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

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.

The G protein α subunit variant XLαs promotes inositol 1,4,5-trisphosphate signaling and mediates the renal actions of parathyroid hormone in vivo.

GNAS, which encodes the stimulatory G protein (heterotrimeric guanine nucleotide-binding protein) α subunit (Gαs), also encodes a large variant of Gαs termed extra-large α subunit (XLαs), and alterations in XLαs abundance or activity are implicated in various human disorders. Although XLαs, like Gαs, stimulates generation of the second messenger cyclic adenosine monophosphate (cAMP), evidence suggests that XLαs and Gαs have opposing effects in vivo. We investigated the role of XLαs in mediating signaling by parathyroid hormone (PTH), which activates a G protein-coupled receptor (GPCR) that stimulates both Gαs and Gαq/11 in renal proximal tubules to maintain phosphate and vitamin D homeostasis. At postnatal day 2 (P2), XLαs knockout (XLKO) mice exhibited hyperphosphatemia, hypocalcemia, and increased serum concentrations of PTH and 1,25-dihydroxyvitamin D. The ability of PTH to reduce serum phosphate concentrations was impaired, and the abundance of the sodium phosphate cotransporter Npt2a in renal brush border membranes was reduced in XLKO mice, whereas PTH-induced cAMP excretion in the urine was modestly increased. Basal and PTH-stimulated production of inositol 1,4,5-trisphosphate (IP3), which is the second messenger produced by Gαq/11 signaling, was repressed in renal proximal tubules from XLKO mice. Crossing of XLKO mice with mice overexpressing XLαs specifically in renal proximal tubules rescued the phenotype of the XLKO mice. Overexpression of XLαs in HEK 293 cells enhanced IP3 generation in unstimulated cells and in cells stimulated with PTH or thrombin, which acts through a Gq/11-coupled receptor. Together, our findings suggest that XLαs enhances Gq/11 signaling to mediate the renal actions of PTH during early postnatal development.