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.     

Megalin antagonizes activation of the parathyroid hormone receptor

Parathyroid hormone (PTH) is predominantly cleared from the circulation by glomerular filtration and degradation in the renal proximal tubules. Here, we demonstrate that megalin, a multifunctional endocytic receptor in the proximal tubular epithelium, mediates the uptake and degradation of PTH. Megalin was purified from kidney membranes as the major PTH-binding protein and shown in BIAcore analysis to specifically bind full-length PTH and amino-terminal PTH fragments (Kd 0.5 microM). Absence of the receptor in megalin knockout mice resulted in 4-fold increased levels of amino-terminal PTH fragments in the urine. In F9 cells expressing both megalin and the PTH/PTH-related peptide receptor (PTH/PTHrP receptor), uptake and lysosomal degradation of the hormone was mediated through megalin. Blocking megalin-mediated clearance of PTH resulted in 3-fold increased stimulation of the PTH/PTHrP receptor. These data provide evidence that megalin is involved in the renal catabolism of PTH and potentially antagonizes PTH/PTHrP receptor activity in the proximal tubular epithelium.     

Binding of parathyroid hormone to bovine kidney-cortex plasma membranes

1. Plasma membranes were purified from bovine kidney cortex, with a fourfold increase in specific activity of parathyroid hormone-sensitive adenylate cyclase over that in the crude homogenate. The membranes were characterized by enzyme studies. 2. Parathyroid hormone was labelled with (125)I by an enzymic method and the labelled hormone shown to bind to the plasma membranes and to be specifically displaced by unlabelled hormone. Parathyroid hormone labelled by the chloramine-t procedure showed no specific binding. (75)Se-labelled human parathyroid hormone, prepared in cell culture, also bound to the membranes. 3. Parathyroid hormone was shown to retain biological activity after iodination by the enzymic method, but no detectable activity remained after chloramine-t treatment. 4. High concentration of pig insulin inhibited binding of labelled parathyroid hormone to plasma membranes and partially inhibited the hormone-sensitive adenylate cyclase activity in a crude kidney-cortex preparation. 5. EDTA enhanced and Ca(2+) inhibited binding of labelled parathyroid hormone to plasma membranes. 6. Whereas rat kidney homogenates were capable of degrading labelled parathyroid hormone to trichloroacetic acid-soluble fragments, neither crude homogenates nor purified membranes from bovine kidney showed this property. 7. Binding of parathyroid hormone is discussed in relation to metabolism and initial events in hormone action.     

Molecular characterization of a ligand-tethered parathyroid hormone receptor

It was recently shown that the covalent tethering of the N-terminus of parathyroid hormone (PTH) to the seventh helical bundle of the G-protein coupled PTH-receptor (PTH1R) leads to autoactivation [Shimizu et al., J. Biol. Chem. 275 (2000) 19456-19460]. Here, we have developed molecular models for the interaction of PTH(1-11) tethered to PTH1R and refined them with molecular dynamics simulations. The starting structure of the ligand/receptor complex is based on experimental data from a series of spectroscopic structural studies of PTH(1-34) and the extracellular domains of PTH1R and intermolecular contact points derived from photoaffinity labeling. The resulting PTH1R/[Arg(11)]PTH(1-11) complex has the N-terminus of PTH interacting with residues of the third extracellular loop of PTH1R, as a possible mode for receptor activation. The hydrophobic residues leucine-5 and methionine-8, centrally located in the N-terminal alpha-helix of PTH(1-11), are located in deep, well-defined hydrophobic pockets in the central core of the seventh helical bundle, consistent with the requirement of these amino acids for autoactivation. We postulate that the improved signaling properties of [Arg(11)]PTH(1-11) over wild type PTH(1-11) is due to a stable hydrogen bond between Arg(11) and E444, at the beginning of TM7. The model provides atomic insight into currently available biochemical data as well as numerous putative ligand/receptor interactions, and thereby may further the rational design of reduced-size PTH agonists at the PTH1 receptor.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24°C. Within 20 min most of the cell lines destroyed 50% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37°C. B_max and K_D for the different lymphocytes was 5.3–19 · 10¹¹ M and 1.8–18.5 · 10¹¹ M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.     

