Structure-function studies of analogues of parathyroid hormone (PTH)-1-34 containing beta-amino acid residues in positions 11-13

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with beta-amino acid residues around the putative hinge located at position 12: I. [Nle(8,18),beta-Ala(11,12),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); II. [Nle(8,18),beta-Ala(12,13),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); III. [Nle(8,18),beta-Ala(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); IV. [Nle(8,18),beta-hLeu(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); V. [Nle(8,18),beta-Ala(12), Nal(23),Tyr(34)]bPTH-(1-34)NH(2); VI. [Nle(8,18),beta-Ala(13), Nal(23),Tyr(34)]bPTH-(1-34)NH(2) (beta-hLeu = beta-homo-leucine; beta-Ala = beta-alanine; Nal = L-2-naphthyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12, or 13 in analogues III-VI does not favor a disordered structure in this portion of the sequence.     

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.     

Sulfur-free parathyroid hormone analogues containing D-amino acids: biological properties in vitro and in vivo

Three sulfur-free analogues of bovine parathyroid hormone (bPTH) containing D-amino acids were synthesized by the solid-phase method and their biological properties compared in an in vitro bioassay (rat renal adenylate cyclase assay), a receptor assay for parathyroid hormone (PTH) (canine renal membranes), and an in vivo bioassay (chick hypercalcemia assay). The analogue [Nle8,Nle18,D-Tyr34]-bPTH-(1-34)-amide, which was found to be more than 4 times as potent in vitro as unsubstituted PTH, is the most potent analogue of PTH yet synthesized. The enhanced potency was largely attributable to increased affinity for the PTH receptor. In vivo, however, this analogue was only one-third as potent as bPTH-(1-34). Cumulative evidence suggests that the nearly 15-fold decline in the relative potency when the compound was assayed in vivo is due to the substitution of norleucine for methionine. The other analogues, [D-Val2,Nle8,D-Tyr34]bPTH-(1-34)-amide and [D-Val2,Nle8,Nle18,D=Tyr34]bPTH-(2-34)-amide, were only weakly active in vitro and in vivo, indicating that substitution with D-amino acids at the NH2 terminus of PTH causes markedly diminished receptor affinity. In fact, the placement of a D-amino acid at the NH2 terminus is more deleterious to biological activity than is omission of amino acids at positions 1 and 2.     

Inhibition of uterine contraction by synthetic parathyroid hormone fragment

The inhibitory effect of synthetic bovine parathyroid hormone fragment [bPTH-(1-34)] on rat uterine contraction was studied $\text{in vitro}$. Oxytocin, prostaglandin F_2α and acetylcholine produced log dose-related contraction. The addition of bPTH-(1-34) shifted the dose-response curves of the three agonists to the right. Two doses of bPtH-(1-34) were tested. The higher dose (400 ng/ml) caused a greater inhibition of the agonists than did the lower dose (40 ng/ml). bPTH-(1-34) also inhibited the uterine contraction elicited by electrical stimulation of the tissue. We suggest that bPTH-(1-34) has a non-specific depressing effect on the contractile mechanism of the uterine tissue.     

Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing alpha-amino-iso-butyric acid (Aib) residues

The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.     

Uterine relaxing action of parathyroid hormone: effect of oxidation and methionine substitution

