RT Journal Article
SR Electronic
T1 Identification of Protein Kinase C Activation as a Novel Mechanism for RGS2 Protein Upregulation through Phenotypic Screening of Natural Product Extracts
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 406
OP 416
DO 10.1124/mol.114.092403
VO 86
IS 4
A1 Raveh, Avi
A1 Schultz, Pamela J.
A1 Aschermann, Lauren
A1 Carpenter, Colleen
A1 Tamayo-Castillo, Giselle
A1 Cao, Shugeng
A1 Clardy, Jon
A1 Neubig, Richard R.
A1 Sherman, David H.
A1 Sjögren, Benita
YR 2014
UL http://molpharm.aspetjournals.org/content/86/4/406.abstract
AB Biochemical high-throughput screening is widely used in drug discovery, using a variety of small molecule libraries. However, broader screening strategies may be more beneficial to identify novel biologic mechanisms. In the current study we used a β-galactosidase complementation method to screen a selection of microbial-derived pre-fractionated natural product extracts for those that increase regulator of G protein signaling 2 (RGS2) protein levels. RGS2 is a member of a large family of proteins that all regulate signaling through G protein–coupled receptors (GPCRs) by accelerating GTPase activity on active Gα as well as through other mechanisms. RGS2−/− mice are hypertensive, show increased anxiety, and are prone to heart failure. RGS2 has a very short protein half-life due to rapid proteasomal degradation, and we propose that enhancement of RGS2 protein levels could be a beneficial therapeutic strategy. Bioassay-guided fractionation of one of the hit strains yielded a pure compound, Indolactam V, a known protein kinase C (PKC) activator, which selectively increased RGS2 protein levels in a time- and concentration-dependent manner. Similar results were obtained with phorbol 12-myristate 13-acetate as well as activation of the Gq-coupled muscarinic M3 receptor. The effect on RGS2 protein levels was blocked by the nonselective PKC inhibitor Gö6983 (3-[1-[3-(dimethylamino)propyl]-5-methoxy-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione), the PKCβ-selective inhibitor Ruboxastaurin, as well as small interfering RNA-mediated knockdown of PKCβ. Indolactam V-mediated increases in RGS2 protein levels also had functional effects on GPCR signaling. This study provides important proof-of-concept for our screening strategy and could define a negative feedback mechanism in Gq/Phospholipase C signaling through RGS2 protein upregulation.