Characterization of the beta-adrenergic receptor mediating secretion of parathyroid hormone

In vitro incubation studies with bovine parathyroid gland slices compared the relative responsiveness of parathyroid hormone (PTH) secretion to isoprotherenol, epinephrine or norepinephrine. Isoproterenol was the most potent and norepinephrine the least potent of the three stimuli, suggesting a beta 2 type of an adrenergic response. However in this in vitro system, tazalol, a selective beta 1 adrenergic agonist significantly stimulated PTH secretion, whereas terbutaline, a selective beta 2 agonist had no effect. In addition, practolol, a selective beta 1 adrenergic antagonist blocked isoproterenol- or tazolol-stimulated PTH secretion. In vivo studies in normal human subjects showed that injection of te nonselective beta agonist, isoproterenol, (0.15 mg s.c.) significantly increased, whereas injection of the selective beta 2 agonist, terbulatine (0.3 mg s.c.) had no effect on serum PTH levels. These latter studies with putative selective beta adrenergic agents suggest that the beta adrenergic receptor mediating PTH secretion is of the beta 1 type (in contrast to the studies above with nonselective agents). The studies suggest that the beta adrenergic receptor mediating PTH secretion apparently differs from the classical beta 1 receptor described in th myocardium or the classical beta 2 receptor described in the bronchial smooth muscle.     

Photoaffinity labeling of the canine renal receptor for parathyroid hormone

Studies were undertaken to identify and characterize components of the parathyroid hormone receptor. An analog of the bovine parathyroid hormone(1-34) sequence was derivatized at the 23-tryptophan position with the photoreactive reagent 2-nitro-5-azidophenylsulfenyl chloride. 2-Nitro-5-azidophenylsulfenyl-bovine parathyroid hormone (NAPS-bPTH) analog retained full biological activity with respect to receptor binding and activation of adenylate cyclase in canine renal cortical plasma membranes. 125I-bPTH(1-34) analog was also derivatized without loss of receptor-binding activity. When 125I-NAPS-bPTH(1-34) analog was incubated with canine renal plasma membranes and then photolyzed, at least two 125I-labeled membrane components were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The incorporation of 125I radioactivity into one of these components (Mr congruent to 60,000) was inhibited when incubation and photolysis were performed in the presence of excess, unlabeled bPTH(1-34). Furthermore, photolysis of membranes in the presence of NAPS-bPTH(1-34) analog led to activation of adenylate cyclase which persisted following washing to remove noncovalently bound peptide, suggesting a functional, covalent hormone-receptor complex had been formed. We conclude that the M r congruent to 60,000 membrane component may be a part of the renal receptor for parathyroid hormone.     

Binding of bovine parathyroid hormone to surface receptors of cultured B-lymphocytes.

Binding of parathyroid hormone onto B-lymphocytes is detected by the utilization of the labelled antibody membrane assay. The amount of parathyroid hormone bound to the receptor sites was depending on the quantity of cells in the incubation milieu. Each cell line showed typical characteristics in time course of parathyroid hormone binding and maximal receptor capacity. Fragmentation of intact parathyroid hormone, also varying with the cell line tested, was very rapid, even at 24 degrees C. Within 20 min most of the cell lines destroyed 20% of the native hormone in the incubation mixture, indicating a fragmentation rate of up to 2.25 ng/min at 37 degrees C. Bmax and KD for the different lymphocytes was 5.3--19 . 10(11) M and 1.8--18,5 . 10(11) M, respectively. These values are in the range of reported plasma concentrations and may therefore represent more physiological values for the capacity and affinity of membrane receptors.     

Development of a rat parathyroid hormone 2 receptor antagonist

The parathyroid hormone 2 (PTH2) receptor is a Family B G-protein coupled receptor most highly expressed within the brain. Current evidence suggests that tuberoinfundibular peptide of 39 residues (TIP39) is the PTH2 receptor's endogenous ligand. To facilitate investigation of the physiological function of the PTH2 receptor/TIP39 system, we have developed a novel PTH2 receptor antagonist, by changing several residues within the amino terminal domain of TIP39. Histidine(4), tyrosine(5), tryptophan(6), histidine(7)-TIP39 binds the PTH2 receptor with high affinity, has over 30-fold selectivity for the rat PTH2 receptor over the rat PTH1 receptor and displays no detectable agonist activity. This ligand should be useful for in vivo investigation of PTH2 receptor function.     

Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptor

The scales of bony fish represent a significant reservoir of calcium but little is known about their contribution, as well as of bone, to calcium balance and how calcium deposition and mobilization are regulated in calcified tissues. In the present study we report the action of parathyroid hormone-related protein (PTHrP) on calcium mobilization from sea bream (Sparus auratus) scales in an in vitro bioassay. Ligand binding studies of piscine 125I-(1-35(tyr))PTHrP to the membrane fraction of isolated sea bream scales revealed the existence of a single PTH receptor (PTHR) type. RT-PCR of fish scale cDNA using specific primers for two receptor types found in teleosts, PTH1R, and PTH3R, showed expression only of PTH1R. The signalling mechanisms mediating binding of the N-terminal amino acid region of PTHrP were investigated. A synthetic peptide (10(-8) M) based on the N-terminal 1-34 amino acid residues of Fugu rubripes PTHrP strongly stimulated cAMP synthesis and [3H]myo-inositol incorporation in sea bream scales. However, peptides (10(-8) M) with N-terminal deletions, such as (2-34), (3-34) and (7-34)PTHrP, were defective in stimulating cAMP production but stimulated [3H]myo-inositol incorporation. (1-34)PTHrP induced significant osteoclastic activity in scale tissue as indicated by its stimulation of tartrate-resistant acid phosphatase. In contrast, (7-34)PTHrP failed to stimulate the activity of this enzyme. This activity could also be abolished by the adenylyl cyclase inhibitor SQ-22536, but not by the phospholipase C inhibitor U-73122. The results of the study indicate that one mechanism through which N-terminal (1-34)PTHrP stimulates osteoclastic activity of sea bream scales, is through PTH1R and via the cAMP/AC intracellular signalling pathway. It appears, therefore, that fish scales can act as calcium stores and that (1-34)PTHrP regulates calcium mobilization from them; it remains to be established if this mechanism contributes to calcium homeostasis in vivo.     

Molecular cloning and functional characterization of the canine parathyroid hormone/parathyroid hormone related peptide receptor (PTH1)

Parathyroid hormone (PTH) is a major mediator of calcium and phosphate metabolism through its interactions with receptors in kidney and bone. PTH binds with high affinity to PTH1 and PTH2, members of the superfamily of G protein-coupled receptors. In order to clone the canine PTH1 receptor, a canine kidney cDNA library was screened using the human PTH1 receptor cDNA and two clones were further characterized. The longest clone was 2177 bp and contained a single open reading frame of 1785 bp, potentially encoding a protein of 595 amino acids with a predicted molecular weight of 66.4 kD. This open reading frame exhibits >91% identity to the human PTH1 receptor cDNA and >95% identity when the putative canine and human protein sequences are compared. Competition binding following transfection of the canine PTH1 receptor into CHO cells demonstrated specific displacement of ¹²⁵I-human PTH 1-34 by canine PTH 1-34, human PTH 1-34, and canine/human parathyroid hormone related peptide (PTHrP) 1-34. Treatment of canine PTH1 receptor transfected cells, but not mock transfected cells, with these ligands also resulted in increased levels of intracellular cAMP. In contrast, the non-related aldosterone secretion inhibiting factor 1-35 neither bound nor activated the canine PTH1 receptor. Northern blot analysis revealed high levels of PTH1 receptor mRNA in the kidney, with much lower, but detectable, levels in aorta, heart, lung, prostate, testis, and skeletal muscle. Together, these data indicate that we have cloned the canine PTH1 receptor and that it is very similar, both in sequence and in functional characteristics, to the other known PTH1 receptors.     

Internalization determinants of the parathyroid hormone receptor differentially regulate beta-arrestin/receptor association.

beta-Arrestins have been implicated in regulating internalization of the parathyroid hormone receptor (PTHR), but the structural features in the receptor required for this effect are unknown. In the present study performed in HEK-293 cells, we demonstrated that different topological domains of PTHR are implicated in agonist-dependent receptor internalization; truncation of the cytoplasmic tail (PTHR-TR), selective mutations of the cytoplasmic tail to remove the sites of parathyroid hormone (PTH)-stimulated phosphorylation (PTHR-PD), and mutations in the third transmembrane helix (N289A) or in the third cytoplasmic loop (K382A) resulted in a 30-60% reduction in (125)I-PTH-related protein internalization. To better define the role of these internalization determinants, we have tested the ability of these mutant PTHRs to associate with beta-arrestins by using three different methodological approaches: 1) ability of overexpression of beta-arrestins to restore the internalization of (125)I-PTH-related protein for the mutant PTHRs; 2) visualization of PTH-mediated trafficking of beta-arrestin1 and -2 fused to the green fluorescent protein with receptors by confocal microscopy; 3) quantification of beta-arrestin1-green fluorescent protein translocation by Western blot. Our data reveal that the receptor' cytoplasmic tail contains determinants of beta-arrestin interaction that are distinct from the phosphorylation sites and are sufficient for transient association of beta-arrestin2, but stable association requires receptor phosphorylation. Determinants in the receptor's core (Asn-289 and Lys-382) appear to regulate internalization of the receptor/beta-arrestin complex toward early endocytic endosomes during the initial step of endocytosis.     

