Ligand-Receptor Interactions at the Parathyroid Hormone Receptors: Subtype Binding Selectivity Is Mediated via an Interaction between Residue 23 on the Ligand and Residue 41 on the Receptor

Abstract

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) bind and activate the PTH/PTHrP receptor (PTH-1R). However, while the related receptor PTH-2R responds potently to PTH, it is not activated by PTHrP. Two hormone sites are known to be responsible for these different potencies. First, the absence of efficacy for PTHrP at PTH-2R is due to the presence of His-5 in PTHrP (Ile-5 in PTH), which interacts with the receptor's juxtamembrane domain. Second, PTHrP has lower affinity than PTH for PTH-2R because of the presence of Phe-23 (Trp-23 in PTH), which interacts with the receptor's N-terminal extracellular domain. We used these different receptor subtype properties to demonstrate that residue 41 in PTH-1R, when either the native Leu or substituted by Ile or Met, can accommodate either Phe or Trp at position 23 of the ligand. However, when Leu-41 is substituted by a smaller side chain, either Ala or Val (its equivalent residue in PTH-2R), the receptor becomes highly selective for those peptide ligands with Trp-23. Hence, despite the conservative nature of the substitutions found in the native ligands (Phe for Trp) and receptors (Leu for Val), they nevertheless enable a significant degree of selectivity to be achieved. Analysis of this functionally important ligand-receptor contact, within the context of the recent X-ray structure of the peptide-bound PTH-1R N domain, reveals the nature of the selectivity filter and how it is by-passed in PTH-1R.Parathyroid hormone (PTH) and parathyroid hormonerelated peptide (PTHrP) mediate many of their physiological effects via the same receptor, PTH-1R (Lanske et al., 1996). Although PTH has 84 residues and PTHrP has 141 residues, the biological effects they mediate via PTH-1R can be replicated by synthetic peptides equivalent to the first 34 amino acids of each peptide (e.g., Segre et al., 1979). These shorter analogs bind and activate PTH-1R in an manner analogous to that of the "two-site model" seen at other related family B G protein-coupled receptors (GPCRs) (Bergwitz et al., 1996;López de Maturana et al., 2003;Castro et al., 2005). In this model, the ligand's C-terminal region interacts with the receptor's N-terminal extracellular domain (N domain) to generate affinity. This first interaction positions the N-terminal region of the peptide such that it activates the receptor via a second interaction with the receptor's juxtamembrane "core" domain, comprising the transmembrane helices and connecting loops. The nature of the first interaction has been investigated via a number of mutagenic and cross-linking studies (Mannstadt et al., 1998;Gensure et al., 2001;Pham and Sexton, 2004) and more recently has been the focus of several structural studies that defined the structures of the peptidebound N domains of the receptors for corticotropin-releasing factor, pituitary adenylyl cyclase-activating polypeptide, glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1 (GLP-1) and, very recently, PTH itself (Grace et al.,

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

“…The Trp23 side-chain is also seen to be in close proximity to residues in the N-terminal a-helix, including Gln37 (Fig. 8), which is in agreement with prior cross-linking data with Bpa23-labeled PTH analogs as well as mutagenesis data that indicated proximity between these two sites (Mannstadt et al, 1998;Mann et al, 2008).…”

“…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...…”

Structure of the full-length glucagon class B G-protein-coupled receptor

“…While it remains to be determined what role the stalk region plays in the conformation of the peptide-bound active GCGR structure, it is clear that the observed orientation of the ECD in the mAb1-bound GCGR-FL crystal structure is not compatible with the class B GPCR two-state peptide binding model where the C-terminal region of the peptide ligand targets the ECD as observed in hormone-bound ECD crystal structures of class B GPCRs, and the N-terminal region of the peptide ligand targets the TMD binding pocket 2,5,16 (Extended Data Fig. 4c).…”

N-Terminal Phosphorylation of Parathyroid Hormone (PTH) Abolishes Its Receptor Activity

“…32 In the control experiment we examined the hPTH(1−84)-mediated cAMP signaling cascade via PTH1R ( Figure 4A) and PTH2R ( Figure 4B). This activation led to an elevation of the cAMP level inside the cells in a concentrationdependent fashion (black in Figure 4) similar to previously reported PTH1R receptor activation.…”