Mutational analysis of the receptor-activating region of human parathyroid hormone.

The first 4 residues of parathyroid hormone (PTH) are highly conserved in evolution and are important for biological activity. We randomly mutated codons 1-4 of human PTH (hPTH) with degenerate oligonucleotides and, after expression in COS cells, screened the mutants for receptor binding and cAMP-stimulating activity using ROS 17/2.8 cells. This survey identified Glu4 and Val2 as important determinants of receptor binding and activation, respectively. Positions 1 and 3 were more tolerant of substitutions indicating that these sites are less vital to hormone function. Activities of synthetic hPTH(1-34) analogs further demonstrated the importance of positions 2 and 4. The binding affinity of [Ala4,Tyr34] hPTH(1-34)NH2 was 100-fold reduced relative to [Tyr34]hPTH(1-34)NH2 (Kd values = 653 +/- 270 and 4 +/- 1 nM, respectively), and [Arg2, Tyr34]hPTH(1-34)NH2 was a weak partial agonist which bound well to the ROS cell receptor (Kd = 31 +/- 10 nM). The Arg2 analog was nearly as potent as PTH(3-34) as an in vitro PTH antagonist in osteoblast derived cells. However, unlike PTH(3-34), [Arg2]PTH was a full agonist in opossum kidney (OK) cells. These observations suggest that the activation domains of the OK and ROS cell PTH receptors are different. Thus, amino-terminal PTH analogs may be useful as probes for distinguishing properties of PTH receptors.     

Stimulation by defined parathyroid hormone fragments of cell proliferation in skeletal-derived cell cultures.

We have reported previously that parathyroid hormone (PTH) acts on cultured bone cells to stimulate creatine kinase (CK) activity and [3H]thymidine incorporation into DNA via phosphoinositide turnover, in addition to its other actions via increased cyclic AMP production. We also found that mid-region fragments of PTH stimulate [3H]thymidine incorporation into avian chondrocytes. In the present study of mammalian systems, we demonstrate differential effects of defined synthetic PTH fragments on CK activity and DNA synthesis, as compared with cyclic AMP production, in osteoblast-enriched embryonic rat calvaria cell cultures, in an osteoblast-like clone of rat osteosarcoma cells (ROS 17/2.8) and in chondroblasts from rat epiphysial cartilage cell cultures. Unlike full-length bovine (b)PTH-(1-84) or the fully effective shorter fragment human (h)PTH-(1-34), fragments lacking the N-terminal region of the hormone did not increase cyclic AMP formation, whereas they did stimulate increases in both DNA synthesis and CK activity. Moreover, the PTH fragment hPTH-(28-48) at 10 microM inhibited the increase in cyclic AMP caused by 10 nM-bPTH-(1-84). The increase of CK activity in ROS 17/2.8 cells caused by bPTH-(1-84) or hPTH-(28-48) was completely inhibited by either cycloheximide or actinomycin D, as was shown previously for rat calvaria cell cultures. These results indicated the presence of a functional domain of PTH in the central part of the molecule which exerts its mitogenic-related effects on osteoblast- and chondroblast-like cells in a cyclic AMP-independent manner. Since cyclic AMP formation by PTH leads to bone resorption, specific mid-region fragments of PTH might prove suitable for use in vivo to induce bone formation without concomitant resorption.     

Dissociation of cAMP accumulation and phosphate uptake in opossum kidney (OK) cells with parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP)