The effects of bPTH-(1-34), oxidized bPTH-(1-34),[Nle8,Nle18, Tyr34] bPTH-(1-34) amide, and oxidized [Nle8,Nle18,Tyr34]bPTH-(1-34)amide were tested in an in vitro rat uterine assay. When bPTH-(1-34) was treated with hydrogen peroxide (H2O2), the ability of this peptide to reduce oxytocin-stimulated uterine contraction in vitro was no longer evident. An analogue of bPTH-(1-34), in which the methionines at positions 8 and 18 were replaced with norleucine ([Nle8,Nle18,Tyr34]bPTH-(1-34)amide), was capable of reducing oxytocin-stimulated uterine contraction. However, when the [Nle8,Nle18,Tyr34]bPTH-(1-34)amide was oxidized, it retained the ability to reduce uterine contraction. Since we have previously shown that H2O2 oxidation affected only the methionines, these results suggest that the methionines are not necessary for the uterine activity of bPTH-(1-34). We have previously shown that oxidation of bPTH-(1-34) also destroys its blood vessel relaxing activity but has no effect in the rat or the Japanese quail hypercalcemic assays. These data combined with the results of the present studies suggest that the uterine and vascular smooth muscle relaxing properties of bPTH-(1-34) may require the same structural conformation and that this conformation is different from that required for the hypercalcemic action of the peptide.     

Cardiovascular effects of bPTH-(1-34) and isoproterenol in the frog, Rana tigrina.

1. The cardiovascular effects of bovine parathyroid hormone fragment [bPTH-(1-34)] and isoproterenol (ISO) on frog (Rana tigrina) isolated atria and helical strips of blood vessels were examined since PTH produces a beta-adrenergic-like effect in the mammal. 2. Data showed that both bPTH-(1-34) and ISO were vasorelaxant in KCl and arginine vasoctocin (AVT) preconstricted dorsal aorta, iliac and femoral arteries. 3. They both relaxed extracellular calcium-dependent contrations. 4. There was no additive nor synergistic effect between them in AVT preconstricted strips. 5. Both bPTH-(1-34) and ISO were positively inotropic but differed in their chronotropic effects, being negative and positive. 6. In the tiger frog, bPTH-(1-34) shows beta-adrenergic like contractile responses in both the cardiac and vascular smooth muscle as in the mammal, but not in the heart rate.     

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.     

Distribution of parathyroid hormone-sensitive adenylate cyclase in isolated rabbit renal cortex microvessels and glomeruli

The effect of the synthetic amino-terminal fragment of bovine parathyroid hormone, bPTH-(1-34), on the adenylate cyclase of microvessels and glomeruli isolated from rabbit kidney cortex was studied in the presence and absence of guanosine triphosphate (GTP). bPTH-(1-34) stimulated the vascular and glomerular adenylate cyclase in a dose-dependent manner with apparent ED50 values of 11.5 nM and 64 nM respectively, in the absence of GTP. 10(-4)M GTP greatly amplified the vascular response to bPTH-(1-34) while, in the glomeruli, both GTP and bPTH-(1-34) had only additive effects. In the presence of GTP, vascular and glomerular apparent ED50 were 190 nM and 64 nM respectively. [Nle8, Nle18, Tyr34] -bPTH-(3-34) amide, described as a PTH antagonist, inhibited the action of bPTH-(1-34) in the microvessels and to a lesser extent in the glomeruli. PTH is therefore a potent stimulator of adenylate cyclase in rabbit renal microvessels and glomeruli, and may play a role in the regulation of renal blood flow and glomerulo-tubular feedback control.     

Cerebral Vascular Adenylate Cyclase: Evidence for Coupling to Receptors for Vasoactive Intestinal Peptide and Parathyroid Hormone

