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Mol Pharmacol. 2014 Oct;86(4):406-16. doi: 10.1124/mol.114.092403. Epub 2014 Aug 1.

Identification of protein kinase C activation as a novel mechanism for RGS2 protein upregulation through phenotypic screening of natural product extracts.

Author information

1
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.).
2
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan (A.R., P.J.S., D.H.S.); Department of Pharmacology & Toxicology, Michigan State University, East Lansing, Michigan (L.A., R.R.N., B.S.); Department of Pharmacology (C.C.), Department of Medicinal Chemistry (D.H.S.), Department of Microbiology and Immunology (D.H.S.), Department of Chemistry (D.H.S.), Center for Chemical Genomics, University of Michigan, Ann Arbor, Michigan (D.H.S.); Unidad Estrategica de Bioprospección, Instituto Nacional de Biodiversidad, Santo Domingo de Heredia, Costa Rica & CIPRONA, Escuela de Química, Universidad de Costa Rica, San Pedro, Costa Rica (G.T-C.); Harvard Medical School, Boston, Massachusetts (S.C., J.C.); and University of Hawaii Cancer Center, Honolulu, Hawaii (S.C.) sjogren1@msu.edu.

Abstract

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.

PMID:
25086086
PMCID:
PMC6067637
DOI:
10.1124/mol.114.092403
[Indexed for MEDLINE]
Free PMC Article

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