Bovine parathyroid hormone (PTH) 1-34 [bPTH(1-34)] and human PTH related protein [hPTHrP(1-34)] stimulated cAMP accumulation in opossum kidney (OK) cells with Km of 5 x 10(-9) M, but inhibition of phosphate uptake was obtained with 17-fold lower Km of 3 x 10(-10) M. Phosphate uptake was partially inhibited with [Nle8.18Tyr34]bPTH(3-34)NH2 without concomitant cAMP stimulation. With hPTHrP(7-34)NH2, cAMP accumulation was increased in parallel to inhibition of phosphate uptake. [D-Trp12Tyr34]bPTH(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 had no agonist activity on cellular cAMP and inhibition of phosphate uptake. bPTH(1-34)-stimulated cAMP accumulation was antagonized by [Nle8.18Tyr34]bPTH(3-34)NH2, [D-Trp12Tyr34]bPTH(7-34)NH2, hPTHrP(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 with Ki of 1.4 x 10(-7), 2 x 10(-7), 4.7 x 10(-7) and 3.7 x 10(-6) M, respectively. But [Nle8.18Tyr34]bPTH(3-34)NH2 and [D-Trp12Tyr34]bPTH(7-34)NH2 reversed the inhibition of phosphate uptake only marginally, and hPTHrP(7-34)NH2 and [Tyr34]hPTH(7-34)NH2 were inactive. With hPTHrP(1-34) the Ki for cAMP accumulation of [Nle8,18Tyr34]bPTH(3-34)NH2 and hPTHrP(7-34)NH2 were 1.9 x 10(-7) and 7.2 x 10(-7) M, and inhibition of phosphate uptake was partially reversed with [Nle8,18Tyr34]bPTH(3-34)NH2, but not with hPTHrP(7-34)NH2. The present results indicate that truncated hPTHrP(7-34)NH2, unlike [Tyr34]hPTH(7-34)NH2 and [D-Trp12Tyr34]bPTH(7-34)NH2, elevates cellular cAMP and inhibits phosphate uptake. bPTH(1-34)- and hPTHrP(1-34)-evoked cAMP accumulation is suppressed by PTH and PTHrP fragments while inhibition of phosphate uptake remains largely unaltered.     

Zebrafish express the common parathyroid hormone/parathyroid hormone-related peptide receptor (PTH1R) and a novel receptor (PTH3R) that is preferentially activated by mammalian and fugufish parathyroid hormone-related peptide.

To further explore the evolution of receptors for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), we searched for zebrafish (z) homologs of the PTH/PTHrP receptor (PTH1R). In mammalian genes encoding this receptor, exons M6/7 and M7 are highly conserved and separated by 81-84 intronic nucleotides. Genomic polymerase chain reaction using degenerate primers based on these exons led to two distinct DNA fragments comprising portions of genes encoding the zPTH1R and the novel zPTH3R. Sequence comparison of both full-length teleost receptors revealed 69% similarity (61% identity), but less homology with zPTH2R. When compared with hPTH1R, zPTH1R showed 76% and zPTH3R 67% amino acid sequence similarity; similarity with hPTH2R was only 59% for both teleost receptors. When expressed in COS-7 cells, zPTH1R bound [Tyr(34)]hPTH-(1-34)-amide (hPTH), [Tyr(36)]hPTHrP-(1-36)-amide (hPTHrP), and [Ala(29),Glu(30), Ala(34),Glu(35), Tyr(36)]fugufish PTHrP-(1-36)-amide (fuguPTHrP) with a high apparent affinity (IC(50): 1.2-3.5 nM), and was efficiently activated by all three peptides (EC(50): 1.1-1.7 nM). In contrast, zPTH3R showed higher affinity for fuguPTHrP and hPTHrP (IC(50): 2.1-11.1 nM) than for hPTH (IC(50): 118.2-127.0 nM); cAMP accumulation was more efficiently stimulated by fugufish and human PTHrP (EC(50): 0.47 +/- 0.27 and 0.45 +/- 0.16, respectively) than by hPTH (EC(50): 9.95 +/- 1.5 nM). Agonist-stimulated total inositol phosphate accumulation was observed with zPTH1R, but not zPTH3R.     

