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

Abstract

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...

Binding specificity of the ectodomain of the parathyroid hormone receptor

“…This is consistent with the results of an Ala scanning analysis described by Dean et al [31] and of the recently published crystal structure of a PTH (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) peptide in complex with the PTH1R ectodomain [12]. Here, we extend these investigations by testing the whole PTH ligand for interactions.…”

“…Furthermore, this analysis showed that nPTH1R binding requires Arg20 and Trp23 in a certain distance [28]. This could be the reason for a slightly detectable binding to a stretch of the reversed sequence of PTH (34-1) where…”

Multiple Sites of Contact between the Carboxyl-terminal Binding Domain of PTHrP-(1–36) Analogs and the Amino-terminal Extracellular Domain of the PTH/PTHrP Receptor Identified by Photoaffinity Cross-linking

“…In contrast, other mutationally intolerant ligand residues nearby (e.g. at positions 23, 28, and 31 (16,17)) tolerated the Bpa substitution with no apparent reduction in binding affinity. At position 23, Bpa replaced a tryptophan, and the structural similarity between these two residues may explain why the substitution was tolerated.…”

“…1 below). Ligand residue 23 (Phe in PTHrP and Trp in PTH) has been found to be important for high affinity binding to the P1R and to the PTH-2 receptor (16,17). Replacement of this residue with a Bpa photoreactive group results in affinity cross-linking to residues 23-40 of the rat P1R, at the extreme end of the amino-terminal extracellular domain.…”

Anatomical and physiological evidence for involvement of tuberoinfundibular peptide of 39 residues in nociception

“…It is present at greatest levels in the nervous system, and unlike the PTH1 receptor, it is found at low density and in only a few cells in kidney and bone (1,6,7). PTH-related peptide has low affinity for PTH2 receptors, and does not significantly activate them (8). Unlike the human PTH2 receptor, which was initially characterized and led to the receptor's name, rat and zebrafish PTH2 receptors are poorly activated by PTH (9,10).…”