Abstract
Opioid peptides exert diverse physiological functions through their cognate receptors. One subtype of the opioid receptors, κ-opioid receptor, is endogenously expressed in human monocytic THP-1 cells. Stimulation of the THP-1 cells with a κ-opioid receptor-selective agonist exerted a Gi-dependent activation of c-Jun N-terminal kinase (JNK). To further investigate the signaling mechanism by which the κ-opioid receptor regulates JNK activity, heterologous expression assays in COS-7 cells were utilized. Overexpression of Gαt in COS-7 cells clearly suppressed κ-opioid receptor-stimulated JNK activity, indicating that the pathway is primarily regulated by Gβγ. In both THP-1 and transfected COS-7 cells, pretreatment of the selective Src family kinase inhibitor pyrazolopyrimidine PP1 abolished the JNK activation, whereas the epidermal growth factor receptor inhibitor AG1478 [N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinanine] failed to do that. Furthermore, the JNK activation in response to κ-opioid receptor was suppressed by an autophosphorylation-resistant mutant of focal adhesion kinase (FAK). Consistently, activated κ-opioid receptor induced Src stimulation and FAK autophosphorylation and promoted the formation of Src-FAK complex. The participation of small GTPases as well as a guanine nucleotide exchange factor was also implicated because dominant-negative mutants of Rac, Cdc42, and Son-of-sevenless (Sos) attenuated the agonist-induced activation of JNK. These studies demonstrate that the activation of JNK by κ-opioid receptors is routed via Gβγ, Src, FAK, Sos, Rac, and Cdc42.
Footnotes
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This work was supported in part by grants from the University Grants Committee of Hong Kong (AoE/B-15/01), the Research Grants Council of Hong Kong (HKUST 6115/00M and 2/99C), and the Hong Kong Jockey Club. Y.H.W. was a recipient of the Croucher Senior Research Fellowship.
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DOI: 10.1124/jpet.104.065078.
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ABBREVIATIONS: IL, interleukin; U50,488H, (±)-trans-U-50,488 methanesulfonate; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated protein kinase; JNK, c-Jun N-terminal kinase; GPCR, G protein-coupled receptor; EGF, epidermal growth factor; FAK, focal adhesion kinase; GEF, guanine nucleotide exchange factors; Sos, Son-of-sevenless; HA, hemagglutinin; PBD, p21-binding domain; PTX, pertussis toxin; AG1478, N-(3-chlorophenyl)-6,7-dimethoxy-4-quinazolinanine; nor-BNI, nor-binaltorphimine; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; LPA, lysophosphatidic acid; SH, Src homology; DH, Dbl homology; PI3K, phosphatidylinositol 3-kinase.
- Received January 1, 2004.
- Accepted March 1, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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