Synergistic effects of high dietary calcium and exogenous parathyroid hormone in promoting osteoblastic bone formation in mice.

In the present study, we investigated whether high dietary Ca and exogenous parathyroid hormone 1-34 fragments (PTH 1-34) have synergistic effects on bone formation in adult mice, and explored the related mechanisms. Adult male mice were fed a normal diet, a high-Ca diet, a PTH-treated diet, or a high-Ca diet combined with subcutaneously injected PTH 1-34 (80 µg/kg per d) for 4 weeks. Bone mineral density, trabecular bone volume, osteoblast number, alkaline phosphatase (ALP)- and type I collagen-positive areas, and the expression levels of osteoblastic bone formation-related genes and proteins were increased significantly in mice fed the high-Ca diet, the PTH-treated diet, and, even more dramatically, the high-Ca diet combined with PTH. Osteoclast number and surface and the ratio of receptor activator for nuclear factor-κB ligand (RANKL):osteoprotegerin (OPG) were decreased in the high-Ca diet treatment group, increased in the PTH treatment group, but not in the combined treatment group. Furthermore, third-passage osteoblasts were treated with high Ca (5 mM), PTH 1-34 (10⁻8 M) or high Ca combined with PTH 1-34. Osteoblast viability and ALP activity were increased in either the high Ca-treated or PTH-treated cultures and, even more dramatically, in the cultures treated with high Ca plus PTH, with consistent up-regulation of the expression levels of osteoblast proliferation and differentiation-related genes and proteins. These results indicate that dietary Ca and PTH play synergistic roles in promoting osteoblastic bone formation by stimulating osteoblast proliferation and differentiation.

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.

Role of calcium channels in carboxyl-terminal parathyroid hormone receptor signaling.

Parathyroid hormone (PTH), an 84-amino acid polypeptide, is a major systemic regulator of calcium homeostasis that activates PTH/PTHrP receptors (PTH1Rs) on target cells. Carboxyl fragments of PTH (CPTH), secreted by the parathyroids or generated by PTH proteolysis in the liver, circulate in blood at concentrations much higher than intact PTH-(1-84) but cannot activate PTH1Rs. Receptors specific for CPTH fragments (CPTHRs), distinct from PTH1Rs, are expressed by bone cells, especially osteocytes. Activation of CPTHRs was previously reported to modify intracellular calcium within chondrocytes. To further investigate the mechanism of action of CPTHRs in osteocytes, cytosolic free calcium concentration ([Ca(2+)](i)) was measured in the PTH1R-null osteocytic cell line OC59, which expresses abundant CPTHRs but no PTH1Rs. [Ca(2+)](i) was assessed by single-cell ratiometric microfluorimetry in fura-2-loaded OC59 cells. A rapid and transient increase in [Ca(2+)](i) was observed in OC59 cells in response to the CPTH fragment hPTH-(53-84) (250 nM). No [Ca(2+)](i) signal was observed in COS-7 cells, in which CPTHR binding also cannot be detected. Neither hPTH-(1-34) nor a mutant CPTH analog, [Ala(55-57)]hPTH-(53-84), that does not to bind to CPTHRs, increased [Ca(2+)](i) in OC59 cells. The [Ca(2+)](i) response to hPTH-(53-84) required the presence of extracellular calcium and was blocked by inhibitors of voltage-dependent calcium channels (VDCCs), including nifedipine (100 nM), omega-agatoxin IVA (10 nM), and omega-conotoxin GVIA (100 nM). We conclude that activation of CPTHRs in OC59 osteocytic cells leads to a rapid increase in influx of extracellular calcium, most likely through the opening of VDCCs.

Analogues of human parathyroid hormone (1-31)NH(2): further evaluation of the effect of conformational constraint on biological activity.

