Abstract
BackgroundMedium-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.MethodsTo 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 β-arrestin-2 recruitment assays. For phenotypical analyses, we used spheroid cell culture models.ResultsWe 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 β-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 β-arrestin-2 recruitment.ConclusionsIn 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.9ZbwrhpX5Yj3XBGuPG7oW-Video .Graphical abstract
Highlights
Medium-chain fatty acids and their 3-hydroxy derivatives are metabolites endogenously produced in humans, food-derived or originating from bacteria
Results Cyclic adenosine monophosphate (cAMP) inhibitory signal of hydroxycarboxylic acid receptor 3 (HCA3) and GPR84 First, we confirmed that HCA3 and GPR84 induce cAMP inhibitory signaling in our experimental setup
Comparing signaling components involved upon stimulation of HCA3 and GPR84 with 3-hydroxydecanoic acid (3HDec), we found that while the HCA3-mediated extracellular-signal regulated kinase (ERK) activation is dependent on caveolin, PI3K, rac1 and ras/rho, the GPR84-mediated ERK activation only involves PI3K (Figs. 4b, 6a, c)
Summary
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. G protein-coupled receptors (GPCRs) activated by metabolites originating from diet, host- and microbiota metabolism gain more and more attention due to their role as regulators of the host (patho)-physiological state [1,2,3,4]. HCA3 and GPR84, are Gi protein-coupled receptors and expressed in immune cells, such as neutrophils, macrophages and monocytes [6, 7, 25,26,27]. HCA3 is rather suggested to elicit hypo-responsiveness of the immune system through mediation of anti-inflammatory processes [25, 28]
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