Parathyroid Hormone (PTH)-PTH-related Peptide Hybrid Peptides Reveal Functional Interactions between the 1–14 and 15–34 Domains of the Ligand

Gardella, Thomas J.; Luck, Michael D.; Wilson, Andrew; Keutmann, Henry T.; Nussbaum, Samuel R.; Potts, John T.; Kronenberg, Henry M.

doi:10.1074/jbc.270.12.6584

Cited by 60 publications

(67 citation statements)

References 28 publications

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“…Peptides and Reagents-The preparation and initial characterization of PTH/PTHrP hybrid ligands was described previously (15 hPTHrP-(7-34)NH 2 , was purchased from Peninsula Laboratories Belmont, CA. All other peptides were prepared by the biopolymer synthesis facility at Massachusetts General Hospital (Boston, MA) using solid-phase chemistry and Fmoc (N-(9-fluorenyl)methoxycarbonyl)protecting groups.…”

Section: Methodsmentioning

confidence: 99%

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Converting Parathyroid Hormone-related Peptide (PTHrP) into a Potent PTH-2 Receptor Agonist

Gardella

Luck

Jensen

et al. 1996

Journal of Biological Chemistry

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Most of the bone and kidney-related functions of parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) are thought to be mediated by the PTH/PTHrP receptor. Recently, a homologous receptor, the PTH-2 receptor, was obtained from rat and human brain cDNA libraries. This receptor displayed the remarkable property of responding potently to PTH, but not to PTHrP. To begin to define residues involved in the ligand specificity of the PTH-2 receptor, we studied the interaction of several PTH/PTHrP hybrid ligands and other related peptide analogs with the human PTH-2 receptor. The results showed that two sites in PTH and PTHrP fully account for the different potencies that the two ligands exhibited with PTH-2 receptors; residue 5 (His in PTHrP and Ile in PTH) determined signaling capability, while residue 23 (Phe in PTHrP and Trp in PTH) determined binding affinity. By changing these two residues of PTHrP to the corresponding residues of PTH, we were able to convert PTHrP into a ligand that avidly bound to the PTH-2 receptor and fully and potently stimulated cAMP formation. Changing residue 23 alone yielded [Trp 23 ]hPTHrP-(1-36), which was an antagonist for the PTH-2 receptor, but a full agonist for the PTH/PTHrP receptor. Residues 5 and 23 in PTH and PTHrP thus play key roles in signaling and binding interactions, respectively, with the PTH-2 receptor. Receptor-selective agonists and antagonists derived from these studies could help to identify the biological role of the PTH-2 receptor and to map specific sites of ligand-receptor interaction.The pharmacological profiles of PTH 1 and PTHrP are nearly identical in most in vitro assay systems; and elevated blood levels of PTH (i.e. primary hyperparathyroidism) or PTHrP (i.e. humoral hypercalcemia of malignancy syndrome) have comparable effects on mineral ion homeostasis (1, 2). The similarities in the biological activities of the two ligands can be explained by their interaction with a common receptor, the PTH/PTHrP receptor, which is expressed abundantly in bone and the kidney (3). The binding of either radiolabeled PTH-(1-34) or PTHrP-(1-36) to the PTH/PTHrP receptor is competitively inhibited by either unlabeled ligand (4, 5); thus, the recognition sites for the two ligands in the PTH/PTHrP receptor probably overlap. In both PTH and PTHrP the 15-34 region contains the principal determinants of binding to the PTH/PTHrP receptor, and although these regions show only minimal sequence homology with only three amino acid identities, each 15-34 peptide can block the binding of either PTH-(1-34) or PTHrP-(1-34) (6 -8). Furthermore, the amino-terminal portion of each ligand is required for bioactivity, and these probably interact with the PTH/PTHrP receptor in similar ways, since 8 of the first 13 residues are identical in PTH and PTHrP.The PTH/PTHrP receptor is a member of a distinct family of G protein-coupled receptors (9, 10) that includes receptors for other peptide hormones such as secretin (11), calcitonin (12), and glucagon (13). Using degenera...

