Cellular Localization Of Beta-arrestin - Asia And Of Tlr-il-1r Pathway Regulation

The activation of immune responses through Toll-like receptor (TLR)-interleukin 1 receptor (IL-1R) signaling is critical for host defense and anti-pathogen. However, over activation of TLR-IL-1R signaling will lead to generation of certain autoimmune diseases and hence is harmful to the host. To study the regulation of TLR-IL-1R is useful for us to further understand the immune system and the development of autoimmune diseases.β-Arrestins are multifunctional molecules, which in addition to their well-established roles in desensitization and endocytosis of cell surface G-protein-coupled-receptors participate in various signaling pathways to modulate phosphorylation, ubiquitination, and/or subcellular localization of related signaling molecules. Here we show thatβ-arrestins directly interacted with TRAF6, which prevented the activation of NF-κB and subsequently decreased the TLR-IL-1R mediated cytokine expression. After LPS stimulation,β-arrestin-deficient MEFs produced more cytokines, like Ccl2, TNFαand IκBδ, in transcriptional level than wild type MEFs. When transfected back with eitherβ-arrestin1 orβ-arrestin2 plasmid, the cytokine expression after LPS stimulation was suppressed. Furthermore, the expression of eitherβ-arrestin1 orβ-arrestin2 in Hela cells also suppressed the cytokine expression after IL-1βstimulation. And when the expression level of endogenousβ-arrestin 1 is decreased by siRNA, THP-1 cells showed enhanced expression of cytokines in protein level after LPS stimulation. Notably, endotoxin treatedβ-arrestin2-deficient mice expressed higher amounts of cytokines, like Ccl2, TNFα, IL-1β, IL-6, and IκBδcompared with wild type littermates. Thusβ-arrestins are negative regulators of TLR-IL-1R signaling and suppress the expression of downstream cytokines. The gene family of arrestin is highly conserved during evolution. It includes two sub families in mammalian: one is the visual arrestin which expressed only in specific tissues while the other one named asβ-arrestin is ubiquitously expressed. Previous studies ofβ-arrestin gene family are mainly focused in mammalian. Recently,β-arrestin2 in zebrafish has been found to regulate the smoothen pathway during embryonic development of zebrafish. However, by our knowledge, there is no report aboutβ-arrestin1 gene member in non-mammalian species of vertebrate lineage. Our study for the first time cloned a new gene from zebrafish, which showed a high sequence and functional similarity with mammalianβ-arrestin1 gene. Like mammalianβ-arrestin1 gene, multi-sequence alignment and phylogenetic analysis showed the existence of the conserved clathrin binding domain, AP-2 binding site and MAKP docking site on this new gene, the sub-cellular localization analysis of GFP-β-arrestin1 showed the even distribution of this gene-encoded protein in both cytoplasm and nucleus, and the assay ofβ2AR internalization demonstrated that this gene-encoded protein also mediated the internalization ofβ2AR, which is the conserved and classical function ofβ-arrestin gene family. The RT-qPCR data showed that bothβ-arrestin1 andβ-arrestin2 from zebrafish have the time specific expression profile during embryonic development of zebrafish, indicating thatβ-arrestin maybe play important roles in zebrafish embryonic development. Thus, we show the cloning and functional characterization of theβ-arrestin1 gene from zebrafish and indicate its potential biological function.