Wu et al. report a novel IL-17–mediated cascade via the IL-17R–TRAF4–ERK5 axis that directly stimulates keratinocyte proliferation and skin tumor formation in mice.

Arrestins mediate phosphorylation-dependent desensitization, internalization, and initiation of signaling cascades for the majority of G protein-coupled receptors (GPCRs). Many GPCRs undergo agonist-mediated internalization through arrestin-dependent mechanisms, wherein arrestin serves as an adapter between the receptor and endocytic proteins. To understand the role of arrestins in N-formyl peptide receptor (FPR) trafficking, we stably expressed the FPR in a mouse embryonic fibroblast cell line (MEF) that lacked endogenous arrestin 2 and arrestin 3 (arrestin-deficient). We compared FPR internalization and recycling kinetics in these cells to congenic wild type MEF cell lines. Internalization of the FPR was not altered in the absence of arrestins. Since the FPR remains associated with arrestins following internalization, we investigated whether the rate of FPR recycling was altered in arrestin-deficient cells. While the FPR was able to recycle in the wild type cells, receptor recycling was largely absent in the arrestin double knockout cells. Reconstitution of the arrestin-deficient line with either arrestin 2 or arrestin 3 restored receptor recycling. Confocal fluorescence microscopy studies demonsrated that in arrestin-deficient cells the FPR may become trapped in the perinuclear recycling compartment. These observations indicate that, although the FPR can internalize in the absence of arrestins, recycling of internalized receptors to the cell surface is prevented. Our results suggest a novel role for arrestins in the postendocytic trafficking of GPCRs.

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs) activate numerous cellular signals through the combined actions of G proteins, GPCR kinases, and arrestins. Although arrestins have traditionally been thought of as mediating GPCR desensitization, they have now been shown to play important roles in the internalization, trafficking, and signaling of many GPCRs. We demonstrate that in cells devoid of arrestins, the stimulation of numerous GPCRs including the N-formyl peptide receptor (FPR) initiates rapid cell rounding, annexin V positivity, and caspase activation followed by cell death. The apoptotic response is initiated by G protein signaling and involves activation of phosphoinositide 3-kinase, mitogen-activated protein kinases, and c-Src resulting in cytochrome c release from mitochondria and ultimately caspase 9 and caspase 3 activation. Reconstitution with either arrestin-2 or arrestin-3 is completely sufficient to prevent FPR-mediated apoptosis. Surprisingly, a non-desensitizing and non-internalizing mutant of the FPR is unable to initiate apoptosis, indicating that receptor phosphorylation and internalization, but not solely chronic activation due to a lack of desensitization, are critical determinants for the induction of apoptosis by the FPR. We further demonstrate that this response is not unique to the FPR with numerous additional GPCRs, including the V2 vasopressin, angiotensin II (type 1A), and CXCR2 receptors, capable of initiating apoptosis upon stimulation, whereas GPCRs such as the ␤ 2 -adrenergic receptor and CXCR4 are not capable of initiating apoptotic signaling. These data demonstrate for the first time that arrestins play a critical and completely unexpected role in the suppression GPCR-mediated apoptosis, which we show is a common consequence of GPCR-mediated cellular activation in the absence of arrestins.

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 ± 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 ± 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2−/−/arrestin 3−/− murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.