Requirement of Gβγ and c-Src in D2 Dopamine Receptor-Mediated Nuclear Factor-κB Activation

Abstract

The D2 dopamine receptor (D2R) was examined for its ability to mediate nuclear factor-B (NF-B) activation through G proteins. Stimulation of D2R-transfected HeLa cells with its agonist quinpirole induced the expression of a NF-B luciferase reporter and formation of NF-B-DNA complex. This response was blocked by pertussis toxin, and by the G␤␥ scavengers transducin and ␤-adrenergic receptor kinase 1 carboxyl-terminal fragment. Unlike G i -coupled chemoattractant receptors, D2R activated NF-B without an increase in phospholipase C-␤ activity, and the response was only slightly affected by the phosphoinositide 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). In contrast, treatment with genistein and 4-amino-1-tert-butyl-3-(p-methylphenyl)pyrazolo [3,4-d] pyrimidine abolished the induced NF-B activation, suggesting involvement of protein tyrosine kinases. Activation of D2R led to phosphorylation of c-Src at Tyr-418, and expression of a kinase-deficient c-Src inhibited D2R-mediated NF-B activation. The D2R-mediated NF-B activation was not dependent on epidermal growth factor (EGF) receptor transactivation since 4-(3Ј-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an EGF receptor-selective tyrphostin used at 1 M, blocked EGF-induced NF-B activation but not the quinpirole-induced response. In addition, the D2R-mediated NF-B activation was enhanced by over-expression of ␤-arrestin 1. These results suggest that D2R-mediated NF-B activation requires G␤␥ and c-Src, and possibly involves ␤-arrestin 1.The transcription factor nuclear factor B (NF-B) regulates the expression of a large number of genes coding for cytokines, growth factors, inducible effector enzymes, and regulators of apoptosis (reviewed in Ghosh et al., 1998). Activation of NF-B can be induced by a variety of environmental factors such as UV, as well as by cytokines such as TNF␣ and interleukin-1␤. The prototypical NF-B activation pathway, as seen in cells stimulated with TNF␣, involves inducible phosphorylation of IB␣ at Ser-32 and Ser-36 and subsequent degradation of this and similar inhibitory proteins.

Identification of a Nuclear Localization Sequence in β-Arrestin-1 and Its Functional Implications

“…DNA Constructs-All constructs were generated using previously described human ␤-arrestin-1 cDNA (NM_0020251) as a template (16). ␤-Arrestin-1 ⌬107-191 was generated by PCR amplification of fragments encoding amino acids 1-107 and 191-409, with a C-terminal FLAG tag incorporated in the sequence.…”

“…HA-CBP was a kind gift from Dr. J. R. Lundblad (Vollum Institute, Oregon Health Sciences University, Portland, OR). The constructs for the B2 bradykinin receptor (B2BKR) and the NF-B luciferase reporter were described previously (16).…”

“…It is reported that both ␤-arrestins work in the cytosol to stabilize IB␣ at resting state, thus minimizing NF-B activation. However, other published data have shown an enhancement of NF-B activity in cells stimulated by GPCR ligands (7,16,17), suggesting that the scaffolding functions of the ␤-arrestins are able to overcome the IB␣ stabilizing effect and promote NF-B activation. In addition to its role in scaffolding signaling pathways within the cytosol, emerging evidence suggests that ␤-arrestin-1 mediates signaling within the nucleus.…”

Role of G protein-coupled receptors in inflammation

“…In resting cells, β-arrestins bind to IκBα and protect it from phosphorylation and proteosome degradation [80,81] . Upon GPCR activation, β-arrestins participate in intracellular signaling leading to activation of multiple pathways that favor NF-κB activation [82][83][84] . Several intracellular signaling molecules that were initially identified for immune cell functions, including CARMA3 and Bcl10, were found to regulate GPCR signaling leading to NF-κB activation [85,86] .…”

Association of Nucleophosmin Negatively Regulates CXCR4-Mediated G Protein Activation and Chemotaxis

“…There is increasing evidence that proteins that regulate receptor signaling and trafficking associate with cytosolic domains of GPCRs (Bockaert et al, 2004). ␤-Arrestin functions as a scaffold for cytoplasmic complexes, including the association of Akt and PP2A with the dopamine receptor (Beaulieu et al, 2005), and formation of ␤-arrestin, c-src, and dopamine receptor complex is a potential mechanism that links D2 dopamine receptor to nuclear factor B activation (Yang et al, 2003). After CXCL12 binding, CXCR4 undergoes down-modulation and ubiquitination of the C terminus (C-ter) by E3 ubiquitin ligase, thereby promoting targeting of the receptor for degradation rather than recycling via the endosomal pathway (Marchese and Benovic, 2001;Marchese et al, 2003).…”