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Section: Methodsmentioning

confidence: 99%

“…Previously, we had prepared a series of PTH/PTHrP hybrid ligands for the purpose of investigating whether homologous domains of PTH and PTHrP could be interchanged (15). In the present study, we have used these hybrid ligands and derivative peptide analogs to map regions of PTH and PTHrP that contribute to the ligand selectivity of the PTH-2 receptor.…”

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confidence: 99%

Converting Parathyroid Hormone-related Peptide (PTHrP) into a Potent PTH-2 Receptor Agonist

Gardella

Luck

Jensen

et al. 1996

Journal of Biological Chemistry

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“…Residues Phe 23, Leu24, and Ile28 are close to the receptor, and photolabeling of Leu24 caused a 10-fold reduction in binding. (20) When this is considered together with the fact that residues Phe23, Leu24, and Ile28 are intolerant to substitution by polar residues, (21,22) the fPTH sequence is in keeping with that of other PTH and PTHrP homologs. Furthermore, the Arg20 and Leu24 of fPTH are in accord with the strict conservation of these residues throughout all known PTH and PTHrP sequences.…”

Section: Identification Of Fish Pth From Fugu Rubripesmentioning

confidence: 99%

Identification of a Parathyroid Hormone in the Fish Fugu rubripes

Danks

Notini

et al. 2003

Journal of Bone and Mineral Research

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A PTH gene has been isolated from the fish Fugu rubripes. The encoded protein of 80 amino acid has the lowest homology with any of the PTH family members. Fugu PTH(1-34) had 5-fold lower potency than human PTH(1-34) in a mammalian cell system. Introduction: Parathyroid hormone (PTH) is the major hypercalcemic hormone in higher vertebrates. Fish lack parathyroid glands, but there have numerous attempts to identify and isolate PTH from fish. Materials and Methods: Polymerase chain reaction (PCR) was performed with primers based on preliminary data from the Joint Genome Institute database. PCR amplification was performed on genomic DNA isolated from Fugu rubripes. PCR products were purified and DNA was sequenced. All sequence was confirmed from more than one independently amplified PCR product. Multiple sequence alignments were carried out, and the percentage of identities and similarities were calculated. An unrooted phylogenetic tree, using all the known PTH and PTH-related protein (PTHrP) amino acid sequences, was determined. Synthetic peptides were tested in a biological assay that measured cyclic adenosine 3Ј,5Ј-monophosphate formation in UMR106.1 cells. Rabbit polyclonal antisera specific for N-terminal human PTHrP and one rabbit polyclonal antiserum specific for N terminus hPTH were used to test the cross-reactivity with fPTH(1-34) in immunoblots.

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“…It has been established that the 34 residues at the N terminus of the 84-amino acid protein, hPTH- , are sufficient for high affinity binding to the PTH receptor (a G-protein-coupled receptor) and retain PTH-like bone-related activities (2,3). Biological activity studies on hybrid peptides of PTH and the parathyroid hormone-related protein (PTHrP), which binds to and activates the same receptor, indicate a functional interaction between the 1-14 and 15-34 domains (4).…”

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Addressing the Tertiary Structure of Human Parathyroid Hormone-(1–34)

Pellegrini

Royo

Rosenblatt

et al. 1998

Journal of Biological Chemistry

130

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Parathyroid hormone (PTH) regulates mineral metabolism and bone turnover by activating specific receptors located on osteoblastic and renal tubular cells and is fully functional as the N-terminal 1-34 fragment, PTH-(1-34). Previously, a "U-shaped" conformation with Nand C-terminal helices brought in close proximity by a turn has been postulated. The general acceptance of this hypothesis, despite limited experimental evidence, has altered the direction of the design of PTH-analogs. Examining the structure of human PTH-(1-34) under conditions that encompass the different environments the hormone may experience in the approach to and interaction with the G-protein-coupled receptor (including benign aqueous and saline solutions and in the presence of dodecylphosphocholine), we observe no evidence for a U-shape conformation or any tertiary structure. Instead, the N-and C-terminal helical domains, which vary in length and stability depending on the condi-

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Parathyroid Hormone (PTH)-PTH-related Peptide Hybrid Peptides Reveal Functional Interactions between the 1–14 and 15–34 Domains of the Ligand

Cited by 60 publications

References 28 publications

Converting Parathyroid Hormone-related Peptide (PTHrP) into a Potent PTH-2 Receptor Agonist

Converting Parathyroid Hormone-related Peptide (PTHrP) into a Potent PTH-2 Receptor Agonist

Identification of a Parathyroid Hormone in the Fish Fugu rubripes

Addressing the Tertiary Structure of Human Parathyroid Hormone-(1–34)

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