A series of conformationally-restricted analogues of hPTH was prepared, based on the parent peptide agonist, cyclo(Lys(18)-Asp(22))[Ala(1),Nle(8),Lys(18),Asp(22),Leu(27)]hPTH(1-31)NH(2) (2, EC(50)=0.29nM). Truncation of 2 at either the N- or C-termini resulted in peptides with reduced agonist activity as measured by stimulation of adenylate cyclase activity in the rat osteosarcoma cell line (ROS 17/2.8). Alanine- and glycine-scanning at the N-terminus of 2 was consistent with data previously obtained on linear hPTH(1-34). Other locations within the primary sequence of hPTH(1-31)NH(2) were evaluated by the placement of the [i, i+4] lactam constraining element. Ring size and lactam orientations at the 18-22 positions were also examined.

The parathyroid hormone 2 (PTH2) receptor.

The human PTH2 receptor binds and is activated at high potency by PTH and by the recently discovered peptide tuberoinfundibular peptide of 39 residues (TIP39). Rat and zebrafish PTH2 receptors are more weakly activated by PTH, suggesting that TIP39 may be the natural ligand for the PTH2 receptor. Unlike the PTH1 receptor, the PTH2 receptor interacts extremely weakly with parathyroid hormone-related peptide (PTHrP). The PTH2 receptor is strongly coupled to stimulation of cAMP accumulation, and more weakly, in a cell-specific manner to increases in intracellular calcium concentration. A variety of evidence supports the general model of receptor amino terminal sequences binding ligand carboxyl terminal sequences with high affinity, and ligand amino terminal sequences activating the receptor through interaction with the "juxtamembrane" portion of the receptor. The receptor is present at greatest levels in the nervous system. It is expressed in scattered cells in the cerebral cortex and basal ganglia and at relatively high abundance in the septum, midline thalamic nuclei, several hypothalamic nuclei, and the dorsal horn of the spinal cord. Peripherally, expression in pancreatic islet somatostatin cells is most dramatic. Functional hypotheses based on the receptor's distribution are being tested. Recent data support involvement in hypothalamic releasing-factor secretion and pain.

Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate.

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR-GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR-GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR-GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR-GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR-GFP back to the plasma membrane was complete within 1-2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding consistent with a role for receptor recycling in functional resensitization.

Domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs).

To investigate the domains of the parathyroid hormone (PTH) receptor required for regulation by G protein-coupled receptor kinases (GRKs), we created mutant PTH receptors lacking potential GRK-phosphorylation sites. Mutant #1 was truncated at amino acid 544 and, therefore, lacked nine hydroxyl group-containing amino acids at the C-terminus. In mutant #2, we replaced threonines 392 and 399 in the third intracellular loop with glycines. Co-transfection of HEK293 cells with the wild-type receptor and either GRK2, GRK3, or GRK5 inhibited PTH-induced cyclic (cAMP) generation; co-transfection of GRK4 or GRK6 had no effect on PTH receptor responsiveness. GRK2-mediated inhibition of PTH receptor signaling was associated with enhanced phosphorylation receptor proteins. Co-expression of GRK2 similarly reduced PTH-induced cAMP generation by the wild-type receptor and mutant #1, and caused phosphorylation of receptor proteins to a similar extent. Co-expression of GRK2 had little effect on PTH-induced cAMP generation by mutant #2 but enhanced agonist-induced phosphorylation of mutant #2 compared with that of either the wild-type receptor or mutant #1. Enhanced phosphorylation of mutant #2 was associated with a reduction in agonist-induced internalization of mutant #2 compared with the wild-type receptor. Thus, phosphorylation of mutant #2 failed to cause receptor desensitization and inhibited receptor internalization. These data are consistent with the notion that: (a) GRKs contribute to regulating PTH receptor responsiveness, and (b) domains in the third intracellular loop are not required for agonist-induced phosphorylation of PTH receptors, but are critical for both agonist-induced internalization of PTH receptors and GRK2-mediated regulation of PTH receptor signaling.

Identification of determinants of inverse agonism in a constitutively active parathyroid hormone/parathyroid hormone-related peptide receptor by photoaffinity cross-linking and mutational analysis.

