G16-mediated Activation of Nuclear Factor κB by the Adenosine A1 Receptor Involves c-Src, Protein Kinase C, and ERK Signaling*
- Department of Biochemistry, Molecular Neuroscience Center, and Biotechnology Research Institute, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
- ‡ Recipient of the Croucher Senior Research Fellowship. To whom correspondence should be addressed: Dept. of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. Tel.: 852-2358-7328; Fax: 852-2358-1552; E-mail: boyung{at}ust.hk.
Abstract
The Gi-linked adenosine A1 receptor has been shown to mediate anti-inflammatory actions, possibly via modulation of the transcription factor nuclear factor-κB (NFκB). Here we demonstrate that an adenosine A1 agonist, N6-cyclohexyladenosine (CHA), activated IKKα/β phosphorylation through PTX-insensitive G proteins in human lymphoblastoma Reh cells. To delineate the mechanism of action, different PTX-insensitive G proteins were expressed in human embryonic kidney 293 cells. Only Gα16 supported the CHA-induced IKK phosphorylation and NFκB-driven luciferase activity in time-dependent, dose-dependent, and PTX-insensitive manners. Gβγ subunits also modulated IKK/NFκB, as indicated by the stimulatory actions of Gβ1γ2 and the abrogation of CHA-induced response by transducin. The participation of phospholipase Cβ, protein kinase C, and calmodulin-dependent kinase II in CHA-induced IKK/NFκB activation were demonstrated by employing specific inhibitors and dominant-negative mutants. Inhibition of c-Src and numerous intermediates along the extracellular signal-regulated (ERK) kinase cascade including Ras, Raf-1 kinase, and MEK1/2 abolished the CHA-induced IKK/NFκB activation. Although c-Jun N-terminal kinase and p38 MAPK were also activated by CHA, they were not required for the IKK/NFκB regulation. Similar results were obtained using Reh cells. These data suggest that the G16-mediated activation of IKK/NFκB by CHA required a complex signaling network composed of multiple intermediates.
Footnotes
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↵1 The abbreviations used are: NFκB, nuclear factor-κB; A1R, adenosine A1 receptor; CaMKII, calmodulin-dependent protein kinase II; CHA, N6-cyclohexyladenosine; ERK, extracellular signal-regulated kinase; GPCRs, G protein-coupled receptors; HEK 293, human embryonic kidney 293; IKK, IκB kinase; JNK, c-Jun N-terminal kinase; MAPKs, mitogen-activated protein kinases; MEK, MAPK/ERK kinase; PKC, protein kinase C; PLCβ, phospholipase Cβ; PTX, pertussis toxin; c-Src, cellular Src; APQ, 6-amino-4-(4-phenoxyphenylethylamino)quinazoline; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine.
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↵* This work was supported in part by Grants from the Research Grants Council of Hong Kong (HKUST 6095/01M, 2/99C, and 3/03C), the University Grants Committee (AoE/B-15/01), and the Hong Kong Jockey Club. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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- Received September 7, 2004.
- Revision received October 8, 2004.
- The American Society for Biochemistry and Molecular Biology, Inc.
