Protocol to characterize Gi/o and Gs protein-coupled receptors in transiently transfected cells using ELISA and cAMP measurements.

Activation of Gs or Gi/o protein-coupled receptors (GPCRs) leads to changes of intracellular cyclic adenosine monophosphate (cAMP) levels. This protocol describes steps for cloning HA- and FLAG-tagged GPCRs, transient transfection of CHO-K1 or HEK293-T cells, and determination of basal and ligand-induced changes in intracellular cAMP levels. We detail enzyme-linked immunosorbent assays to determine relative GPCR plasma membrane and total expression levels. For complete details on the use and execution of this protocol, please refer to Schulze et al. (2022).1.

Evolutionary analyses reveal immune cell receptor GPR84 as a conserved receptor for bacteria-derived molecules.

The G protein-coupled receptor 84 (GPR84) is found in immune cells and its expression is increased under inflammatory conditions. Activation of GPR84 by medium-chain fatty acids results in pro-inflammatory responses. Here, we screened available vertebrate genome data and found that GPR84 is present in vertebrates for more than 500 million years but absent in birds and a pseudogene in bats. Cloning and functional characterization of several mammalian GPR84 orthologs in combination with evolutionary and model-based structural analyses revealed evidence for positive selection of bear GPR84 orthologs. Naturally occurring human GPR84 variants are most frequent in Asian populations causing a loss of function. Further, we identified cis- and trans-2-decenoic acid, both known to mediate bacterial communication, as evolutionary highly conserved ligands. Our integrated set of approaches contributes to a comprehensive understanding of GPR84 in terms of evolutionary and structural aspects, highlighting GPR84 as a conserved immune cell receptor for bacteria-derived molecules.

Combining metabolic phenotype determination with metabolomics and transcriptional analyses to reveal pathways regulated by hydroxycarboxylic acid receptor 2.

BACKGROUND: The adaptation of cellular metabolism is considered a hallmark of cancer. Oncogenic signaling pathways support tumorigenesis and cancer progression through the induction of certain metabolic phenotypes associated with altered regulation of key metabolic enzymes. Hydroxycarboxylic acid receptor 2 (HCA2) is a G protein-coupled receptor previously shown to act as a tumor suppressor. Here, we aimed to unveil the connection between cellular metabolism and HCA2 in BT-474 cells. Moreover, we intend to clarify how well this metabolic phenotype is reflected in transcriptional changes and metabolite levels as determined by global metabolomics analyses. METHODS: We performed both, siRNA mediated knockdown of HCA2 and stimulation with the HCA2-specific agonist monomethyl fumarate. Seahorse technology was used to determine the role of HCA2 in BT-474 breast cancer cell metabolism and its potential to induce a switch in the metabolic phenotype in the presence of different energy substrates. Changes in the mRNA expression of metabolic enzymes were detected with real-time quantitative PCR (RT-qPCR). Untargeted liquid chromatography-mass spectrometry (LC-MS) metabolic profiling was used to determine changes in metabolite levels. RESULTS: Knockdown or stimulation of HCA2 induced changes in the metabolic phenotype of BT474 cells dependent on the availability of energy substrates. The presence of HCA2 was associated with increased glycolytic flux with no fatty acids available. This was reflected in the increased mRNA expression of the glycolytic enzymes PFKFB4 and PKM2, which are known to promote the Warburg effect and have been described as prognostic markers in different types of cancer. With exogenous palmitate present, HCA2 caused elevated fatty acid oxidation and likely lipolysis. The increase in lipolysis was also detectable at the transcriptional level of ATGL and the metabolite levels of palmitic and stearic acid. CONCLUSIONS: We combined metabolic phenotype determination with metabolomics and transcriptional analyses and identified HCA2 as a regulator of glycolytic flux and fatty acid metabolism in BT-474 breast cancer cells. Thus, HCA2, for which agonists are already widely used to treat diseases such as psoriasis or hyperlipidemia, may prove useful as a target in combination cancer therapy.

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

G protein-coupled receptors (GPCRs) are key regulatory proteins of immune cell function inducing signaling in response to extracellular (pathogenic) stimuli. Although unrelated, hydroxycarboxylic acid receptor 3 (HCA3) and GPR84 share signaling via Gαi/o proteins and the agonist 3-hydroxydecanoic acid (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-1ß. 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.

Succinate receptor 1 inhibits mitochondrial respiration in cancer cells addicted to glutamine.

