Binding Selectivity of Abaloparatide for PTH-Type-1-Receptor Conformations and Effects on Downstream Signaling

Abstract

The PTH receptor type 1 (PTHR1) mediates the actions of two endogenous polypeptide ligands, PTH and PTHrP, and thereby plays key roles in bone biology. Based on its capacity to stimulate bone formation, the peptide fragment PTH (1-34) is currently in use as therapy for osteoporosis. Abaloparatide (ABL) is a novel synthetic analog of human PTHrP (1-34) that holds promise as a new osteoporosis therapy, as studies in animals suggest that it can stimulate bone formation with less of the accompanying bone resorption and hypercalcemic effects that can occur with PTH (1-34). Recent studies in vitro suggest that certain PTH or PTHrP ligand analogs can distinguish between two high-affinity PTHR1 conformations, R(0) and RG, and that efficient binding to R(0) results in prolonged signaling responses in cells and prolonged calcemic responses in animals, whereas selective binding to RG results in more transient responses. As intermittent PTH ligand action is known to favor the bone-formation response, whereas continuous ligand action favors the net bone-resorption/calcemic response, we hypothesized that ABL binds more selectively to the RG vs the R(0) PTHR1 conformation than does PTH (1-34), and thus induces more transient signaling responses in cells. We show that ABL indeed binds with greater selectivity to the RG conformation than does PTH (1-34), and as a result of this RG bias, ABL mediates more transient cAMP responses in PTHR1-expressing cells. The findings provide a plausible mechanism (ie, transient signaling via RG-selective binding) that can help account for the favorable anabolic effects that ABL has on bone.

Figure 1.

PTH and PTHrP ligand analogs and binding to the ECD. A, Shown are the amino acid sequences of the ligands assessed in this study. Residues native to human PTH are colored blue, residues native to human PTHrP are colored green, substituted residues in LA-PTH are colored purple, and substituted residues in ABL are colored red. Amino acids are indicated in one-letter code, X indicates Aib. B, Structural model of the PTHR1-ECD region (gray) in complex with the (13–34) segments of PTHrP (gold), superimposed with the 13–34 region ABL (magenta with side chains of substituted residues: F22E, F23L, H25E, H26K, I28L, E30K, and I31L in atomic CPK colors; the A29Aib substitution is not depicted). The model is derived from the crystal structure of the PTHR1-ECD•PTHrP (13–34) complex (14). The enlargement of the boxed area identifies five of the PTHrP residues substituted in ABL.

Figure 2.

Ligand binding to the RG and R⁰ conformations of the PTHR1. The binding of ABL, PTH (1–34), PTHrP (1–36) and LA-PTH to two distinct PTHR1 conformations RG (A) and R⁰ (B) was assessed by competition methods in membranes prepared from GP-2.3 cells stably expressing the PTHR1. RG reactions used ¹²⁵I-M-PTH (1–15) as tracer radioligand, which binds selectively to the G protein–coupled receptor conformation (RG). R⁰ reactions used ²⁵I-PTH (1–34) as tracer radioligand and contained an excess concentration (1 × 10⁻⁵ M) of GTPγS, which enriches for the G protein–uncoupled receptor conformation (R⁰). Data are means (± SEM) of six experiments, each performed in duplicate. Curve fitting parameters are reported in Table 1.

Figure 3.

Ligand potency for cAMP signaling in GP-2.3 cells. The potency of ligand-induced cAMP signaling was assessed in GP-2.3 cells, which are HEK-293-derived cells that were stably transfected to express the luciferase-based glosensor cAMP reporter as well as the human PTHR1. The cells were preloaded with luciferin and then treated with varying concentrations of ABL, PTH (1–34), PTHrP (1–36), or LA-PTH, and luminescence was assessed. At each ligand dose, the peak luminescence response, which typically occurred 10–15 minutes after ligand addition (luminescence was assessed at 2-min intervals for ∼60 min), was plotted vs ligand concentration. Data are means (± SEM) of five or four (LA-PTH) experiments, each performed in duplicate. Curve fitting parameters are reported in Table 2.

Figure 4.

Duration of PTH ligand-induced cAMP-signaling responses in GP-2.3 cells. GP-2.3 cells preloaded with luciferin were treated with a near-EC₅₀ concentration of PTH (1–34) (0.3nM), PTHrP (1–36) (1nM), ABL (0.1nM), or LA-PTH (0.3nM) and luminescence was assessed at 2-min intervals for 14 min (A). The cells were then removed from the plate reader, rinsed twice with media to remove unbound ligand, and then fresh media containing luciferin but lacking ligand was added and luminescence was assessed for additional 150 min (B). Data are means (± SEM) of six experiments, each performed in triplicate. Curve fitting parameters are reported in Table 3.

Figure 5.

Ligand potency for ERK-1/2 signaling in GP-2.3 cells. The potency of ligand-induced signaling via the ERK-1/2 pathway was assessed in GP-2.3 cells. The cells were treated with varying concentrations of ABL, PTH (1–34), PTHrP (1–36), or LA-PTH for 5 min, and then lysed and the levels of phosphorylated ERK-1/2 proteins in the lysates were assessed using the antibody-based Surefire assay system. At each ligand dose, the peak fluorescence signal observed was plotted vs ligand concentration. Data are means (± SEM) of three experiments, each performed in duplicate.