Abstract
FPR2, a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to β-arrestin recruitment. β-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of β-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent β-arrestin/AP2 interaction and β-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/β-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of β-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in β-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis revealed that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of β-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of β-arrestin, while Barbadin selectively augments FPR2-mediated ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/β-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from neutrophils.
Highlights
The inflammatory response is initiated when microorganisms or trauma inflicts tissue injury
chronic granulomatous disease (CGD) neutrophils are deficient in PMA-triggered neutrophil formation of extracellular traps (NETosis), apoptosis and autophagy [54] and most patients are characterized by high sensitivity to fungal and bacterial infections, an increased susceptibility thought to be due to impaired Reactive oxygen species (ROS) dependent killing of phagocytosed microbes
No transient rise in the concentration of cytosolic Ca2+ accompanies receptor reactivation [240]. This type of reactivation phenomena is not exclusive to FPRs, as the C5aR seems to be regulated in a similar way, whereas free fatty acid receptor 2 (FFAR2), platelet activating factor (PAFR) and CXCR2 are not be reactivated/resensitizated by cytoskeleton disrupting drugs [243, 244]. Taken together these data suggest that the actin cytoskeleton plays important roles in receptor desensitization in neutrophils, and in contrast to what have been shown for other G-protein coupled receptors (GPCRs) and cells, there is no direct link between β-arrestin and desensitization or ERK signaling in neutrophils [245]
Summary
Vi träffar dagligen mikroorganismer som kan orsaka sjukdom och i värsta fall död, men trots det är vi sällan allvarligt sjuka, det beror på ett effektivt immunförsvar som har utvecklats för att skydda oss mot sjukdomsframkallande mikroorganismer som bakterier, virus, svamp och parasiter. Detta medfödda immunförsvarets celler känner igen ett begränsat antal strukturer som finns hos många olika mikroorganismer men ibland också hos skadade kroppsegna celler och vävnader. En receptorgrupp kallas för sjutransmembran eller G-protein kopplade receptorer (GPCRs) beroende på att de har en gemensam struktur (de passerar det membran de sitter i sju gånger) och de vidarebefordrar informationen med hjälp av ett speciellt signalprotein (G-protein). Målsättningen med avhandlingsarbetet har varit att undersöka hur receptorer som uttrycks av neutrofiler och som tillhör GPCR familjen, känner igen agonister/antagonister och sedan för information vidare till cellen. De nya kunskaperna kan förhoppningsvis vara till hjälp vid framtida utveckling av läkemedel för behandling av inflammatoriska sjukdomar, men också bidra till förståelsen av hur andra receptorer som tillhör GPCR-familjen fungerar och hur aktiviteten hos dessa kan styras
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