PTH-receptors regulate norepinephrine release in human heart and kidney

Recent data suggests that chronic renal failure and hyperparathyroidism are associated with sympathetic overactivity. Since peptide hormones are known to modulate norepinephrine (NE) release by activating prejunctional receptors, this study investigates whether parathyroid hormone fragment (1-34) (hPTH(1-34)) increases neuronal NE release in human heart and kidney. Using specific PTH-receptor agonists and antagonists, this study furthermore highlights functional differences between PTH1 and PTH2 receptors. Human atrial and renal tissues were incubated with [(3)H]-NE and superfused. Three electrical stimulations (5Hz, 1min) induced a stable [(3)H]-NE release which was taken as an index of endogenous NE release. RT-PCR with specific primers for PTH1- and PTH2-receptor was performed in heart and kidney. hPTH(1-34) (0.01-0.1μmol/L) and a stable analog of its second messenger cAMP (8-bromo-cAMP) increased [(3)H]-NE release in human atria. This facilitatory effect of PTH was also observed in human renal cortex. The PTH1-receptor antagonist (D-Trp(12), Tyr(34))-pTH-(7-34) (0.5μmol/L) abolished the effect of hPTH(1-34). This data was verified using isolated perfused mouse kidneys. Tuberoinfundibular peptide of 39 residues (TIP-39) (0.1nmol/L-0.1μmol/L) decreased [(3)H]-NE release in atria. PTH1- and PTH2-receptor expressions were demonstrated in human heart and kidney. Moreover, a splice variant of the PTH2-receptor was detected in human kidney. In conclusion, PTH is able to facilitate NE release in human atria and renal cortex by activation of PTH1-receptors. The highly increased PTH levels that can be observed in chronic renal failure might be one contributor for the elevated sympathetic nerve activity and the associated cardiovascular mortality in patients with end stage renal disease.     

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.     

Existence of parathyroid hormone binding sites on murine hemopoietic blast cells

We demonstrated that 125I-labeled human parathyroid hormone (1-34;8,18-Nle,34-Tyr)[[125I]hPTH(1-34)] bound specifically to hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. Half-maximal inhibition of binding was achieved at concentrations of unlabeled hPTH(1-34) of about 5 x 10(-9)M. Insulin and hPTH(39-68) did not compete for PTH binding sites. Specific binding of hPTH(1-34) was detected in neither macrophages nor multinucleated cells (MNC's). Furthermore, treatment of hemopoietic blast cells with hPTH(1-34) stimulated MNC formation, and the range of concentrations (10(-10)-10(-8)M) over which hPTH(1-34) caused these effects was similar to that which inhibited the binding of [125I]hPTH(1-34). These findings suggest the presence of a PTH receptor on osteoclast precursors and the direct effect of PTH on them, resulting in osteoclast-mediated bone resorption.     

A G protein-coupled receptor from zebrafish is activated by human parathyroid hormone and not by human or teleost parathyroid hormone-related peptide. Implications for the evolutionary conservation of calcium-regulating peptide hormones.

Genomic and cDNA clones encoding portions of a putative catfish parathyroid hormone (PTH) 2 receptor (PTH2R) led to the isolation of a cDNA encoding a full-length zebrafish PTH2R (zPTH2R). The zPTH2R shared 63 and 60% amino acid sequence identity with human and rat PTH2Rs, respectively, 47-52% identity with mammalian and frog PTH/PTHrP receptors (PTH1R), and less than 37% with other members of this family of G protein-coupled receptors. COS-7 cells expressing zPTH2R(43), a 5' splice variant that lacked 17 amino acids in the amino-terminal extracellular domain, showed cAMP accumulation when challenged with [Tyr(34)]hPTH(1-34)-amide (hPTH) (EC(50), 1.64 +/- 0. 95 nM) and [Ile(5),Trp(23),Tyr(36)]hPTHrP-(1-36)-amide ([Ile(5), Trp(23)]hPTHrP) (EC(50), 46.8 +/- 12.1 nM) but not when stimulated with [Tyr(36)]hPTHrP-(1-36)-amide (hPTHrP), [Trp(23), Tyr(36)]hPTHrP-(1-36)-amide ([Trp(23)]hPTHrP), or [Ala(29),Glu(30), Ala(34),Glu(35),Tyr(36)]fugufish PTHrP-(1-36)amide (fuguPTHrP). FuguPTHrP also failed to activate the human PTH2R but had similar efficiency and efficacy as hPTH and hPTHrP when tested with cells expressing the human PTH1R. Agonist-dependent activation of zPTH2R was less efficient than that of zPTH2R(43), and both receptor variants showed no cAMP accumulation when stimulated with either secretin, growth hormone-releasing hormone, or calcitonin. The zPTH2R thus has ligand specificity similar to that of the human homolog, which raises the possibility that a PTH-like molecule exists in zebrafish, species which lack parathyroid glands.     

