GPCRs in health and disease

Kathleen M. Caron, University of North Carolina at Chapel Hill, USA

Illuminating Orphan GPCRs in Cardiovascular Development and Function

Since approximately 30% of all clinically-available drugs target G-protein coupled receptors (GPCRs), there is great interest in learning more about this family of proteins in order to discover new therapeutic targets. When considering the different classes of proteins encoded by the human genome that are predicted to be pharmacologically-tractable, a remarkable one quarter represent GPCRs. And yet, of the ~345 non-odorant GPCRs a large proportion remain orphan—with no known ligand—or have uncharacterized physiological functions. Since we have a dearth of pharmacological targets for lymphatic vessels, it stands to reason that focused efforts to explore and characterize the lymphatic “GPCRome” is a worthwhile endeavor. Using multimodal genomic approaches, we have identified 4 orphan GPCRs that have enriched expression in lymphatic endothelial cells: GPRC5B, GPR116, FZD8 and GPR61. Using cell-based and model organism approaches we have characterized the expression and functions of these receptors in the lymphatic vasculature during development and adulthood. Our results provide novel physiological insights into several orphan GPCRs and have the potential to illuminate therapeutic targets for the modulation of lymphatics, which remains an understudied research area with unmet clinical needs.

Karina A. Peña,

University of Pittsburgh, USA

Location Bias in cAMP and PTHR Biology

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). Our recent studies 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.

Mirja Harms,

Ulm University Medical Center, Germany

The CXCR4 inhibitor EPI-X4 – from discovery to drug design

The CXCR4/CXCL12 axis plays an important role for several processes in our body, including development, stem cell homeostasis and immune cell function. However, aberrant CXCR4/CXCL12 signaling is involved in diverse pathologies such as cancer and inflammatory diseases. The Endogenous Peptide Inhibitor of CXCR4 (EPI-X4) is a 16 amino acid long fragment of human serum albumin, which has previously been identified in our lab. The peptide specifically binds to CXCR4, blocks CXCL12-induced signaling and migration and acts as an inverse receptor agonist. EPI-X4 is a promising candidate for the development of improved analogues for the therapy of CXCR4-

associated diseases. We optimized the antagonistic activity of EPI-X4 by combining computational approaches and rational drug design. In addition, we applied different methods to prevent enzymatic degradation and to prolong systemic circulation time in vivo. Compared to the wild type peptide these newly developed EPI-X4 derivatives have a more than 1000-fold increased activity, are stable for several hours in blood plasma and circulation in vivo, and are therapeutically active in different mouse models of inflammatory diseases and cancer.

Mercedes Alfonso-Prieto, Forschungszentrum Jülich, Germany

Structure-function effects of a disease-linked variant of the oxytocin receptor

The oxytocin receptor (OXTR) is a GPCR involved in the regulation of multiple social and emotional behaviors. Hence, single nucleotide polymorphisms (SNPs) in the oxtr gene have been linked to several psychological traits. In particular, the non-synonymous SNP rs4686302, resulting in an A218T mutation in the OXTR protein, has been associated with autism spectrum disorder (ASD). Here we have analyzed the structural, functional and intracellular effects of the OXTR A218T variant by combining in vitro and in silico approaches. Structural modeling indicates that the mutant A218T OXTR is more stable than the wild-type, in line with protein degradation assays. The mutation is also predicted to alter the flexibility of transmembrane helix 5, which in turn could affect receptor activation. A systems biology-based model of the OXTR-mediated signaling cascade, integrating the aforementioned molecular information with calcium imaging data, further supports the impact of the A218T variant on receptor activation. Subsequent whole genome and RNA sequencing analyses reveal differentially regulated genes, some of which are associated with ASD. Altogether, integration of computational modeling and experimental data provides insights into the molecular and cellular mechanisms by which the OXTR rs4686302 SNP leads to ASD-associated genetic dysregulations.

References: Meyer et al. Mol. Psychiatry (2022), doi:10.1038/s41380-021-01241-8

Claudia Stäubert,

University Leipzig, Medical Faculty, Germany

Hydroxycarboxylic acid receptor 3 and GPR84 – two metabolite-sensing G protein-coupled receptors with opposing functions in innate immune cells

Authors: Anna Peters, Philipp Rabe, Aenne-Dorothea Liebing, Petra Krumbholz, Anders Nordström, Elisabeth Jäger, Robert Kraft, Claudia Stäubert

Affiliation: Rudolf Schönheimer Institute of Biochemistry, Faculty of Medicine, Leipzig University, Johannisallee 30, 04103 Leipzig, Germany

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G protein-coupled receptors (GPCRs) are key regulatory proteins of immune cell function inducing signaling in response to extracellular (pathogenic) stimuli. Hydroxycarboxylic acid receptor 3 (HCA3) and GPR84 share signaling via Gαi/o proteins and the agonist 3-hydroxydecanoate (3HDec). Both receptors are abundantly expressed in monocytes, macrophages and neutrophils but have opposing functions in these innate immune cells. Detailed insights into the molecular mechanisms and signaling components involved in immune cell regulation by GPR84 and HCA3 are still lacking.

Here, we report that GPR84-mediated pro-inflammatory signaling depends on coupling to the hematopoietic cell-specific Gα15 protein in human macrophages, while HCA3 exclusively couples to Gαi protein. We show that activated GPR84 induces Gα15-dependent ERK activation, increases intracellular Ca2+ and IP3 levels as well as ROS production. In contrast, HCA3 activation shifts macrophage metabolism to a less glycolytic phenotype, which is associated with anti-inflammatory responses. This is supported by an increased release of anti-inflammatory IL-10 and a decreased secretion of pro-inflammatory IL-1β. In primary human neutrophils, stimulation with HCA3 agonists counteracts the GPR84-induced neutrophil activation. Our analyses reveal that 3HDec acts solely through GPR84 but not HCA3 activation in macrophages.

In summary, this study shows that HCA3 mediates hyporesponsiveness in response to metabolites derived from dietary lactic acid bacteria and uncovers that GPR84, which is already targeted in clinical trials, promotes pro-inflammatory signaling via Gα15 protein in macrophages.


Peters A, Liebing AD, Rabe P, Krumbholz P, Nordström A, Jäger E, Kraft R, Stäubert C (2022) Hydroxycarboxylic acid receptor 3 and GPR84 – two metabolite-sensing G protein-coupled receptors with opposing functions in innate immune cells Pharmacol Res. Feb;176:106047

Peters A, Rabe P, Krumbholz P, Kalwa H, Kraft R, Schöneberg T, Stäubert C (2020) Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84 Cell Commun Signal. 18(1):31.

Peters A, Krumbholz P, Jäger E, Heintz-Buschart A, Çakir MV, Gaudl A, Ceglarek U, Schöneberg T, Stäubert C (2019) Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3. PLoS Genet. 15(5):e1008145