We have investigated receptor structural components responsible for ligand-dependent inverse agonism in a constitutively active mutant of the human parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP) receptor type 1 (hP1R). This mutant receptor, hP1R-H223R (hP1R(CAM-HR)), was originally identified in Jansen's chondrodysplasia and is altered in transmembrane domain (TM) 2. We utilized the PTHrP analog, [Bpa(2),Ile(5),Trp(23),Tyr(36)]PTHrP-(1-36)-amide (Bpa(2)-PTHrP-(1-36)), which has valine 2 replaced by p-benzoyl-l-phenylalanine (Bpa); this substitution renders the peptide a photoreactive inverse agonist at hP1R(CAM-HR). This analog cross-linked to hP1R(CAM-HR) at two contiguous receptor regions as follows: the principal cross-link site (site A) was between receptor residues Pro(415)-Met(441), spanning the TM6/extracellular loop three boundary; the second cross-link site (site B) was within the TM4/TM5 region. Within the site A interval, substitution of Met(425) to Leu converted Bpa(2)-PTHrP-(1-36) from an inverse agonist to a weak partial agonist; this conversion was accompanied by a relative shift of cross-linking from site A to site B. The functional effect of the M425L mutation was specific for Bpa(2)-containing analogs, as inverse agonism of Bpa(2)-PTH-(1-34) was similarly eliminated, whereas inverse agonism of [Leu(11),d-Trp(12)]PTHrP-(5-36) was not affected. Overall, our data indicate that interactions between residue 2 of the ligand and the extracellular end of TM6 of the hP1R play an important role in modulating the conversion between active and inactive receptor states.

Zinc(II)-mediated enhancement of the agonist activity of histidine-substituted parathyroid hormone(1-14) analogues.

Previous studies on parathyroid hormone (PTH)(1-14) revealed that residues (1-9) played a dominant role in stimulating PTH-1 receptor-mediated increases in cAMP formation. In the present study, we examined the effects of installing a metal-binding motif in the (10-14) region of rat PTH(1-14) on the peptide's agonist activity. We found that substitution of histidine for the native asparagine at position 10 of PTH(1-14) provided a peptide that was approx. 8-fold more potent as an agonist in the presence of divalent zinc salts than it was in the absence of the metal. This enhancement in potency was dependent on the native histidine at position 14, the concentration of Zn(II) utilized, and did not occur with other divalent metal ions. The zinc-activated [His(10)]-PTH(1-14) peptide was blocked by a classical PTH-1 receptor antagonist, PTHrP(7-36), and did not activate the PTH-2 receptor. The zinc-mediated enhancing effect did not require the large N-terminal extracellular domain of the PTH-1 receptor. Although we were able to demonstrate that [His(10)]-PTH(1-14) binds Zn(II) using (1)H-NMR, our spectroscopic studies (circular dichroism and nuclear magnetic resonance) were not consistent with the notion that zinc enhanced the activity of [His(10)]-PTH(1-14) simply by inducing a helical structure in the 10-14 region. Rather, the data suggest that the enhancement in cAMP potency arises from the formation of a ternary complex between [His(10)]-PTH(1-14), a zinc atom, and the extracellular loop/transmembrane domain region of the PTH-1 receptor.

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.

Tuberoinfundibular peptide (7-39) [TIP(7-39)], a novel, selective, high-affinity antagonist for the parathyroid hormone-1 receptor with no detectable agonist activity.

