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J Biomol Screen. 2015 Dec;20(10):1294-9. doi: 10.1177/1087057115596326. Epub 2015 Jul 20.

A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

Author information

1
Lankenau Institute for Medical Research Chemical Genomics Center, Wynnewood, PA, USA.
2
BellBrook Labs, Madison, WI, USA.
3
BellBrook Labs, Madison, WI, USA bob.lowery@bellbrooklabs.com.

Abstract

Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs.

KEYWORDS:

GDP; GEF; GTPase; Rho; Transcreener

PMID:
26195453
DOI:
10.1177/1087057115596326
[Indexed for MEDLINE]

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