Parathyroid hormone stimulates protein kinase C but not adenylate cyclase in mouse epidermal keratinocytes.

Intact human parathyroid hormone, hPTH [1-84], and the hPTH [1-34] fragment stimulated membrane-associated protein kinase C (PKC) activity in immortalized (but still differentiation-competent) murine BALB/MK-2 skin keratinocytes. Unexpectedly, the hormone and its fragment did not stimulate adenylate cyclase. The failure of PTH to stimulate adenylate cyclase activity was not due to the lack of a functioning receptor-cyclase coupling mechanism because the cells were stimulated to synthesize cyclic adenosine monophosphate (cyclic AMP) by the beta-adrenergic drug isoproterenol. Thus, skin keratinocytes seem to have an unconventional PTH receptor that is coupled to a PKC-activating mechanism but not to adenylate cyclase. These observations suggest that normal and neoplastic skin keratinocytes respond to the PTH-related peptide that they make and secrete.     

Age, strain and species differences in circulating parathyroid hormone

A new, sensitive parathyroid hormone (PTH) radioimmunoassay that appears specific for the intact hormone, and its validation for measuring rat PTH are described. The assay is based on antibody C2-7 from chicken immunized with bovine PTH; it has a detection limit of 6 pg of bPTH per assay tube and measures basal PTH in most rats; it is responsive to provoked changes in endogenous PTH concentration, and the intra-assay and inter-assay coefficients of variation are 6.0% and 7.2%, respectively. Multiple dilutions of rat serum and parathyroid gland extract, result in competitive inhibition curves that are parallel to that of highly purified bPTH. Under our assay conditions the C2-7 antibody cross-reacts well with intact PTH but synthetic fragments of the hormone (1-34bPTH, 1-34hPTH, 28-48hPTH, 44-68hPTH, 53-84hPTH) do not depress tracer (125l-bPTH) binding to the antibody. Studies designed to validate the assay gave predictable results such as enhanced secretion of the hormone in response to EDTA infusion, and failure to detect the hormone in serum following thyroparathyroidectomy. In addition, we made the novel observation that in F344 rats circulating immunoreactive PTH increases progressively with aging.     

Carboxy-terminal PTH fragments stimulate [3H]thymidine incorporation in vascular endothelial cells

We have previously reported that the intact PTH molecule (1-84) stimulates proliferation of human umbilical vein endothelial cells (HUVECs). To define the bioactive portion of the PTH molecule we utilized amino, mid and carboxy-terminal PTH fragments. Carboxy- but not amino-terminal fragments were equivalent to the intact PTH molecule in stimulating [3H]thymidine incorporation in HUVEC. Carboxy- but not amino-terminal PTH fragments increased intracellular calcium. Blocking the rise in intracellular calcium with calcium chelators abolished PTHs proliferative effect on HUVEC. In contrast to PTH 1-84, the carboxy-terminal fragment effect on [3H]thymidine incorporation was blocked by KN-93 an inhibitor of CaM kinase II. Taken together, these data suggest that the carboxy-terminal PTH is (or contains) the bioactive fragment responsible for the changes in intracellular calcium and thymidine incorporation in HUVEC stimulated with the intact PTH molecule.     