The parathyroid hormone (PTH)-1 receptor mediates the pathophysiological effects of PTH in hyperparathyroidism and PTH-related protein (PTHrP) in humoral hypercalcemia of malignancy. A PTH1 receptor antagonist may be of therapeutic utility in these disorders. We recently identified a novel antagonist, tuberoinfundibular peptide (7-39) [TIP(7-39)], derived from the likely endogenous ligand for the PTH2 receptor TIP39. In this study its in vitro profile is evaluated and compared with that of [D-Trp(12),Tyr(34)]bPTH(7-34) and PTHrP(7-34), representing the two previously known structural classes of PTH1 receptor antagonists. TIP(7-39) binds with higher affinity (6.2 nM) to the PTH1 receptor than [D-Trp(12),Tyr(34)]bPTH(7-34) (45 nM) and PTHrP(7-34) (65 nM) and displays a 5.5-fold greater PTH1/PTH2 receptor selectivity. TIP(7-39) does not stimulate cAMP accumulation via the PTH1 receptor [in a sensitive assay that detects the activity of the weak partial agonist [Nle(8,18),Tyr(34)]bPTH(3-34)] and does not increase intracellular calcium. Schild analysis for TIP(7-39) was consistent with purely competitive antagonism of PTH(1-34)'s stimulation of cAMP accumulation (slope = 0.99 +/- 0.24). The pK(B) for TIP(7-39) (7.1 +/- 0.3) was higher than that for [D-Trp(12),Tyr(34)]bPTH(7-34) (6.5 +/- 0.0) and PTHrP(7-34) (6.0 +/- 0.1). Binding of (125)I-TIP(7-39) to the PTH1 receptor could be measured (K(D) = 1.3 +/- 0.1 nM, B(max) = 1.3 +/- 0.1 pmol/mg), whereas binding of (125)I-[Nle(8,18),D-Trp(12),Tyr(34)]bPTH(7-34) could not be detected. Kinetic analysis indicated that (125)I-TIP(7-39) dissociates much more slowly (t(1/2) = 14 min) than [D-Trp(12),Tyr(34)]bPTH(7-34) (13 s) and PTHrP(7-34) (9 s). The novel antagonist TIP(7-39) therefore displays a more favorable in vitro pharmacological profile than antagonists derived from PTH and PTHrP and may be useful for demonstrating the utility of PTH1 receptor antagonism in the treatment of hypercalcemia.

Minimization of parathyroid hormone. Novel amino-terminal parathyroid hormone fragments with enhanced potency in activating the type-1 parathyroid hormone receptor.

The amino-terminal and carboxyl-terminal portions of the 1-34 fragment of parathyroid hormone (PTH) contain the major determinants of receptor activation and receptor binding, respectively. We investigated how the amino-terminal signaling portion of PTH interacts with the receptor by utilizing analogs of the weakly active fragment, rat (r) PTH(1-14)NH(2), and cells transfected with the wild-type human PTH-1 receptor (hP1R-WT) or a truncated PTH-1 receptor which lacked most of the amino-terminal extracellular domain (hP1R-delNt). Of 132 mono-substituted PTH(1-14) analogs, most having substitutions in the (1-9) region were inactive in assays of cAMP formation in LLC-PK1 cells stably expressing hP1R-WT, whereas most having substitutions in the (10-14) region were active. Several substitutions (e.g. Ser(3) --> Ala, Asn(10) --> Ala or Gln, Leu(11) --> Arg, Gly(12) --> Ala, His(14) --> Trp) enhanced activity 2-10-fold. These effects were additive, as [Ala(3),(10,12),Arg(11), Trp(14)] rPTH(1-14)NH(2) was 220-fold more potent than rPTH(1-14)NH(2) (EC(50) = 0.6 +/- 0.1 and 133 +/- 16 micrometer, respectively). Native rPTH(1-11) was inactive, but [Ala(3,10), Arg(11)]rPTH(1-11)NH(2) achieved maximal cAMP stimulation (EC(50) = 17 micrometer). The modified PTH fragments induced cAMP formation with hP1R-delNt in COS-7 cells as potently as they did with hP1R-WT; PTH(1-34) was 6,000-fold weaker with hP1R-delNt than with hP1R-WT. The most potent analog, [Ala(3,10,12),Arg(11), Trp(14)]rPTH(1-14)NH(2), stimulated inositol phosphate production with hP1R-WT. The results show that short NH(2)-terminal peptides of PTH can be optimized for considerable gains in signaling potency through modification of interactions involving the regions of the receptor containing the transmembrane domains and extracellular loops.

Inhibition of chondrogenesis by parathyroid hormone in vivo during repair of full-thickness defects of articular cartilage.