We have studied the responsiveness of vascular adenylate cyclase to vasoactive intestinal peptide (VIP) and parathyroid hormone (PTH) using preparations of cerebral microvessels and arteries. Cerebral microvessels obtained from rats, guinea-pigs, cattle, and pigs all responded potently to bovine (b) PTH-(1-34), whereas considerable between-species variability was observed in the responsiveness to VIP. The homologous peptide to VIP, PHI (porcine heptacosapeptide), stimulated adenylate cyclase in both rat microvessels and a broken-cell preparation of bovine arteries. The ED50 values for activation of bovine arterial adenylate cyclase by VIP, PHI, and bPTH-(1-34) were 6.9 nM, 10 nM, and 100 nM, respectively, with the following order of efficacy: VIP = PHI greater than bPTH-(1-34). The other related peptides, hpGRF (human pancreatic growth hormone releasing factor), secretin, and glucagon, and the fragment VIP-(10-28) were inactive. The PTH antagonist, [Nle8, Nle18, Tyr34]bPTH-(3-34) amide, inhibited bPTH-(1-34) activation of vascular adenylate cyclase but did not affect activation by VIP using either microvessels or arteries. VIP or PHI demonstrated an additive effect with bPTH-(1-34) on vascular adenylate cyclase activity. However, the effects of VIP and PHI were nonadditive with each other. These data suggest that VIP and bPTH-(1-34) activate cerebral vascular adenylate cyclase by interacting with pharmacologically distinct receptors, whereas PHI and VIP likely interact with a common receptor.     

Chemical and biological properties of synthetic, sulfur-free analogues of parathyroid hormone.

Several analogues of the biologically active fragment of bovine parathyroid hormone (bPTH), based on the sequence of the NH2-terminal 34 amino acids, were prepared by solid phase synthesis and bioassayed in the in vitro adenylyl cyclase assay to provide further information concerning structure-activity relations in parathyroid hormone. In two analogues both methionines of the natural hormone were replaced with the sulfur-free and closely isosteric amino acid norleucine (Nle). The synthetic analogue [Nle-8, Nle-18]bPTH-(1-34) was highly active in the in vitro rat adenylyl cyclase bioassay, thus demonstrating that neither of the methionines, found in the native sequence, is indispensable for biological activity. Tyrosine was substituted for phenylalanine at position 34 in the synthesis of two other hormone analogues, [Try-34]bPTH-(1-34) and [Nle-8,Nle-18,Tyr-34]bPTH-(1-34). Both derivatives were exposed to conventional iodination procedures involving use of the oxidant chloramine T. Although iodination of [Try-34]bPTH-(1-34) resulted in virtually complete loss of biological activity, [Nle-8,Nle-18,Tyr-34]-bPTH-(1-34), which lacks methionine, could be exposed to oxidants and labeled efficiently with iodine with retention of nearly complete biological activity. These findings confirm that the loss of biological activity after oxidation of bPTH, as previously observed with the native hormone, is indeed attributable to the oxidation lability of methionine rather than to any other modifications. This sulfur-free, radioiodinated, biologically active analogue of parathyroid hormone may prove useful in studies of interaction of the hormone with the membrane receptors of target tissues and in studies of the metabolism of parathyroid hormone.     

Structure-function relationship studies of PTH(1-11) analogues containing sterically hindered dipeptide mimetics.

The N-terminal 1-34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and reproduces all biological responses characteristic of the native intact PTH. In order to develop safer and non-parenteral PTH-like bone anabolic agents, we have studied the effect of introducing conformationally constrained dipeptide mimetics into the N-terminal portion of PTH in an effort to generate miniaturized PTH-mimetics. To this end, we have synthesized and conformationally and biologically characterized PTH(1-11) analogues containing 3R-carboxy-6S-amino-7,5-bicyclic thiazolidinlactam (7,5-bTL), a rigidified dipeptide mimetic unit. The wild type sequence of PTH(1-11) is H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-NH(2). The following pseudo-undecapeptides were prepared: [Ala(1), 7,5-bTL(3, 4), Nle(8), Arg(11)]hPTH(1-11)NH(2) (I); [Ala(1), 7,5-bTL(6, 7), Nle(8), Arg(11)]hPTH(1-11)NH(2) (II); [Ala(1), Nle(8), 7,5-bTL(9, 10), Arg(11)]hPTH(1-11)NH(2) (III). In aqueous solution containing 20% TFE, only analogue I exhibited the typical CD pattern of the alpha-helical conformation. NMR experiments and molecular dynamics calculations located the alpha-helical stretch in the sequence Ile(5)-His(9). The dipeptide mimetic unit 7,5-bTL induces a type III beta-turn, occupying the positions i - 1 and i of the turn. Analogue II exhibited an equilibrium between a type I beta-turn and an alpha-helix, and analogue III did not show any ordered structure. Biological tests revealed poor activity for all analogues (EC(50) > 0.1 mM). Apparently, the relative side-chain orientation of Val(2), Ile(5) and Met(8) can be critical for effective analogue-receptor interaction. Considering helicity as an essential property to obtain active PTH agonists, one must decorate the correctly positioned dipeptide mimetic azabicycloalkane scaffold with substitutions corresponding to the displaced amino acids.     

