Toll-like receptors (TLRs) are a recently described receptor class involved in the regulation of innate and adaptive immunity. Here, we demonstrate that arrestin-2 and GRK5 (G protein-coupled receptor kinase 5), proteins that regulate G protein-coupled receptor signaling, play a negative role in TLR4 signaling in Raw264.7 macrophages. We find that lipopolysaccharide (LPS)-induced ERK1/2 phosphorylation is significantly enhanced in arrestin-2 and GRK5 knockdown cells. To elucidate the mechanisms involved, we tested the effect of arrestin-2 and GRK5 knockdown on LPS-stimulated signaling components that are upstream of ERK phosphorylation. Upon LPS stimulation, IB kinase promotes phosphorylation and degradation of NFB1 p105 (p105), which releases TPL2 (a MAP3K), which phosphorylates MEK1/2, which in turn phosphorylates ERK1/2. We demonstrate that knockdown of arrestin-2 leads to enhanced LPS-induced phosphorylation and degradation of p105, enhanced TPL2 release, and enhanced MEK1/2 phosphorylation. GRK5 knockdown also results in enhanced IB kinase-mediated p105 phosphorylation and degradation, whereas GRK2 and GRK6 knockdown have no effect on this pathway. In vitro analysis demonstrates that arrestin-2 directly binds to the COOH-terminal domain of p105, whereas GRK5 binds to and phosphorylates p105. Taken together, these results suggest that p105 phosphorylation by GRK5 and binding of arrestin-2 negatively regulates LPS-stimulated ERK activation. These results reveal that arrestin-2 and GRK5 are important negative regulatory components in TLR4 signaling.Pathogen-associated molecular patterns in microbes serve as ligands to activate a recently described class of pattern recognition receptors called Toll-like receptors (TLRs).3 13 TLRs have been identified to date, with TLR1, -2, -4, -5, -6, and -11 displayed on the cell surface and TLR3, -7, -8, and -9 localized intracellularly (1). Activation of TLR signaling constitutes one of the earliest responses of an organism to microbe invasion and understanding the signaling pathways stimulated by these TLRs is an area of intense interest. The TLRs as well as interleukin receptors have a conserved ϳ200-amino acid region in their cytoplasmic tails, known as the Toll/interleukin-1 receptor domain. Within the TIR domain are conserved regions that are crucial for signaling. After ligand binding, the TLRs dimerize and undergo conformational changes that are required for the recruitment of downstream signaling molecules (2). These include the adaptor molecule Myd88 (myeloid differentiation primary response protein 88), interleukin-1 receptor-associated kinases, TAK1 (transforming growth factor--activated kinase), TAK1-binding protein (TAB1 and -2), and TRAF-6 (tumor necrosis factor (TNF) receptor-associated factor 6) (3-6).TLRs primarily activate two major signaling pathways, nuclear factor -B (NFB) and mitogen-activated protein kinase (ERK, JNK, and p38) pathways. The NFB pathway results in activation of transcription factors that are thought to act as a "master switch" for inf...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.