Cancer cells display metabolic alterations to meet the bioenergetic demands for their high proliferation rates. Succinate is a central metabolite of the tricarboxylic acid (TCA) cycle, but was also shown to act as an oncometabolite and to specifically activate the succinate receptor 1 (SUCNR1), which is expressed in several types of cancer. However, functional studies focusing on the connection between SUCNR1 and cancer cell metabolism are still lacking. In the present study, we analyzed the role of SUCNR1 for cancer cell metabolism and survival applying different signal transduction, metabolic and imaging analyses. We chose a gastric, a lung and a pancreatic cancer cell line for which our data revealed functional expression of SUCNR1. Further, presence of glutamine (Gln) caused high respiratory rates and elevated expression of SUCNR1. Knockdown of SUCNR1 resulted in a significant increase of mitochondrial respiration and superoxide production accompanied by an increase in TCA cycle throughput and a reduction of cancer cell survival in the analyzed cancer cell lines. Combination of SUCNR1 knockdown and treatment with the chemotherapeutics cisplatin and gemcitabine further increased cancer cell death. In summary, our data implicates that SUCNR1 is crucial for Gln-addicted cancer cells by limiting TCA cycle throughput, mitochondrial respiration and the production of reactive oxygen species, highlighting its potential as a pharmacological target for cancer treatment.

Natural biased signaling of hydroxycarboxylic acid receptor 3 and G protein-coupled receptor 84.

BACKGROUND: Medium-chain fatty acids and their 3-hydroxy derivatives are metabolites endogenously produced in humans, food-derived or originating from bacteria. They activate G protein-coupled receptors, including GPR84 and HCA3, which regulate metabolism and immune functions. Although both receptors are coupled to Gi proteins, share at least one agonist and show overlapping tissue expression, GPR84 exerts pro-inflammatory effects whereas HCA3 is involved in anti-inflammatory responses. Here, we analyzed signaling kinetics of both HCA3 and GPR84, to unravel signal transduction components that may explain their physiological differences. METHODS: To study the signaling kinetics and components involved in signal transduction of both receptors we applied the label-free dynamic mass redistribution technology in combination with classical cAMP, ERK signaling and ß-arrestin-2 recruitment assays. For phenotypical analyses, we used spheroid cell culture models. RESULTS: We present strong evidence for a natural biased signaling of structurally highly similar agonists at HCA3 and GPR84. We show that HCA3 signaling and trafficking depends on dynamin-2 function. Activation of HCA3 by 3-hydroxyoctanoic acid but not 3-hydroxydecanoic acid leads to ß-arrestin-2 recruitment, which is relevant for cell-cell adhesion. GPR84 stimulation with 3-hydroxydecanoic acid causes a sustained ERK activation but activation of GPR84 is not followed by ß-arrestin-2 recruitment. CONCLUSIONS: In summary, our results highlight that biased agonism is a physiological property of HCA3 and GPR84 with relevance for innate immune functions potentially to differentiate between endogenous, non-pathogenic compounds and compounds originating from e.g. pathogenic bacteria. Video Abstract.

Correction: Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3.

[This corrects the article DOI: 10.1371/journal.pgen.1008145.].

Metabolites of lactic acid bacteria present in fermented foods are highly potent agonists of human hydroxycarboxylic acid receptor 3.

The interplay of microbiota and the human host is physiologically crucial in health and diseases. The beneficial effects of lactic acid bacteria (LAB), permanently colonizing the human intestine or transiently obtained from food, have been extensively reported. However, the molecular understanding of how LAB modulate human physiology is still limited. G protein-coupled receptors for hydroxycarboxylic acids (HCAR) are regulators of immune functions and energy homeostasis under changing metabolic and dietary conditions. Most mammals have two HCAR (HCA1, HCA2) but humans and other hominids contain a third member (HCA3) in their genomes. A plausible hypothesis why HCA3 function was advantageous in hominid evolution was lacking. Here, we used a combination of evolutionary, analytical and functional methods to unravel the role of HCA3 in vitro and in vivo. The functional studies included different pharmacological assays, analyses of human monocytes and pharmacokinetic measurements in human. We report the discovery of the interaction of D-phenyllactic acid (D-PLA) and the human host through highly potent activation of HCA3. D-PLA is an anti-bacterial metabolite found in high concentrations in LAB-fermented food such as Sauerkraut. We demonstrate that D-PLA from such alimentary sources is well absorbed from the human gut leading to high plasma and urine levels and triggers pertussis toxin-sensitive migration of primary human monocytes in an HCA3-dependent manner. We provide evolutionary, analytical and functional evidence supporting the hypothesis that HCA3 was consolidated in hominids as a new signaling system for LAB-derived metabolites.