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Cardiotonic Steroids Stabilize Regulator of G Protein Signaling 2 Protein Levels
Benita Sjögren, Sergio Parra, Lauren J. Heath, Kevin B. Atkins, Zie-Jian Xie and Richard R. Neubig
Molecular Pharmacology September 2012, 82 (3) 500-509; DOI: https://doi.org/10.1124/mol.112.079293
Benita Sjögren
Departments of Pharmacology (B.S., S.P., L.J.H., R.R.N.) and Internal Medicine (K.B.A., R.R.N.), and Center for Chemical Genomics (R.R.N.), University of Michigan, Ann Arbor, Michigan; and Department of Physiology and Pharmacology, College of Medicine, University of Toledo, Toledo, Ohio (Z.-J.X.)
Sergio Parra
Lauren J. Heath
Kevin B. Atkins
Zie-Jian Xie
Richard R. Neubig
Regulator of G protein signaling 2 (RGS2), a Gq-specific GTPase-activating protein, is strongly implicated in cardiovascular function. RGS2(−/−) mice are hypertensive and prone to heart failure, and several rare human mutations that accelerate RGS2 degradation have been identified among patients with hypertension. Therefore, pharmacological up-regulation of RGS2 protein levels might be beneficial. We used a β-galactosidase complementation method to screen several thousand compounds with known pharmacological functions for those that increased RGS2 protein levels. Several cardiotonic steroids (CTSs), including ouabain and digoxin, increased RGS2 but not RGS4 protein levels. CTSs increased RGS2 protein levels through a post-transcriptional mechanism, by slowing protein degradation. RGS2 mRNA levels in primary vascular smooth muscle cells were unaffected by CTS treatment, whereas protein levels were increased 2- to 3-fold. Na+/K+-ATPase was required for the increase in RGS2 protein levels, because the effect was lost in Na+/K+-ATPase-knockdown cells. Furthermore, we demonstrated that CTS-induced increases in RGS2 levels were functional and reduced receptor-stimulated, Gq-dependent, extracellular signal-regulated kinase phosphorylation. Finally, we showed that in vivo treatment with digoxin led to increased RGS2 protein levels in heart and kidney. CTS-induced increases in RGS2 protein levels and function might modify several deleterious mechanisms in hypertension and heart failure. This novel CTS mechanism might contribute to the beneficial actions of low-dose digoxin treatment in heart failure. Our results support the concept of small-molecule modulation of RGS2 protein levels as a new strategy for cardiovascular therapy.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grant DA023252]; the Swedish Heart and Lung Foundation [Grant 20110193]; and Bristol-Meyers-Squibb (to R.R.N.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
http://dx.doi.org/10.1124/mol.112.079293.
cardiotonic steroid
G protein-coupled receptor
regulator of G protein signaling
VSMC
vascular smooth muscle cell
phosphoinositide
extracellular signal-regulated kinase
Dulbecco's modified Eagle's medium
human embryonic kidney
TBS-T
Tris-buffered saline-Tween 20
short hairpin RNA
hemagglutinin
ProLabel
N-(benzyloxycarbonyl)leucinylleucinylleucinal
4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine
2-morpholin-4-yl-8-phenylchromen-4-one.
Accepted June 13, 2012.
Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.
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CTSs Stabilize RGS2 Protein Levels
Molecular Pharmacology September 1, 2012, 82 (3) 500-509; DOI: https://doi.org/10.1124/mol.112.079293
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