Minimization of Parathyroid Hormone

Abstract

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 3 Ala, Asn 10 3 Ala or Gln, Leu 11 3 Arg, Gly 12 3 Ala, His 14 3 Trp) enhanced activity 2-10-fold. These effects were additive, as [Ala In mammals, parathyroid hormone (PTH) 1 plays a vital role in regulating blood calcium concentrations, and PTH-related peptide (PTHrP) plays a critical role in the development of the fetal skeleton (1). The biological actions of both of these peptides are mediated by the PTH/PTHrP receptor (or PTH-1 receptor) (2), a family B G protein-coupled receptor (3) that strongly activates the adenylyl cyclase/protein kinase A-signaling cascade (2), and more weakly the phospholipase C protein kinase C-signaling pathway (4). The mechanisms by which parathyroid hormone and PTHrP bind to the PTH-1 receptor and induce receptor activation are poorly understood but appear to involve multiple sites of intermolecular interaction. Early studies of PTH fragment analogs assigned the major determinants of receptor-binding affinity and cAMP-stimulating potency to the COOH-terminal and NH 2 -terminal portions of the fully active PTH(1-34) peptide, respectively (5, 6). PTH(1-34)-based analogs with NH 2 -terminal deletions, such as PTH(3-34) and PTH(7-34), bind efficiently to the receptor and are severely defective in stimulating a cAMP response; such peptides thus function as PTH-1 receptor antagonists (7-9). The dominant role that the NH 2 -terminal residues of PTH and PTHrP play in receptor activation is further reflected by their high level of evolutionary conservation.The anabolic effects of PTH on bone density (10, 11) have prompted considerable interest in the development of new PTH-1 receptor agonist analogs. Recently PTH(1-28) was shown to be an effective agonist for cAMP production in cellbased assays, although potency was ϳ10-fold reduced from that of PTH(1-34) (12, 13). Recently we found that in COS-7 or LLC-PK1 cells transfected with high levels of rat or human PTH-1 receptors, a fragment as short as PTH(1-14) elicited ϳ20-fold increases in cAMP formation levels (14). Although the potency of PTH(1-14) in these transfected cells was weak compared with PTH(1-34) (EC 50 ϭ 1 nM and 100 M, respectively), the response was sufficient for us to perform an initial structure-activity relationship analysis...

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

“…Consistent with these mutational data, Bpa introduced at position 1 of PTH- or position 2 of either PTH- or PTHrP-(1-36) was found to cross-link to methionine 425, at the extracellular end of TM6 in the P1R (9,10). Interactions between the aminoterminal portion of the ligand and the transmembrane domains/extracellular loops of the receptor are also suggested by a study showing that PTH- (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) can stimulate cAMP accumulation in a mutant P1R missing most of the amino-terminal extracellular domain as efficiently as it does in the wild-type P1R (11). Furthermore, if a PTH fragment comprising the first 9 amino acids is covalently attached to the amino-terminal end of such a truncated receptor, the resulting ligand-receptor chimera displays constitutive activity, indicative of an intramolecular stimulation of the receptor's activation domain by the tethered ligand fragment (12).…”

Backbone-methylated Analogues of the Principle Receptor Binding Region of Human Parathyroid Hormone

“…This analogue has a helix-bend-helix structure in aqueous media at near-physiological pH and ionic strength (14), similar to that found in previous studies of PTH-(1-34) (13). A C-terminal amphiphilic ␣-helix, extending from Ser 17 to Gln 29 , is the major structure within this receptorbinding region, and this structure is thought to bind via its hydrophobic face to the receptor (21)(22)(23). Thus, binding and AC-stimulating activities are very sensitive to replacement of residues on the nonpolar face of this helix, including Leu 24 , Leu 28 , and to a lesser extent Val 31 , whereas residues on the polar face are less sensitive to substitution (22,24).…”

“…A deletion analogue of the P1R (P1R-delNT) that lacks the N-terminal domain (Ref. 16 and citations, therein) can activate the AC and PLC signaling pathways on stimulation with PTH-(1-34) (17), although with much reduced potency, whereas N-terminal PTH analogues as short as PTH-(1-11) exhibit equivalent potencies on P1R-delNt and the intact P1R (16,17). The presence of PLC-independent protein kinase C activity determinants in the C-terminal region of PTH- implies that this region also binds to the extracellular loop/transmembrane domain region of the receptor, but only one report directly supports such an interaction (18).…”

Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism

“…Extensive studies have demonstrated that [Aib 1,3 , M]PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) and other PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) analogs (8,(19)(20)(21) mainly, if not exclusively, bind to the PTHR J-domain in contrast to PTH(1-34), which binds to both receptor's N-and J-domains (22). When [Aib 1,3 , M]PTH (1-14) is bound, it prevents PTH analogs from occupying their binding site at the J-domain of the receptor (8,18).…”