at least 50-fold higher affinity than does the rat receptor homolog. In contrast, PTH-(1-34) binding affinities are similar for both receptor homologs. To map those areas of the PTH/PTHrP receptors that determine the binding specificity for carboxyl-terminal fragments of PTH-(1-34), we constructed chimeric rat/human PTH/PTHrP receptors. These bound PTH-(1-34) with normal affinity and, therefore, must have an overall conformation that resembles that of native receptors. Chimeras with the amino-terminal extracellular domain of the human PTH/PTHrP receptor have a considerably higher binding affinity for PTH-(7-34), PTH-(10-34), and PTH-(15-34) than do the reciprocal receptor constructs in which the amino-terminal region is from the rat PTH/PTHrP receptor. The opossum PTH/PTHrP receptor homolog also binds PTH-(7-34) with higher affinity than the rat receptor, and studies of rat/opossum chimeras confirm the importance of the amino-terminal extracellular domain in determining the PTH-(7-34) binding specificity. Mutant rat and human PTH/PTHrP receptors in which either residues 61-105 of the extracellular region or most of the intracellular tail were deleted have PTH-(7-34) binding characteristics indistinguishable from those of either wild-type receptor. These findings indicate that the amino-terminal extracellular region of the PTH/PTHrP receptor contains a domain(s) that largely determines the binding affinity of amino-terminally truncated PTH analogs. This region, therefore, is likely to constitute a site for ligand-receptor interaction.

Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia.

A single homozygous nucleotide exchange in exon E3 of the gene encoding the parathyroid hormone receptor type 1 (PTHR1) was identified in an infant with Blomstrand chondrodysplasia born to consanguineous parents. This alteration changes a strictly conserved proline residue at position 132 in the receptor's amino terminal extracellular domain to leucine. COS-1 cells expressing the mutant receptor did not accumulate cyclic adenosine 3',5'-monophosphate in response to PTH or PTH-related peptide (PTHrP) and did not bind the radiolabeled ligand. Expression of the mutant protein on the cell surface of transiently transfected COS-1 cells and in growth plate chondrocytes derived from the affected infant suggests that proline 132 is critical for the receptor's intrinsic binding activity. These findings suggest that the Blomstrand form of human short-limbed dwarfism arises from defective PTHR1 signaling in the developing cartilaginous skeleton.

Design and applications of parathyroid hormone analogues.

The endocrine parathyroid hormone (PTH) is the major regulator of serum calcium levels. In contrast, the autocrine/paracrine parathyroid hormone-related peptide (PTHrP) has been associated with organism development. Both are secreted as much larger molecules but have their major functions associated with their N-terminal 34 residues. They share a common receptor expressed in organs critical to PTH function - bone, kidney, and intestine. PTH and PTHrP receptor activation stimulates adenylyl cyclase (AC), phospholipase C (PLC), and phospholipase D (PLD) in target cells. It has been possible to separate the AC-stimulation from that of PLC. AC-stimulation requires at least the N-terminal 28 residues of PTH and PLC-stimulation requires a minimum of residues 29-32-NH2. Intermittent administration of PTH stimulates bone growth and requires AC-stimulation. The shortest linear sequence of hPTH with essentially full anabolic activity for bone growth-stimulation is hPTH(1-31)NH2. Two applications are postulated for PTH and PTHrP-based pharmaceuticals - treatment of bone loss due to osteoporosis and reversal of the hypercalcemic effect of malignancy. PTHrP analogues which strongly inhibit PTHrP AC-stimulation showed promise for the treatment of malignancy-associated hypercalcemia in animal trials but failed in human ones. However, both animal and human trials of hPTH have shown significant bone growth-stimulating effects. New deletion, substitution and cyclized analogues of PTH show great promise both for greater in vitro activity and possibly for improved delivery and greater specificity as agents for restoration of bone loss in osteoporosis.

Parathyroid hormone and parathyroid hormone-related protein: model systems for the development of an osteoporosis therapy.

The parathyroid hormone (PTH) plays a vital role in the homeostasis of calcium within the blood stream. Given its unique ability to increase bone density, an understanding of the molecular mechanism by which the hormone is recognized and binds to its receptor should provide targets for the development of PTH-based, anabolic agents for the treatment of osteoporosis. Parathyroid hormone related protein (PTHrP), a genetically and structurally distinct hormone which displays similar binding and activation profiles as PTH, has greatly facilitated the effort to establish a structure-biological function relationship by allowing for direct comparisons. In an analogous manner, the presence of two receptors, PTH/PTHrP (PTH1) and PTH2, which differ in their ligand selectivity (PTH2 is activated by PTH, not PTHrP) has provided a unique vehicle for probing the structural motifs of the receptor required for ligand recognition and binding. Recent photo-affinity cross-linking studies of PTH and PTHrP binding to PTH1 have produced direct points of contact between the ligand and receptor. Here, we review each of the components involved in this important hormone system, with particular emphasis on the structural features of each: the ligands (PTH and PTHrP), the receptors (PTH1 and PTH2), and the interaction between ligand and receptor. Although the current understanding of the molecular mechanism of ligand binding and receptor activation does not allow for the rational design of drug candidates, and indeed contains much conjecture, significant strides have been made towards this end.