title: Arrestin-2 and G protein-coupled receptor kinase 5 interact with NFkappaB1 p105 and negatively regulate lipopolysaccharide-stimulated ERK1/2 activation in macrophages.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Narayanan
authors.initials: N
authors.lastname: Parameswaran
authors.forename: Christina S
authors.initials: CS
authors.lastname: Pao
authors.forename: Kristi S
authors.initials: KS
authors.lastname: Leonhard
authors.forename: Dong Soo
authors.initials: DS
authors.lastname: Kang
authors.forename: Michelle
authors.initials: M
authors.lastname: Kratz
authors.forename: Steven C
authors.initials: SC
authors.lastname: Ley
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Benovic
mesh_terms: D000818:Animals; D019393:Arrestin / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D047468:Immunoprecipitation; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D019393:Arrestin; D008070:Lipopolysaccharides; D052002:NF-kappa B p50 Subunit; C489548:NFKB1 protein, human; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D017346:Protein Serine-Threonine Kinases; D051550:I-kappa B Kinase; D054774:G-Protein-Coupled Receptor Kinase 5; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; C517898:GRK5 protein, human; C517897:Grk5 protein, rat; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3