Dataset Information

Proteolytic Cleavage of the Extracellular Domain Affects Signaling of Parathyroid Hormone 1 Receptor.

ABSTRACT: Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G_s and increased activation of G_q compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.

SUBMITTER: Klenk C

PROVIDER: S-EPMC8902639 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Proteolytic Cleavage of the Extracellular Domain Affects Signaling of Parathyroid Hormone 1 Receptor.

Klenk Christoph C Hommers Leif L Lohse Martin J MJ

Frontiers in endocrinology 20220222

Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage <i>in vivo</i> that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain u ...[more]

PMID: 35273573

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Project description:IntroductionReceptor activity-modifying proteins (RAMPs) are known to modulate the pharmacology and function of several G-protein-coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R). However, the precise effects of different RAMPs on PTH1R signalling and trafficking remain poorly understood. This study investigated the impact of RAMP2 and RAMP3 on PTH1R function using a range of PTH and PTH-related protein (PTHrP)-derived ligands.MethodsWe employed FRET imaging to assess PTH1R interactions with RAMPs. Cell surface expression of PTH1R was evaluated in the presence of RAMPs. PTH1R-mediated cAMP accumulation, β-arrestin recruitment, and calcium signalling were measured in response to various ligands. Antibody-capture scintillation proximity assays were used to examine G-protein activation patterns.ResultsPTH1R preferentially interacted with RAMP2 and, to a lesser extent, RAMP3, but not with RAMP1. RAMP3 co-expression reduced cell surface expression of PTH1R. RAMP2 significantly enhanced PTH1R-mediated signalling responses to PTH (1-34), PTHrP (1-34), PTH (1-84), and PTH (1-17) analogue ZP2307, while RAMP3 co-expression attenuated or abolished these responses. Full-length PTHrP analogues exhibited lower potency and efficacy than PTHrP (1-34) in activating PTH1R. RAMP2 increased the potency and/or efficacy of these analogues, whereas RAMP3 reduced these responses. RAMP2 differentially modulated G-protein activation by PTH1R in a ligand-dependent manner, with PTH (1-34) and PTHrP (1-34) inducing distinct patterns of G-protein subtype activation.DiscussionThese findings highlight the complex role of RAMPs in regulating PTH1R signalling and trafficking, revealing differential effects of RAMP2 and RAMP3 on receptor function. The data suggest that targeting the PTH1R/RAMP2 complex may be a promising strategy for developing novel bone anabolic therapies by leveraging biased agonism and functional selectivity. Further research using physiologically relevant models is needed to elucidate the therapeutic potential of this approach.

Dataset Information

Proteolytic Cleavage of the Extracellular Domain Affects Signaling of Parathyroid Hormone 1 Receptor.

Publications

Proteolytic Cleavage of the Extracellular Domain Affects Signaling of Parathyroid Hormone 1 Receptor.

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