PTH receptor-1 signalling-mechanistic insights and therapeutic prospects

Abstract

Parathyroid hormone/parathyroid hormone-related protein receptor (PTH/PTHrP type 1 receptor; commonly known as PTHR1) is a family B G-protein-coupled receptor (GPCR) that regulates skeletal development, bone turnover and mineral ion homeostasis. PTHR1 transduces stimuli from PTH and PTHrP into the interior of target cells to promote diverse biochemical responses. Evaluation of the signalling properties of structurally modified PTHR1 ligands has helped to elucidate determinants of receptor function and mechanisms of downstream cellular and physiological responses. Analysis of PTHR1 responses induced by structurally modified ligands suggests that PTHR1 can continue to signal through a G-protein-mediated pathway within endosomes. Such findings challenge the longstanding paradigm in GPCR biology that the receptor is transiently activated at the cell membrane, followed by rapid deactivation and receptor internalization. Evaluation of structurally modified PTHR1 ligands has further led to the identification of ligand analogues that differ from PTH or PTHrP in the type, strength and duration of responses induced at the receptor, cellular and organism levels. These modified ligands, and the biochemical principles revealed through their use, might facilitate an improved understanding of PTHR1 function in vivo and enable the treatment of disorders resulting from defects in PTHR1 signalling. This Review discusses current understanding of PTHR1 modes of action and how these findings might be applied in future therapeutic agents.

Figure 1

Altered modes of cAMP signalling at PTHR1. Structurally distinct PTH and PTHrP ligands can bind preferentially to two different high affinity receptor conformations, R⁰ and R^G, and thereby induce different modes of Gα_S-mediated cAMP signalling (inset, lower panel). R^G-selective ligands (for example PTHrP) induce transient cAMP responses that are derived from signalling complexes localized at the plasma membrane, whereas R⁰-selective ligands (for example certain PTH analogues^,) can also induce prolonged cAMP responses that are derived from complexes associated within endosomes. The internalized signalling complexes contain β-arrestin, which promotes, rather than terminates cAMP signalling by activating ERK1/2, leading to the inhibition of PDE4 enzymes. Termination of endosomal signalling correlates with an exchange at the complex of β-arrestin for retromer sorting proteins, and is promoted by vATPase-mediated vesicle acidification. The vATPases are activated by cAMP-dependent PKA, and thereby establish a negative feedback loop. PTHR1 activation of cAMP signalling that differs in duration and location of origin within the cell provides a potential mechanism for ligand-directed diversification of cellular responses.^– Abbreviations: ERK1/2, extracellular signal-regulated protein kinase 1/2; PDE4, phosphodiesterase 4; PKA, protein kinase A; PTH, parathyroid hormone; PTHR1, PTH/PTHrP type 1 receptor; PTHrP, parathyroid hormone-related protein; R⁰, G-protein-independent conformational state; R^G, G-protein-dependent conformational state; vATPase, vacuolar H⁺-ATPase.

Figure 2

Distinct mechanisms of prolonged PTH analogue action in vivo. Both the receptor conformation-selective PTH analogue, M-PTH(1–34), and the backbone-modified, stabilized PTH analogue, D6, stimulate prolonged calcaemic responses when injected into mice as compared with unmodified PTH(1–34), but the underlying mechanisms are distinct, as reflected by markedly different pharmacokinetic profiles. Injection with either a | M-PTH(1–34) or c | D6 results in an increase in blood Ca²⁺ concentrations that persists longer than the response induced by an identical dose of PTH(1–34); however, b | M-PTH(1–34) disappears more rapidly from the bloodstream than does PTH(1–34) (intravenous injection), whereas d | D6 disappears more slowly than does PTH(1–34) (subcutaneous injection). These results can best be explained by the capacity of M-PTH(1–34) to bind in a pseudo-irreversible fashion to a specific PTHR1 conformation (R⁰) in target cells, and to an enhanced resistance of D6 to degradation by systemic proteases. Blood concentrations of PTH ligands were determined by cAMP-based bioassays; data are means ± SEM. Abbreviations: D6, backbone-modified PTH analogue; M–PTH(1–34), modified parathyroid hormone (1–34); PTH, parathyroid hormone; PTHR1, PTH/PTHrP type 1 receptor; PTHrP, parathyroid hormone-related protein; R⁰, G-protein-independent conformational state. Panels a and b modified with permission from The National Academy of Sciences © Okazaki, M. et al. Proc. Natl Acad. Sci. USA 105, 16525–16530 (2008). Panels c and d modified with permission from NPG © Cheloha, R. W. et al. Nat. Biotech. 32, 653–655 (2014).