We studied the effects of parathyroid hormone (PTH) on differentiation of chondroprogenitor cells during the repair of full-thickness articular cartilage defects. Three-millimeter cylindrical full-thickness articular cartilage defects, which are small enough to be resurfaced spontaneously by hyaline cartilage, were created in the femoral trochlea of the rabbit knee. Recombinant human PTH(1-84) (hPTH[1-84]) (25 ng/h) then was administered into the joint cavity with an osmotic pump, or in control animals, saline alone was administered. The animals were killed at 1, 2, 4, and 8 weeks. At 1 week, the defects were filled with undifferentiated cells, regardless of the PTH treatments. By 8 weeks, well-developed cartilage covered the defects with reconstitution of subchondral bone up to the original bone-articular cartilage junction. In contrast, no evidence of chondrogenic differentiation was seen at any time during the experimental period in the defects treated with PTH. The reparative tissues also were examined immunohistochemically using anti-proliferating cell nuclear antigen (PCNA) and anti-PTH/PTH-related peptide (PTHrP) receptor antibodies. Interestingly, the chondroprogenitor cells that filled the defects expressed PTH/PTHrP receptor, suggesting that these cells are capable of responding to PTH/PTHrP signaling before overt chondrogenesis. Application of PTH did not interfere with proliferation but inhibited chondrogenic differentiation of the cells resulting in the formation of fibrous tissue that lost the expression of PTH/PTHrP receptor within 4 weeks.

Photoaffinity cross-linking identifies differences in the interactions of an agonist and an antagonist with the parathyroid hormone/parathyroid hormone-related protein receptor.

Analogs of parathyroid hormone (PTH)-related protein (PTHrP), singularly substituted with a photoreactive L-p-benzoylphenylalanine (Bpa) at each of the first 6 N-terminal positions, were pharmacologically evaluated in human embryonic kidney cells stably expressing the recombinant human PTH/PTHrP receptor. Two of these analogs, in which the photoreactive residue is either in position 1 or 2 (Bpa(1)- and Bpa(2)-PTHrP, respectively) displayed high affinity binding. Bpa(1)-PTHrP also displayed high efficacy for the stimulation of increased cAMP levels. Surprisingly, Bpa(2)-PTHrP was found to be a potent antagonist, despite the presence of the principal activation domain (sequence 1-6). Analysis of the digestion profiles of the ligand-receptor photoconjugates revealed that both the agonist and the antagonist cross-link to the S-CH(3) group of Met(425) in transmembrane domain 6 of the human PTH/PTHrP receptor. However, the antagonist Bpa(2)-PTHrP also cross-links to a proximal site within the receptor domain Pro(415)-Met(425). Unlike the antagonist Bpa(2)-PTHrP, the potent agonist Bpa(2)-PTH, also bearing the Bpa residue in position 2, cross-links only to the S-CH(3) group of Met(425) (similar to Bpa(1)-PTHrP and Bpa(1)-PTH). Taken together, these results suggest that the antagonist Bpa(2)-PTHrP is able to distinguish between two distinct conformations of the receptor. The comparison between PTHrP analogs substituted by Bpa at two consecutive positions and across PTH and PTHrP reveals insights into the PTH/PTHrP ligand-receptor bimolecular interaction at the level of a single amino acid.

Studies of the N-terminal region of a parathyroid hormone-related peptide (1-36) analog: receptor subtype-selective agonists, antagonists, and photochemical cross-linking agents.

The N-terminal regions of PTH and PTH-related peptide (PTHrP) are involved in receptor-mediated signaling and subtype selectivity. To better understand the molecular basis for these processes, we first prepared a series of [I5,W23,Y36]-PTHrP(1-36)NH2 analogs having stepwise deletions of residues 1-4 and characterized them with the human (h)PTH-1 and hPTH-2 receptor subtypes stably transfected in LLC-PK1 cells. Deletions beyond residue 2 caused progressive and severe losses in cAMP-signaling efficacy without dramatically diminishing receptor-binding affinity; consistent with this, [I5,W23]-PTHrP(5-36) was a potent antagonist for both PTH receptor subtypes. We then prepared and characterized photolabile analogs of [I5,W23,Y36]-PTHrP(1-36)NH2 that were singly modified with parabenzoyl-L-phenylalanine (Bpa) along the first six residues. These full-length analogs exhibited receptor subtype-selective agonism, antagonism, and photochemical cross-linking profiles. In particular, the [Bpa2]- and [Bpa4]-substituted analogs selectively antagonized and preferentially cross-linked to the PTH-1 receptor and PTH-2 receptor, respectively. These results demonstrate that the 1-5 region of [I5,W23]-PTHrP(1-36) is critical for activating the PTH-1 and PTH-2 receptors and suggest that the individual residues in this region play distinct roles in modulating the activation states of the two receptors. The cross-linking of both agonist and antagonist ligands to these PTH receptors lays the groundwork for identifying critical signaling determinants in the ligand binding pocket of the receptor.