Preparation of a photoreactive analog of parathyroid hormone [Nle8, Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]bovine parathyroid hormone-(1-34)NH2, a selective, high-affinity ligand for characterization of parathyroid hormone receptors

Photoaffinity radiolabeling techniques have been widely used to characterize the properties of peptide hormone receptors. However, the identity of authentic receptors is often uncertain because many macromolecules are labeled. These ambiguities are due, in part, to the use of a heterogeneous mixture of photoreactive photoligands, many of which have no or low affinity for the relevant hormone receptor. In this report, we describe the synthesis, purification, and structural analysis of the photoreactive parathyroid hormone analog, [Nle8,Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]-bovine parathyroid hormone-(1-34)NH2. The sulfur-free, oxidation-resistant, synthetic analog of bovine parathyroid hormone (PTH), [Nle8,Nle18,Tyr34]bovine PTH-(1-34)NH2 (NlePTH), was reacted with 4-fluoro-3-nitrophenylazide under nonaqueous conditions to yield several derivatives which were separated by reverse-phase high-performance liquid chromatography and analyzed by amino acid compositional analysis, thin-layer chromatography, and ultraviolet and visible absorption spectroscopy. Among the NlePTH derivatives generated, one of the least hydrophobic was shown to retain the highest potency as assessed in the canine renal cortical membrane radioreceptor assay. Sequence analysis of this peptide, after it had been derivatized with 4-fluoro-3-nitro-[2,6-3H]phenylazide and purified to homogeneity, permitted us to determine that the structure of this analog is [Nle8,Lys(N-epsilon-4-azide-2-nitrophenyl)13,Nle18,Tyr34]bovine PTH-(1-34)NH2. We emphasize the importance of using photoreactive ligands which are purified and subjected to detailed chemical and biological analyses for characterizing the properties of parathyroid hormone receptors and receptors for other peptide hormones.     

Interaction of human parathyroid hormone-related peptide with parathyroid hormone receptors in clonal rat osteosarcoma cells

Synthetic peptides corresponding to the amino-terminal region of the human parathyroid hormone-related peptide (hPTHrp) were used to characterize the interaction of hPTHrp with parathyroid hormone (PTH) receptors in clonal rat osteosarcoma cells (ROS 17/2.8). Both hPTHrp-(1-34) and [Tyr40]hPTHrp-(1-40) showed full agonist activity in stimulating cyclic AMP accumulation in ROS cells; human PTHrp-(1-34) was approximately 2.5-fold as potent as hPTH-(1-34). Both [Tyr-40]hPTHrp-(3-40) and hPTH-(3-34) inhibited the cyclic AMP increase induced by either hPTHrp or PTH with parallel dose-inhibition curves. Binding to intact ROS cells of a 125I-labeled [Tyr40]hPTHrp-(1-40) (125I-[Tyr40]hPTHrp-(1-40)) which retains full biological activity was time- and temperature-dependent and reversible. Binding of 125I-[Tyr40]hPTHrp-(1-40) and 125I-labeled [Nle8, Nle18, Tyr34]bovine PTH-(1-34)NH2 to ROS cells was competed for, to the same extent and with the comparable potency, by either unlabeled hPTHrp or PTH peptides. The binding capacity and affinity of receptors in ROS cells were strikingly similar for hPTHrp and PTH. Affinity cross-linking with either radioligand resulted in high affinity, specific labeling of an apparently identical macromolecule centering at Mr = 80,000, which was detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both reducing and nonreducing conditions. The data indicate that hPTHrp and PTH, their amino-terminal fragments at least, interact with the identical receptors with regard to affinity, capacity, specificity, and physicochemical characteristics in osteoblastic ROS 17/2.8 cells.     