Purification and characterization of a receptor for human parathyroid hormone and parathyroid hormone-related peptide

The human parathyroid hormone (PTH) receptor (hPTH1R), containing a 9-amino acid sequence of rhodopsin at its C terminus, was transiently expressed in COS-7 cells and solubilized with 0.25% n-dodecyl maltoside. Approximately 18 microg of hPTH1R were purified to homogeneity per mg of crude membranes by single-step affinity chromatography using 1D4, a monoclonal antibody to a rhodopsin epitope. The N terminus of the hPTH1R is Tyr(23), consistent with removal of the 22-amino acid signal peptide. Comparisons of hPTH1R by quantitative immunoblotting and Scatchard analysis revealed that 75% of the receptors in membrane preparations were functional; there was little, if any, loss of functional receptors during purification. The binding affinity of the purified hPTH1R was slightly lower than membrane-embedded hPTH1R (K(d) = 16.5 +/- 1.3 versus 11.9 +/- 1.9 nm), and the purified receptors bound rat [Nle(8,21),Tyr(34)]PTH-(1-34)-NH(2) (PTH-(1-34)), and rat [Ile(5),Trp(23),Tyr(36)]PTHrP-(5-36)-NH(2) with indistinguishable affinity. Maximal displacement of (125)I-PTH-(1-34) binding by rat [alpha-aminoisobutyric acid (Aib)(1,3),Nle(8),Gln(10),Har(11),Ala(12),Trp(14),Arg(19),Tyr(21)]PTH-(1-21)-NH(2) and rat [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]PTH-(1-14)-NH(2) of 80 and 10%, respectively, indicates that both N-terminal and juxtamembrane ligand binding determinants are functional in the purified hPTH1R. Finally, PTH stimulated [(35)S]GTP gamma S incorporation into G alpha(s) in a time- and dose-dependent manner, when recombinant hPTH1R, G alpha(s)-, and beta gamma-subunits were reconstituted in phospholipid vesicles. The methods described will enable structural studies of the hPTH1R, and they provide an efficient and general technique to purify proteins, particularly those of the class II G protein-coupled receptor family.     

Osteopontin negatively regulates parathyroid hormone receptor signaling in osteoblasts

Systemic hormonal control exerts its effect through the regulation of local target tissues, which in turn regulate upstream signals in a feedback loop. The parathyroid hormone (PTH) axis is a well defined hormonal signaling system that regulates calcium levels and bone metabolism. To understand the interplay between systemic and local signaling in bone, we examined the effects of deficiency of the bone matrix protein osteopontin (OPN) on the systemic effects of PTH specifically within osteoblastic cell lineages. Parathyroid hormone receptor (PPR) transgenic mice expressing a constitutively active form of the receptor (caPPR) specifically in cells of the osteoblast lineage have a high bone mass phenotype. In these mice, OPN deficiency further increased bone mass. This increase was associated with conversion of the major intertrabecular cell population from hematopoietic cells to stromal/osteoblastic cells and parallel elevations in histomorphometric and biochemical parameters of bone formation and resorption. Treatment with small interfering RNA (siRNA) for osteopontin enhanced H223R mutant caPPR-induced cAMP-response element (CRE) activity levels by about 10-fold. Thus, in addition to the well known calcemic feedback system for PTH, local feedback regulation by the bone matrix protein OPN also plays a significant role in the regulation of PTH actions.     

Extracellular nucleotides enhance agonist potency at the parathyroid hormone 1 receptor

Parathyroid hormone (PTH) activates the PTH/PTH-related peptide receptor (PTH1R) on osteoblasts and other target cells. Mechanical stimulation of cells, including osteoblasts, causes release of nucleotides such as ATP into the extracellular fluid. In addition to its role as an energy source, ATP serves as an agonist at P2 receptors and an allosteric regulator of many proteins. We investigated the effects of concentrations of extracellular ATP, comparable to those that activate low affinity P2X7 receptors, on PTH1R signaling. Cyclic AMP levels were monitored in real-time using a bioluminescence reporter and β-arrestin recruitment to PTH1R was followed using a complementation-based luminescence assay. ATP markedly enhanced cyclic AMP and β-arrestin signaling as well as downstream activation of CREB. CMP – a nucleotide that lacks a high energy bond and does not activate P2 receptors – mimicked this effect of ATP. Moreover, potentiation was not inhibited by P2 receptor antagonists, including a specific blocker of P2X7. Thus, nucleotide-induced potentiation of signaling pathways was independent of P2 receptor signaling. ATP and CMP reduced the concentration of PTH (1–34) required to produce a half-maximal cyclic AMP or β-arrestin response, with no evident change in maximal receptor activity. Increased potency was similarly apparent with PTH1R agonists PTH (1–14) and PTH-related peptide (1–34). These observations suggest that extracellular nucleotides increase agonist affinity, efficacy or both, and are consistent with modulation of signaling at the level of the receptor or a closely associated protein. Taken together, our findings establish that ATP enhances PTH1R signaling through a heretofore unrecognized allosteric mechanism.     

