Location Bias in cAMP Signaling and its effects on Parathyroid Hormone Receptor Biological Effectiveness

Abstract

The parathyroid hormone (PTH) type 1 receptor (PTHR) is a class B G protein-coupled receptor (GPCR) that regulates mineral-ion, vitamin D, and bone homeostasis. PTH-induced activation of the PTHR results in both transient and sustained cAMP production, from the plasma membrane and endosomes, respectively; however, it is not clear whether the spatial (location) or temporal (duration) components of cAMP signaling result in distinct biological outcomes. To answer this question, we generated a location biased ligand via epimerization of a single amino acid in PTH (PTH7d). Here we show that PTH7d induces sustained cAMP responses exclusively from the plasma membrane that are very similar to those observed for PTH1-34 and a long-acting PTH analog (LA-PTH), two previously developed synthetic PTHR ligands that trigger sustained cAMP signaling from endosomes. PTH7d location bias occurs as a result of a unique active PTHR conformation that triggers sustained cAMP signaling exclusively from the plasma membrane due to impaired β-arrestin (βarr) coupling to the receptor. We further demonstrate the physiological importance of subcellular signaling location by the PTHR, as studies in mice show that sustained cAMP from endosomes is required for PTHR-mediated elevations in serum Ca2+ and active vitamin D levels. Additionally, assays in polarized epithelial cells reveal that endosomal cAMP signaling is a determinant for renal upregulation of the rate-limiting hydroxylase that catalyzes the formation of active vitamin D. Together, these results advance our understanding of how location of GPCR signaling can regulate particular biological functions and add new insights into drug design based upon spatiotemporal manipulation of GPCR signaling.