Relaxation of bovine, porcine and human brain arteries by parathyroid hormone

We have studied the relaxant effect of bovine parathyroid hormone (bPTH) on helical strips of branches of bovine and human middle cerebral arteries and bovine and porcine basilar arteries. All arteries were studied after contraction with prostaglandin (PG) F2 alpha or KCl. In the case of all arteries contracted with PGF2 alpha, the ED50 of PTH vasorelaxation related to maximal vasorelaxation induced by papaverine ranged from 9 to 14 nM for bPTH-(1-34) and 100 to 220 ng/ml for native bPTH-(1-84). The PTH inhibitor, [Nle8, Nle18, Tyr34]bPTH-(3-34) amide, attenuated the vasorelaxant effect of both bPTH-(1-34) and bPTH-(1-84). The vasorelaxant effects of PTH which we have observed in this study are consistent with the stimulatory effects of PTH on vascular adenylate cyclase which we had previously reported.     

Second messenger signaling in the regulation of cytosolic pH and DNA synthesis by parathyroid hormone (PTH) and PTH-related peptide in osteoblastic osteosarcoma cells: role of Na+/H+ exchange.

The present study was performed to investigate the regulation of cytosolic pH (pHi) and DNA synthesis by parathyroid hormone(PTH) and PTH-related peptide (PTHrP) in osteoblasts, using osteoblastic osteosarcoma cells, UMR-106 which possessed PTH-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [Ca/PKC]) and amiloride-inhibitable Na+/H+ exchange system. Both human (h)PTH-(1-34) and hPTHrP-(1-34) caused a progressive decrease in pHi and the inhibition of [3H]thymidine incorporation (TdR) to the same degree in a dose-dependent manner with a minimal effective dose of 10(-10) M. Dibutyryl cAMP (10(-4) M and Sp-cAMPS (10(-4) M), a direct stimulator of PKA also caused a progressive decrease in pHi, and calcium ionophores (A23187 and ionomycin, 10(-6) M) caused a transient decrease in pHi. Pretreatment with amiloride (0.3 mM) mostly blocked dbcAMP- and Sp-cAMPS-induced decrease in pHi but did not affect calcium ionophore-induced decrease in pHi. In the presence of amiloride, PTH and PTHrP caused a transient decrease in pHi, which was similar to the pattern of calcium ionophore-induced change in pHi. Amiloride did not affect the inhibition of TdR by PTH or PTHrP as well as that by cAMP analogues or calcium ionophores. The present study indicated that PTH and PTHrP caused cytosolic acidification through PKA-inhibited Na+/H+ exchange and increased cytosolic calcium-induced pathway and that the regulation of DNA synthesis by PTH and PTHrP was not via Na+/H+ exchange system.     

Mapping the bimolecular interface of the parathyroid hormone (PTH)-PTH1 receptor complex: spatial proximity between Lys(27) (of the hormone principal binding domain) and leu(261) (of the first extracellular loop) of the human PTH1 receptor