Evidence that the thyroid-stimulating hormone (TSH) receptor transmembrane domain influences kinetics of TSH binding to the receptor ectodomain

Thyroid-stimulating hormone (TSH)-induced reduction in ligand binding affinity (negative cooperativity) requires TSH receptor (TSHR) homodimerization, the latter involving primarily the transmembrane domain (TMD) but with the extracellular domain (ECD) also contributing to this association. To test the role of the TMD in negative cooperativity, we studied the TSHR ECD tethered to the cell surface by a glycosylphosphatidylinositol (GPI) anchor that multimerizes despite the absence of the TMD. Using the infinite ligand dilution approach, we confirmed that TSH increased the rate of dissociation (k(off)) of prebound (125)I-TSH from CHO cells expressing the TSH holoreceptor. Such negative cooperativity did not occur with TSHR ECD-GPI-expressing cells. However, even in the absence of added TSH, (125)I-TSH dissociated much more rapidly from the TSHR ECD-GPI than from the TSH holoreceptor. This phenomenon, suggesting a lower TSH affinity for the former, was surprising because both the TSHR ECD and TSH holoreceptor contain the entire TSH-binding site, and the TSH binding affinities for both receptor forms should, theoretically, be identical. In ligand competition studies, we observed that the TSH binding affinity for the TSHR ECD-GPI was significantly lower than that for the TSH holoreceptor. Further evidence for a difference in ligand binding kinetics for the TSH holoreceptor and TSHR ECD-GPI was obtained upon comparison of the TSH K(d) values for these two receptor forms at 4 °C versus room temperature. Our data provide the first evidence that the wild-type TSHR TMD influences ligand binding affinity for the ECD, possibly by altering the conformation of the closely associated hinge region that contributes to the TSH-binding site.     

Structural analysis of yoked chorionic gonadotropin-luteinizing hormone receptor ectodomain complexes by circular dichroic spectroscopy

Binding of the heterodimeric glycoprotein hormone, chorionic gonadotropin (CG), occurs to the heptahelical LH receptor N-terminal ectodomain (ECD), a large portion of which has been modeled as a leucine-rich repeat protein. In this study, we expressed and purified three single chain N-CG-ECD-C complexes, one comprising the full-length ECD, 1-341 (encoded by exons 1-10 and a portion of 11), and two C-terminal ECD deletion fragments, 1-294 (encoded by exons 1-10) and 1-180 (encoded by exons 1-7). The fusion proteins, including yoked CG (N-beta-alpha-C), were characterized by Western blot analysis and circular dichroism (CD). Analysis of the CD spectra obtained on the CG-ECD fusion proteins, and of the difference spectrum of each after subtracting the CG contribution, yielded secondary structures consistent with a repeating beta-strand/alpha-helix fold as predicted in the homology model. A marked decrease in helicity was observed when the C-terminal 47 amino acid residues were removed from the ECD. Removal of an additional 114 residues, i.e. the region encoded by exons 8-10, results in the loss of fewer helical residues. These results suggest that the hinge region of the ECD, predicted to contain only limited secondary structure, interacts with and stabilizes the ligand-occupied N-terminal portion. Furthermore, the results support a repeating fold, consistent with the proposed model for the LHR ECD.     

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 ([Ca²⁺]_i) was measured in the PTH1R-null osteocytic cell line OC59, which expresses abundant CPTHRs but no PTH1Rs. [Ca²⁺]_i was assessed by single-cell ratiometric microfluorimetry in fura-2-loaded OC59 cells. A rapid and transient increase in [Ca²⁺]_i was observed in OC59 cells in response to the CPTH fragment hPTH-(53–84) (250 nM). No [Ca²⁺]_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 [Ca²⁺]_i in OC59 cells. The [Ca²⁺]_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), -agatoxin IVA (10 nM), and -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. calcium influx; osteocytes