Analogues of parathyroid hormone modified at positions 3 and 6. Effects on receptor binding and activation of adenylyl cyclase in kidney and bone.

Predictive and spectroscopic methods were used to develop a model of the structures of the 1-34 peptides of parathyroid hormone (PTH) and the PTH-related protein (PTHrP). Circular dichroism (CD) studies of bovine PTH-(1-34) and human PTHrP-(1-34)amide in the presence of trifluoroethanol suggest the presence of 24-26 alpha-helical residues. For both peptides, interactions between amino- and carboxyl-region alpha-helices are predicted to result in a hydrophobic core with externally facing hydrophilic residues that include probable determinants of receptor binding and activation. Two such residues, Ser3 and Gln6, are conserved in all known members of the PTH/PTHrP family. We have synthesized 13 novel analogues of bovine PTH-(1-34) monosubstituted at positions 3 and 6 and have determined their biological activities in renal and bone cell radioreceptor and adenylyl cyclase assays. Position 3 analogues displayed biological activity that was reduced in direct proportion to the volume of the substituent side-chain. Position 6 analogues also displayed reduced biological activity, but no simple correlation with side-chain volume or hydrophobicity was evident. The analogues fully displaced labeled PTH from binding sites in renal membranes and bone cells, but [Phe3]bPTH-(1-34), [Tyr3]bPTH-(1-34), [Phe6] bPTH-(1-34), and [Ser6]bPTH-(1-34) were only partial agonists in one or both adenylyl cyclase assays. Of these, [Phe3]bPTH-(1-34) and [Phe6]bPTH-(1-34) were tested for antagonist activity and were found to inhibit the activation of adenylyl cyclase in response to bPTH-(1-34) or hPTHrP-(1-34)amide. These results indicate that positions 3 and 6 contribute important determinants of PTH receptor binding and activation. Modification at these positions represents a novel approach to the development of antagonists of PTH action.     

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.     

Synthetic peptides comprising the amino-terminal sequence of a parathyroid hormone-like protein from human malignancies. Binding to parathyroid hormone receptors and activation of adenylate cyclase in bone cells and kidney

A tumor-derived protein with a spectrum of biologic activities remarkably similar to that of parathyroid hormone (PTH) has recently been purified and its sequence deduced from cloned cDNA. This PTH-like protein (PLP) has substantial sequence homology with PTH only in the amino-terminal 1-13 region and shows little similarity to other regions of PTH thought to be important for binding to receptors. In the present study, we compared the actions of two synthetic PLP peptides, PLP-(1-34)amide and [Tyr36]PLP-(1-36)amide, with those of bovine parathyroid hormone (bPTH)-(1-34) on receptors and adenylate cyclase in bone cells and in renal membranes. Synthetic PLP peptides were potent activators of adenylate cyclase in canine renal membranes (EC50 = 3.0 nM) and in UMR-106 osteosarcoma cells (EC50 = 0.05 nM). Bovine PTH-(1-34) was 6-fold more potent than the PLP peptides in renal membranes, but was 2-fold less potent in UMR-106 cells. A competitive PTH receptor antagonist, [Tyr34]bPTH-(7-34)amide, rapidly and fully inhibited adenylate cyclase stimulation by the PLP peptides as well as bPTH-(1-34). Competitive binding experiments with 125I-labeled PLP peptides revealed the presence of high affinity PLP receptors in UMR-106 cells IC50 = 3-4 nM) and in renal membranes (IC50 = 0.3 nM). There was no evidence of heterogeneity of PLP receptors. Bovine PTH-(1-34) was equipotent with the PLP peptides in binding to PLP receptors. Likewise, PLP peptides and bPTH-(1-34) were equipotent in competing with 125I-bPTH-(1-34) for binding to PTH receptors in renal membranes. Photoaffinity cross-linking experiments revealed that PTH and PLP peptides both interact with a major 85-kDa and minor 55- and 130-kDa components of canine renal membranes. We conclude that PTH and PLP activate adenylate cyclase by binding to common receptors in bone and kidney. The results further imply that subtle differences exist between PTH and PLP peptides in their ability to induce receptor-adenylate cyclase coupling.     

