Structural Basis for Parathyroid Hormone-related Protein Binding to the Parathyroid Hormone Receptor and Design of Conformation-selective Peptides

Abstract

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic ␣-helix to the same hydrophobic groove in the ECD as occupied by PTH, but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally "unwound." The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECDbinding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics. The parathyroid hormone receptor (PTH1R)3 is a class B G protein-coupled receptor (GPCR) that transduces signals from two related signaling molecules that have distinct functions in biology: parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) (Ref. 1; reviewed in Ref. 2). PTH is an 84-amino acid polypeptide endocrine hormone that is produced by the parathyroid glands and secreted into the circulation in response to low calcium levels (reviewed in Refs. 3-5), to act on bone and kidney cells and thus restore blood calcium to normal levels. In bone, PTH directly stimulates osteoblasts, resulting in bone formation (reviewed in Ref. 6), which in turn activate osteoclasts to induce bone resorption. In the kidney, PTH stimulates the reabsorption of filtered calcium, inhibits the reabsorption of phosphate, and stimulates the synthesis of 1,25-dihydroxyvitamin D3. The paradoxical anabolic/ catabolic actions of PTH on bone can be modulated by exogenous PTH, and provide the molecular basis for the clinical use of PTH as an anabolic therapy for osteoporosis (7). Anabolic PTH therapy requires intermittent administration to minimize bone-resorptive effects, which predominate with sustained administration of PTH. PTHrP is a 141-amino acid polypeptide that was originally isolated as the factor responsible for humoral hyperca...

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

“…Whether such a divergence in binding modes correlates with some of the differences in functional responses that have been observed for the two ligands in cell-based kinetic binding and signaling assays (Dean et al, 2008) is unclear. For both ligands, the guanidinium side-chain makes extensive, mostly polar contacts with a cluster of receptor residues that form a small pocket at the proximal end of the groove Pioszak et al, 2009). The elaborate set of interactions made by the Arg20 side chain is fully consistent with the high level of evolutionary conservation seen for this residue, as arginine is present at this position in all PTH family ligands studied to date (Fig.…”

“…The mode of binding observed for PTHrP was found to be highly similar that for PTH, such that the new structural data combined were fully consistent with and indeed confirmed the prior functional studies that indicated that, despite the glaring sequence variation, the (15-34) domains of PTH and PTHrP bind to the same or at least largely overlapping sites in the receptor (Jüppner et al, 1988;Caulfield et al, 1990). The cocrystal structure thus shows that the PTHrP domain binds as an amphipathic a-helix with ligand residues Phe23, Leu24, and Ile28 along one helical surface forming extensive hydrophobic interactions with receptor residues lining the groove of the ECD (Pioszak et al, 2009). A slight bend, however, is present in the PTHrP helix at position-27, which leads to a modest divergence in the receptor contacts made by the C-terminal portions of the two ligands.…”

“…Soon after the PTH•PTHR1-ECD structure was solved, the same group reported the crystal structure of the PTHR1 ECD in complex with the binding domain fragment of PTHrP (Pioszak et al, 2009). The mode of binding observed for PTHrP was found to be highly similar that for PTH, such that the new structural data combined were fully consistent with and indeed confirmed the prior functional studies that indicated that, despite the glaring sequence variation, the (15-34) domains of PTH and PTHrP bind to the same or at least largely overlapping sites in the receptor (Jüppner et al, 1988;Caulfield et al, 1990).…”

“…The receptor thus has a relatively large amino ECD of about 160 amino acids (minus the 23 amino acids of the N-terminal signal sequence) that are involved in initial ligand binding, the seven helical transmembrane domains and connecting loops that mediate agonist-induced receptor activation and signal transduction events, and a C-terminal tail of about 130 amino acids that contains sites involved in mediating ligand-induced receptor internalization, trafficking, and signal termination events. Key specific amino acids identified include the four pairs of extracellular cysteine (C) residues that form a disulfide bond network that is conserved in the family B GPCRs and maintains receptor structure and function (Lee et al, 1994;Pioszak et al, 2009); four glycosylated asparagine (N) residues in the ECD (Zhou et al, 2000); Thr33 and Gln37, which modulate interaction with tryptophan-23 in the ligand (Mannstadt et al, 1998;Mann et al, 2008); Phe184 and Arg186, which mediate interactions involving ligand residues at or near lysine-13 Carter et al, 1999a); Ser370, Ile371, Met425, Trp437, and Gln440, which contribute interactions involving ligand residues at or near valine-2 and likely play a role in receptor activation Lee et al, 1995;Bisello et al, 1998;Behar et al, 1999;Gensure et al, 2001a); Arg233 and Gln451, which participate in an interhelical interaction network (dashed connectors) that likely helps modulate PTHR activation (Gardella et al, 1996a) and is conserved in the family B GPCRs (Hollenstein et al, 2013); conserved Pro132 in the ECD, which is the site of an inactivating mutation (Leu) in Blomstrand's chondrodysplasia ; His223, Thr410, and Ile458, at which mutations result in constitutive signaling activity and in patients result in Jansen's chondrodysplasia (Schipani et al, 1999); Lys319, at which mutations impair Ga q signaling (Iida-Klein et al, 1997); Lys388, at which mutations impair Ga q and Ga s signaling (Huang et al, 1996). Key residues in the C-tail include the seven serine (S) residues that are phosphorylated upon agonist activation and mediate recruitment of b-arrestins (Malecz et al, 1998;Qian et al, 1998;Tawfeek et al, 2002;Vilardaga et al, 2002;Rey et al, 2006) and the C-terminal ETVM sequence that mediates interaction with the NHERF family of proteins (Mahon et al, 2002(Mahon et al, , 2003Ardura...…”

A tagged parathyroid hormone derivative as a carrier of antibody cargoes transported by the G protein coupled PTH1 receptor

“…Recent crystal structures of agonist-bound PTH 1 R extracellular domain revealed that the Cterminal hormone residue does not form direct contacts with the receptor [16]. This made possible two recent biotechnological forms of PTH 1-34 -based agonists elongated at their Cterminus, either with a linker and trans-membrane tether in one case [6,7], or in the other, with the green fluorescent protein (GFP), leading to a high molecular weight probe suitable for imaging studies in cells that express recombinant PTH 1 R [3] (schematic representation, Fig.…”