In an effort to characterize the bimolecular interface between parathyroid hormone (PTH) and its human receptor PTH1-Rc (hPTH1-Rc), we previously identified two contact sites in the receptor: one for position 1 and another for position 13 (located at the ends of the principal activation domain) in PTH(1-34). The present study reports a third, novel "contact site" between hPTH1-Rc and Lys(27) of PTH(1-34). Lys(27) is located in the principal binding domain of the hormone (residues 25-34). The photoreactive PTH(1-34) analogue K27 contains a benzophenone (BP) moiety on Lys(27). The analogue binds to stably transfected HEK 293/C-21 cells (which express a high level of recombinant hPTH1-Rc) and stimulates adenylyl cyclase activity with a potency similar to PTH(1-34). In addition, (125)I-K27 cross-links effectively and specifically to the hPTH1-Rc. Enzymatic (Glu-C and Lys-C) and chemical (CNBr and BNPS-skatole) digestions of the photoconjugate between (125)I-K27 and hPTH1-Rc were performed. In addition, photoconjugates involving the bioactive mutants [L261M]- and [R262K]-hPTH1-Rc, transiently expressed in COS-7 cells, were also digested. The data obtained clearly identify L(261) or R(262) of the first extracellular loop of hPTH1-Rc as the contact site for Lys(27) in the hormone. On the basis of (i) the similarity in molecular mass between the CNBr digest of the (125)I-K27-[L261M]hPTH1-Rc conjugate and free (125)I-K27 and (ii) the failure to cross-link (125)I-K27 to a bioactive mutant receptor [L261A]hPTH1-Rc, we conclude that L(261) is the cross-linking site. These results provide the first demonstration of an interaction between the principal binding domain of PTH and the first extracellular loop of hPTH1-Rc. Revealing proximity of Lys(27) (in PTH) to L(261) (in hPTH1-Rc) provides additional insight into the nature of the ligand-receptor bimolecular interface and clearly illustrates that the extracellular loops of the receptor contribute to the specificity of the PTH-PTH1-Rc interaction. Taken together with previous studies, the new findings add important constraints on the possible positioning of the C-terminal helix of PTH (which contains the principal binding domain) relative to the first extracellular loop and the distal C-terminal helix of the large extracellular amino terminal domain of the PTH1-Rc.     

Effects of bPTH fragments (1-34), (3-34) and (7-34) on uterine contraction

Synthetic bovine parathyroid hormone containing the NH2 terminal 34 amino acids [bPTH-(1-34)] was recently demonstrated to inhibit oxytocin stimulated uterine contraction in vitro. The parathyroid hormone analogues [Nle8, Nle18, Tyr34]bPTH-(3-34)amide [NTA-(3-34)] and [Tyr34]bPTH-(7-34)amide [NTA-(7-34)] have been reported to act as inhibitors of antagonists of parathyroid hormone (PTH) in numerous assays. In the present study the effects of these PTH analogues on uterine contraction and the ability of these analogues to act as antagonists to the uterine inhibitory action of bPTH-(1-34) in vitro were investigated. The NTA-(3-34) fragment had no effect on oxytocin stimulated uterine contractions. However, the NTA-(3-34) fragment was able to alter the ability of bPTH (1-34) to reduce oxytocin stimulated uterine contraction in a dose-related manner. Bovine PTH(1-34) (0.3 microgram/ml) reduced the contractile response obtained with oxytocin (0.5 mU/ml) by 20%. A dose of 15 micrograms/ml) of NTA-(3-34) abolished this inhibitory action of bPTH-(1-34) on oxytocin stimulated uterine contraction. In contrast the NTA-(7-34) caused a change in itself, stimulated contraction of resting uterine horns in a dose-related manner; 3.0 micrograms/ml of NTA-(7-34) caused a change in gram tension of + 1.5 grams. Bovine PTH-(1-34) was able to reduce the uterine contraction stimulated by NTA-(7-34) and 0.3 microgram/ml of bPTH-(1-34) reduced the contractile response obtained with 3.0 micrograms/ml of NTA-(7-34) by as much as 70%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modifications of position 12 in parathyroid hormone and parathyroid hormone related protein: toward the design of highly potent antagonists