Characterization of soluble and particulate parathyroid hormone receptors using a biotinylated bioactive hormone analog

A bioactive biotin-containing derivative of the synthetic bovine parathyroid hormone analog [Nle8,Nle18,Tyr34]bovine parathyroid hormone-(1-34) (bPTH-(1-34] amide was prepared by reacting the peptide with N-biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide ester. The derivative was incubated with particulate renal plasma membranes or with detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) extracts of renal cortical membranes, and two membrane components were identified. Labeling of these components was competitively inhibited by underivatized bPTH-(1-34) or bPTH-(3-34) but not by insulin, adrenocorticotropin, or oxidized rat PTH-(1-34). PTH-binding components that were immobilized on nitrocellulose could be detected by incubating the membrane with biotinyl-bPTH-(1-34). Binding components of apparent molecular mass 68, 70, and 150 kDa were specifically labeled in plasma membranes derived from canine, human, and porcine renal cortex, rat liver, and human fibroblasts. The 68-kDa binding protein was found to be consistently more acidic than the 70-kDa binding protein in human, porcine, and canine renal membranes analyzed by two-dimensional electrophoresis. The 68-70-kDa receptor doublet could be specifically isolated by streptavidin-agarose chromatography of solubilized membrane extracts that had first been incubated with biotinyl-BPTH-(1-34). Biotinyl-bPTH-(1-34) should be useful as a tool for further characterization and purification of the PTH receptor.     

Formation of water-soluble complex between the 1-34 fragment of parathyroid hormone and dimyristoylphosphatidylcholine

Two biologically active, 34 amino acid fragments of parathyroid hormone interact with dimyristoylphosphatidylcholine to form lipoprotein particles. In the lipid-bound form these parathyroid hormone peptides exhibit an increased amount of folded secondary structure and the tryptophan residue of [Nle8, Nle18, Tyr34] b PTH (1-34) amide appears to become buried in a more hydrophobic environment. The lipoprotein particle which is formed has dimensions of approximately 65 X 7 nm but aggregates to larger structures with increasing temperature. Above the phase transition of the phospholipid the peptides no longer affect the morphology of the lipid and the spectral properties of the peptide are not perturbed by the lipid. This is similar to the behavior of glucagon with dimyristoylphatidylcholine. The results indicate that several nonhomologous peptide hormones have common features which allow them to fold into an amphipathic helix and solubilize phospholipid.     

Synthesis and characterization of extended and deleted recombinant analogues of parathyroid hormone-(1-84): correlation of peptide structure with function

