Role of Amino Acid Side Chains in Region 17–31 of Parathyroid Hormone (PTH) in Binding to the PTH Receptor

Abstract

Parathyroid hormone (PTH)2 plays a key role in calcium and phosphate homeostasis and has potent effects on the bone-remodeling process. PTH interacts with a Class 2 G protein-coupled receptor that is prominently expressed in bone osteoblasts and in cells located in the proximal and distal portions of the renal convoluted tubules. The PTH receptor (PTHR) is also expressed in the primordia of developing long bones, heart, mammary glands, and other tissues, where it mediates the morphogenic actions of PTH-related protein (PTHrP) (1).For both PTH and PTHrP, the bioactive portions of the molecule reside within the first 34 amino acids of the processed polypeptides. Within region 1-34, the principal determinants of receptor-binding affinity and receptor-signaling activity map to the C-and N-terminal domains, respectively (2, 3). Solutionphase NMR studies of PTH-(1-34)-based ligands typically show a well formed ␣-helix in the region of the C-terminal binding domain (4,5 , and Leu 28 , which form the hydrophobic face of this ␣-helix, are particularly important for efficient interaction with the receptor (9 -11).The mechanism by which PTH interacts with its receptor has been investigated via the approaches of ligand analog design, receptor mutagenesis, and photochemical cross-linking (reviewed in Ref. 12). The view that has emerged from these studies is that the overall mechanism consists of two principal and, to some extent, autonomous components: 1) an interaction between the C-terminal helical domain of the ligand and the N-terminal extracellular domain (N domain; spanning Tyr 23 to approximately Ile 190 ) of the mature receptor and 2) an interaction between the N-terminal portion of the ligand and the juxtamembrane domain (J domain) of the receptor containing the extracellular loops and seven transmembrane helices. The N domain component of the interaction is thought to provide the major portion of binding energy and stability to the complex, and the J domain component is thought to mediate the conformational changes involved in receptor activation (13). It now seems likely that most, if not all, of the 15 or so other Class 2 G protein-coupled receptors utilize a similar two-site binding mechanism for interacting with their cognate peptide ligands (14 -16).Consistent with such a two-site binding mechanism for PTH and the PTHR, we have shown that N-terminal PTH peptide fragments, such as PTH-(1-14), bind only extremely weakly to the receptor, but can nevertheless induce at least measurable increases in cAMP levels in PTHR-expressing cells (17). The potency of such N-terminal PTH fragments can be significantly enhanced by introducing substitutions that improve the affinity

Molecular recognition of parathyroid hormone by its G protein-coupled receptor

“…In particular, replacement of the four core residues (R20, W23, L24, and L28) with alanine markedly reduced PTH binding, with mutations in the three hydrophobic residues nearly abolishing PTH binding to the ECD. Our data are in good agreement with the recent study of alanine-scanning of PTH-(17-31) (29). These results thus further support the detailed molecular interactions observed in our crystal structure.…”

“…Crystals of the purified MBP-PTH1R-ECD bound to a synthetic PTH fragment (residues [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34] were grown in hanging drops containing a reservoir solution of 100 mM sodium cacodylate (pH 6.5) and 30% (vol/vol) polypropylene glycol P400 (PPG P400). Diffraction data were collected at 21-ID-D (LS-CAT) of the Advanced Photon Source at Argonne National Laboratory (Argonne, IL).…”

“…Studies with ligand variants and receptor/ligand photo cross-linking indicate that the Nterminal fragment of PTH (residues 1-14) binds to the transmembrane domain, albeit with low affinity, and that it is able of activating the receptor (26,27). The C-terminal fragment of PTH (residues 15-34) binds to the N-terminal ECD of the receptor to confer high affinity and specificity to the receptor (28,29). This ''two-domain'' model of PTH binding and activation is further supported by studies with chimeric ligands and receptors (30)(31)(32) and has since been demonstrated for other class B GPCR molecules (33)(34)(35).…”

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.…”

International Union of Basic and Clinical Pharmacology. XCIII. The Parathyroid Hormone Receptors—Family B G Protein–Coupled Receptors

“…6B). Moreover, structureactivity relationship studies focused on this position have shown that even structurally related amino acid substitutions have significant and deleterious impacts on receptor binding affinity (Barbier et al, 2001;Dean et al, 2006a). Thus the contacts identified in the crystal structures between the Arg20 side chain and the receptor must play a key and critical role in docking the ligand to the ECD and/or maintaining the stability of the ligand/ receptor complex once it is formed.…”