Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages

• Published in: The Journal of biological chemistry 2006-11, Vol.281 (45), p.34159-34170
• Main Authors: Parameswaran, Narayanan, Pao, Christina S, Leonhard, Kristi S, Kang, Dong Soo, Kratz, Michelle, Ley, Steven C, Benovic, Jeffrey L
• Format: Article
• Language: English
• Subjects: Animals; Arrestin - genetics; Arrestin - metabolism; Blotting, Western; Cells, Cultured - drug effects; Cells, Cultured - metabolism; Enzyme Activation - drug effects; G-Protein-Coupled Receptor Kinase 5; G-Protein-Coupled Receptor Kinases; Gene Expression Regulation; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Humans; I-kappa B Kinase - metabolism; Immunoprecipitation; Kidney - drug effects; Kidney - metabolism; Lipopolysaccharides - pharmacology; Macrophages - cytology; Macrophages - drug effects; Macrophages - metabolism; Mitogen-Activated Protein Kinase 1 - metabolism; Mitogen-Activated Protein Kinase 3 - metabolism; NF-kappa B p50 Subunit - genetics; NF-kappa B p50 Subunit - metabolism; Phosphorylation - drug effects; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Rats; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Signal Transduction
• ISSN: 0021-9258

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, IkappaB kinase promotes phosphorylation and degradation of NFkappaB1 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 IkappaB 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.