Recombinant analogues of human parathyroid hormone [hPTH-(1-84)] were expressed in Escherichia coli harboring plasmids containing synthetic genes under the control of the lac promoter. The level of expression of the gene encoding the truncated analogue, hPTH-(3-84), was greater than that of the gene encoding full-length hPTH-(1-84) but less than that of the gene encoding proparathyroid hormone (hProPTH). This may be due in part to the relative efficiency of translation of the mRNA as suggested by secondary structure analysis and in part because of enhanced stability of the extended peptide. Formylmethionyl derivatives of hProPTH and of hPTH-(3-84) and underivatized hPTH-(3-84) were purified by HPLC, and their identity was confirmed by NH2-terminal sequencing and amino acid analysis. The bioactivity of these recombinant peptides was then tested in skeletal and renal adenylate cyclase assays in vitro and in assays examining effects on plasma and urine calcium and phosphate levels and on urine cyclic AMP levels in vivo. The NH2-terminally extended analogue fMet-hProPTH displayed 10% of the in vitro activity of hPTH-(1-84) and was a partial agonist in vivo. The peptides hPTH-(3-84) and fMet-hPTH-(3-84) were inert in vitro and were very weak in vitro antagonists when compared to the NH2-terminal analogue bovine [Nle8,18Tyr34]PTH-(3-34)-NH2. In vivo, hPTH-(3-84) and the bPTH-(3-34) analogue, when assayed at a 10:1 molar ratio relative to bPTH-(1-84), were each inert, and neither demonstrated antagonist activity at these concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor necrosis factor and interleukin 1 inhibit parathyroid hormone-responsive adenylate cyclase in clonal osteoblast-like cells by down-regulating parathyroid hormone receptors.

The effects of the monokines tumor necrosis factor alpha (TNF) and interleukin 1 (IL 1) on parathyroid hormone (PTH)-responsive adenylate cyclase were examined in clonal rat osteosarcoma cells (UMR-106) with the osteoblast phenotype. Recombinant TNF and IL 1 incubated with UMR-106 cells for 48 hr each produced concentration-dependent inhibition of PTH-sensitive adenylate cyclase, with maximal inhibition of PTH response (40% for TNF, 24% for IL 1) occurring at 10(-8) M of either monokine. Both monokines also decreased adenylate cyclase stimulation by the tumor-derived PTH-related protein (PTHrP). In contrast, TNF and IL 1 had little or no inhibitory effect on receptor-mediated stimulation of adenylate cyclase by isoproterenol and nonreceptor-mediated enzyme activation by cholera toxin and forskolin; both monokines increased prostaglandin E2 stimulation of adenylate cyclase. Binding of the radioiodinated agonist mono-[125I]-[Nle8,18, Tyr34]bPTH-(1-34)NH2 to UMR-106 cells in the presence of increasing concentrations of unlabeled [Nle8,18, Tyr34]bPTH-(1-34)NH2 revealed a decline in PTH receptor density (Bmax) without change in receptor binding affinity (dissociation constant, Kd) after treatment with TNF or IL 1. Pertussis toxin increased PTH-sensitive adenylate cyclase activity but did not attenuate monokine-induced inhibition of PTH response. In time course studies, brief (1 hr) exposure of cells to TNF or IL 1 during early culture was sufficient to decrease PTH response but only after exposed cells were subsequently allowed to grow for prolonged periods. Inhibition of PTH response by monokines was blocked by cycloheximide. The results indicate that TNF and IL 1 impair responsiveness to PTH (and PTHrP) by a time- and protein synthesis-dependent down-regulation of PTH receptors linked to adenylate cyclase.     

Cardiac actions of bovine parathyroid hormone (1-34), isoproterenol, and propranolol in turtles.

1. In isolated turtle hearts bovine parathyroid hormone (b-PTH) (1-34) stimulated both force of contraction and rate (beats/min) apparently stimulating beats/min to a larger extent. 2. Isoproterenol stimulated both rate and force, perhaps affecting contractile force more. 3. Propranolol alone clearly decreased contractile force and beats/min. 4. Pre-treatment with propranolol removed stimulatory effects of both bPTH (1-34) and isoproterenol. Propranolol's effect on isoproterenol was expected. Its effect on bPTH (1-34) may be related to beta-stimulation by PTH or to some other non-specific inhibitory effect of propranolol.