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Modification and functional inhibition of regulator of G-protein signaling 4 (RGS4) by 4-hydroxy-2-nonenal.

ABSTRACT: Oxidative stress has been implicated as a component of various pathologies including ischemia/reperfusion injury (IRI) and neurodegenerative diseases such as Parkinson's disease (PD) and schizophrenia. Similarly, regulator of G-protein signaling 4 (RGS4) has been implicated as an important player in each of these pathologies. RGS4, like other RGS proteins, is responsible for temporally regulating G-protein coupled receptor signaling by increasing the intrinsic GTPase activity of G? subunit of the heterotrimeric signaling complex. In this study we evaluated whether modification by 4-hydroxy-2-nonenal (4HNE), a common lipid peroxidation product, inhibits RGS4. Using immunoprecipitation, we first determined RGS4 modification was occurring in cells at concentrations of 4HNE within reported physiological conditions. Following this determination, we evaluated modification of RGS4 by 4HNE by both Western blot and mass spectrometry (MS). Once it was established that covalent modification occurred only on cysteine containing constructs, tryptic digest followed by mass spectrometry analysis revealed modification occurs at cysteine residues 71, 148, and 183. In order to determine the effect 4HNE had on RGS4 activity, a steady-state colorimetric assay was used to analyze the GAP activity of ?51-RGS4 as well as the cysteine null mutant. From the data, we determined that RGS4 activity can be modulated by 4HNE through modification at cysteine residues similar to previously reported small molecule inhibition of RGS4.

SUBMITTER: Monroy CA

PROVIDER: S-EPMC4441825 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Project description:Tubulogenesis by epithelial cells regulates kidney, lung, and mammary development, whereas that by endothelial cells regulates vascular development. Although functionally dissimilar, the processes necessary for tubulation by epithelial and endothelial cells are very similar. We performed microarray analysis to further our understanding of tubulogenesis and observed a robust induction of regulator of G protein signaling 4 (RGS4) mRNA expression solely in tubulating cells, thereby implicating RGS4 as a potential regulator of tubulogenesis. Accordingly, RGS4 overexpression delayed and altered lung epithelial cell tubulation by selectively inhibiting G protein-mediated p38 MAPK activation, and, consequently, by reducing epithelial cell proliferation, migration, and expression of vascular endothelial growth factor (VEGF). The tubulogenic defects imparted by RGS4 in epithelial cells, including its reduction in VEGF expression, were rescued by overexpression of constitutively active MKK6, an activator of p38 MAPK. Similarly, RGS4 overexpression abrogated endothelial cell angiogenic sprouting by inhibiting their synthesis of DNA and invasion through synthetic basement membranes. We further show that RGS4 expression antagonized VEGF stimulation of DNA synthesis and extracellular signal-regulated kinase (ERK)1/ERK2 and p38 MAPK activation as well as ERK1/ERK2 activation stimulated by endothelin-1 and angiotensin II. RGS4 had no effect on the phosphorylation of Smad1 and Smad2 by bone morphogenic protein-7 and transforming growth factor-beta, respectively, indicating that RGS4 selectively inhibits G protein and VEGF signaling in endothelial cells. Finally, we found that RGS4 reduced endothelial cell response to VEGF by decreasing VEGF receptor-2 (KDR) expression. We therefore propose RGS4 as a novel antagonist of epithelial and endothelial cell tubulogenesis that selectively antagonizes intracellular signaling by G proteins and VEGF, thereby inhibiting cell proliferation, migration, and invasion, and VEGF and KDR expression.

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Modification and functional inhibition of regulator of G-protein signaling 4 (RGS4) by 4-hydroxy-2-nonenal.

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