Truncated N-terminal fragments of parathyroid hormone (PTH), [Tyr34]bovine PTH(7-34)NH2, and parathyroid hormone related protein (PTHrP), PTHrP(7-34)NH2, inhibit [Nle8,18,[125I]iodo-Tyr34]-bPTH(1-34)NH2 binding and PTH-stimulated adenylate cyclase in bone and kidney assays. However, the receptor interactions of these peptides are 2-3 orders of magnitude weaker than those of their agonist counterparts. To produce an antagonist with increased receptor-binding affinity but lacking agonist-like properties, structure-function studies were undertaken. Glycine at position 12 (present in all homologues of PTH and in PTHrP), which is predicted in both hormones to participate in a beta-turn, was examined by substituting conformational reporters, such as D- or L-Ala, Pro, and alpha-aminoisobutyric acid (Aib), in both agonist and antagonist analogues. Except for N-substituted amino acids, which substantially diminished potency, substitutions were well tolerated, indicating that this site can accept a wide latitude of modifications. To augment receptor avidity, hydrophobic residues compatible with helical secondary structure were introduced. Incorporation of the nonnatural amino acids D-Trp, D-alpha-naphthylalanine (D-alpha-Nal), or D-beta-Nal into either [Tyr34]bPTH(7-34)NH2 or [Nle8,18,Tyr34]bPTH(7-34)NH2 resulted in antagonists that were about 10-fold more active than their respective 7-34 parent compound. Similarly, [D-Trp12]PTHrP(7-34)NH2 was 6 times more potent than the unsubstituted peptide but retained partial agonistic properties, although markedly reduced, similar to PTHrP(7-34)NH2. The antagonistic potentiating effect was configurationally specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a photoreactive parathyroid hormone antagonist to probe antagonist-receptor bimolecular interaction.

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert their calciotropic activities by binding to a specific seven-transmembrane-helix-containing G protein-coupled receptor mainly located in bone and kidney cells. In order to map in detail the nature of hormone-receptor interaction, we are employing 'photoaffinity scanning' of the bimolecular interface. To this end, we have developed photoreactive benzophenone (BP)-containing PTH analogs which can be specifically and efficiently cross-linked to the human (h) PTH/PTHrP receptor. In this report, we describe the photocross-linking of a BP-containing PTH antagonist, [Nle8,18,D-2-Nal12,Lys13(epsilon-BP),2-Nal23,Tyr34]bPT H(7-34)NH2 (ANT) to the recombinant hPTH/PTHrP receptor stably expressed in human embryonic kidney cells (HEK-293, clone C-21). This photoreactive antagonist has high affinity for the hPTH/PTHrP receptor and inhibits agonist-induced cyclase activity and intracellular calcium release. The photo-induced cross-linking of the radioiodinated antagonist (125I-ANT) to the recombinant hPTH/PTHrP receptor followed by SDS-PAGE analysis reveals a single radiolabeled band of approximately 85kDa, similar to that observed after cross-linking of a radioiodinated BP-containing agonist. The formation of this covalent 125I-ANT - hPTH/PTHrP receptor conjugate is competed dose-dependently by a variety of unlabelled PTH- and PTHrP-derived agonists and antagonists. This is the first report of a specific and efficient photocross-linking of a radioiodinated PTH antagonist to the hPTH/PTHrP receptor. Therefore, it provides the opportunity to study directly the nature of the bimolecular interaction of PTH antagonist with the hPTH/PTHrP receptor.     

Biological activity of parathyroid hormone antagonists substituted at position 13.

Lysine occupies position 13 in the parathyroid hormone (PTH) antagonist, [Nle8,18,Tyr34]bPTH(7-34)NH2. Acylation of the epsilon-amino group in lysine 13 by a hydrophobic moiety is well tolerated in terms of bioactivity: the analog [Nle8,18, D-Trp12,Lys 13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(7-34)NH2 is equivalent to the parent peptide in its affinity for PTH receptors and its ability to inhibit PTH-stimulated adenylate cyclase in both kidney- and bone-based assays. Truncation of this peptide by deletion of phenylalanyl7 with concomitant removal of the amino-terminal alpha-amino group yielded the analog desamino[Nle8,18,D-Trp12,Lys13 (epsilon-3-phenylpropanoyl),Tyr34]bPTH(8-34)NH2, an antagonist of high potency in vitro (Kb = 4 and 9 nM, Ki = 73 and 3.5 nM in kidney- and bone-based assays, respectively). Also this analog is potentially stable to aminopeptidases present in many biological systems.