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CCR2 Signaling Selectively Regulates IFN-?: Role of ?-Arrestin 2 in IFNAR1 Internalization.

ABSTRACT: An integral component of the antiviral response, type I IFNs require regulation to modulate immune activation. We identify ?-arrestin 2 as a key modulator of type I IFN in primary human macrophages, an essential component of the innate immune response. ?-Arrestin 2 was selectively activated by CCL2/CCR2 signaling, which induced a decrease in IFN-?, but not IFN-? expression. Small interfering RNA knockdown of ?-arrestin 2 demonstrated its role in IFNAR1 internalization, as well as STAT1 and IRF3 activation. As a result, cytokine responses were not propagated following HIV infection and TLR3 activation. However, remnants of IFN signaling remained intact, despite ?-arrestin 2 activation, as IFN-?, IFN-?, IFN-?1, IRF7, TRAIL, and MxA expression were sustained. Similar effects of ?-arrestin 2 on IFN signaling occurred in hepatocytes, suggesting that arrestins may broadly modulate IFN responses in multiple cell types. In summary, we identify a novel role of ?-arrestin 2 as an integral regulator of type I IFN through its internalization of IFNAR1 and a subsequent selective loss of downstream IFN signaling.

SUBMITTER: Williams DW

PROVIDER: S-EPMC6310093 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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CCR2 Signaling Selectively Regulates IFN-α: Role of β-Arrestin 2 in IFNAR1 Internalization.

Williams Dionna W DW Askew Lauren C LC Jones Elonna E Clements Janice E JE

Journal of immunology (Baltimore, Md. : 1950) 20181130 1

An integral component of the antiviral response, type I IFNs require regulation to modulate immune activation. We identify β-arrestin 2 as a key modulator of type I IFN in primary human macrophages, an essential component of the innate immune response. β-Arrestin 2 was selectively activated by CCL2/CCR2 signaling, which induced a decrease in IFN-α, but not IFN-β expression. Small interfering RNA knockdown of β-arrestin 2 demonstrated its role in IFNAR1 internalization, as well as STAT1 and IRF3 ...[more]

PMID: 30504423

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Project description:PurposeThe G-protein coupled receptor (GPCR) Dopamine Receptor D4 (DRD4) plays an essential role in cAMP regulation and gap junctional coupling in the photoreceptors, where DRD4 expression is under circadian control. Previous in vitro transfection studies of human DRD4 desensitization have reported that DRD4 is not internalized upon dopamine stimulation when beta-arrestin is co-transfected with DRD4. We hypothesized that the visual arrestins, ARR1 and ARR4, play a modulatory role in DRD4 desensitization in the photoreceptors.MethodsTo test this hypothesis, immunohistochemistry analysis of mouse retinas was used to determine the cellular localization of beta-arrestins and DRD4 in photoreceptors. In vitro studies were performed in HEK293T cells transiently transfected with human DRD4 and arrestins. First, co-immunoprecipitation experiments were executed to test protein-protein interactions and to investigate the effect of dopamine stimulation. Second, immunohistochemistry analysis was implemented to study DRD4 internalization and translocation of ARR4.ResultsImmunohistochemistry studies of mouse retinas confirmed the expression of beta-arrestin 2, ARR1 and ARR4, as well as DRD4 in mouse cone photoreceptor inner segments. Co-immunoprecipitation experiments revealed a dopamine-dependent protein-protein interaction between human DRD4 and ARR4. In vitro internalization experiments showed that no detectable internalization of DRD4 was observed with any single arrestin co-transfected. However, a dopamine-dependent internalization of DRD4 was observed with three out of six sets of two arrestins co-transfected with DRD4. Each of these pairs of arrestins contained one visual arrestin and one beta-arrestin, and no internalization was observed with either two visual arrestins or two beta-arrestins. Additional time-course experiments revealed that in vitro, ARR4 translocates to co-localize with DRD4 at the plasma membrane in response to 30min of dopamine stimulation.ConclusionsThe results have functional implications and we hypothesize that the desensitization and internalization of DRD4 in photoreceptors are synergistically mediated by both visual and beta-arrestins. These results are additionally unique because they demonstrate for the first time that at least one G-protein coupled receptor, DRD4, requires two arrestins for desensitization and internalization, and opens up the possibility that other G-protein coupled receptors may require more than one arrestin for desensitization and/or internalization.

Project description:Background: The atypical chemokine receptor 3 (ACKR3) belongs to the superfamily of G protein-coupled receptors (GPCRs). Unlike classical GPCRs, this receptor does not activate G proteins in most cell types but recruits β-arrestins upon activation. ACKR3 plays an important role in cancer and vascular diseases. As recruitment of β-arrestins is triggered by phosphorylation of the C-terminal tail of GPCRs, we studied the role of different potential phosphorylation sites within the ACKR3 C-tail to further delineate the molecular mechanism of internalization and trafficking of this GPCR. Methods: We used various bioluminescence and fluorescence resonance energy transfer-based sensors and techniques in Human Embryonic Kidney (HEK) 293T cells expressing WT or phosphorylation site mutants of ACKR3 to measure CXCL12-induced recruitment of β-arrestins and G-protein-coupled receptor kinases (GRKs), receptor internalization and trafficking. Results: Upon CXCL12 stimulation, ACKR3 recruits both β-arrestin 1 and 2 with equivalent kinetic profiles. We identified interactions with GRK2, 3 and 5, with GRK2 and 3 being important for β-arrestin recruitment. Upon activation, ACKR3 internalizes and recycles back to the cell membrane. We demonstrate that β-arrestin recruitment to the receptor is mainly determined by a single cluster of phosphorylated residues on the C-tail of ACKR3, and that residue T352 and in part S355 are important residues for β-arrestin1 recruitment. Phosphorylation of the C-tail appears essential for ligand-induced internalization and important for differential β-arrestin recruitment. GRK2 and 3 play a key role in receptor internalization. Moreover, ACKR3 can still internalize when β-arrestin recruitment is impaired or in the absence of β-arrestins, using alternative internalization pathways. Our data indicate that distinct residues within the C-tail of ACKR3 differentially regulate CXCL12-induced β-arrestin recruitment, ACKR3 trafficking and internalization.

Dataset Information

CCR2 Signaling Selectively Regulates IFN-?: Role of ?-Arrestin 2 in IFNAR1 Internalization.

Publications

CCR2 Signaling Selectively Regulates IFN-α: Role of β-Arrestin 2 in IFNAR1 Internalization.

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