Comparison of rat and human parathyroid hormone 2 (PTH2) receptor activation: PTH is a low potency partial agonist at the rat PTH2 receptor.

The human PTH2 receptor, expressed in tissue culture cells, is selectively activated by PTH. Detailed investigation of its anatomical and cellular distribution has been performed in the rat. It is expressed by neurons in a number of brain nuclei; by endocrine cells that include pancreatic islet somatostatin cells, thyroid parafollicular cells, and peptide secreting cells in the gastrointestinal tract; and by cells in the vasculature and heart. The physiological role of the PTH2 receptor expressed by these cells remains to be determined. All pharmacological studies performed to date have used the human receptor. We have now isolated a complementary DNA including the entire coding sequence of the rat PTH2 receptor and compared its pharmacological profile with that of the human PTH2 receptor when each is expressed in COS-7 cells. PTH-based peptides, including rat PTH(1-84), rat PTH(1-34), and human PTH(1-34), have low potency at the rat PTH2 receptor for stimulation of adenylyl cyclase (EC50 = 19-140 nM). When compared with the effect of a bovine hypothalamic extract, PTH-based peptides are partial agonists at the rat PTH2 receptor. This suggests that PTH is unlikely to be a physiologically important endogenous ligand for the PTH2 receptor. A peptide homologous to an activity detected in a bovine hypothalamic extract is a good candidate for the endogenous PTH2 receptor ligand.

Conformational studies of a bicyclic, lactam-constrained parathyroid hormone-related protein-derived agonist.

The N-terminal 1-34 segments of both parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N- and C-termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1-34) analog [(Lys13-Asp17, Lys26-Asp30)]PTHrP(1-34)NH2, which contains lactam-constrained Lys-Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam-constrained analog to aqueous solution. By CD, 2D-NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13-18, and 26-31) with high flexibility around Gly12 and Arg19. Thus, the essential structural features observed in the aqueous-organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side-chain lactam constraints on the alpha-helical structure.

Measurement of agonist and antagonist ligand-binding parameters at the human parathyroid hormone type 1 receptor: evaluation of receptor states and modulation by guanine nucleotide.

Determination of ligand-binding constants for parathyroid hormone (PTH) receptors has been hampered by a lack of suitable experimental systems and mechanistic models for data analysis. In this study, ligand binding to the cloned human PTH-1 receptor was measured using membrane-based radioligand-binding assays. Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) (10 microM) reduced binding of agonist radioligands [125I]rPTH(1-34) and [125I]PTHrP(1-36) but only to a limited extent (by 29 +/- 5 and 42 +/- 3%, respectively). Radiolabeled agonist dissociation was described by three and two phases in the absence and presence of GTPgammaS, respectively. GTPgammaS treatment removed a pseudoirreversible binding phase. Inhibition of radiolabeled antagonist ([125I]bPTH(3-34)) binding was measured using a 90-min incubation, which allowed binding of ligands to closely approach the asymptotic maximum. Agonist/[125I]bPTH(3-34) displacement curves were fitted best by assuming two independent affinity states, both in the presence and absence of GTPgammaS. After a 3-h incubation, binding of PTH agonists in the presence of GTPgammaS was described by a single affinity state, indicating the presence of slow components in the binding reaction. Antagonist binding was described by a single affinity state and was not significantly affected by GTPgammaS. The data were used to evaluate potential receptor-binding models. Although other models could not be excluded, all of the observations could be explained by assuming two binding sites on the receptor that recognize two corresponding sites on agonist ligands. Using the model, it was possible to estimate receptor-ligand-binding constants and to propose a direct method for identifying ligands that interact with a putative antagonist binding region of the receptor.