title: A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.
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.
authors.affiliation: Lankenau Institute for Medical Research Chemical Genomics Center, Wynnewood, PA, USA.
authors.forename: Melvin
authors.initials: M
authors.lastname: Reichman
authors.affiliation: Lankenau Institute for Medical Research Chemical Genomics Center, Wynnewood, PA, USA.
authors.forename: Amanda
authors.initials: A
authors.lastname: Schabdach
authors.affiliation: BellBrook Labs, Madison, WI, USA.
authors.forename: Meera
authors.initials: M
authors.lastname: Kumar
authors.affiliation: BellBrook Labs, Madison, WI, USA.
authors.forename: Tom
authors.initials: T
authors.lastname: Zielinski
authors.affiliation: Lankenau Institute for Medical Research Chemical Genomics Center, Wynnewood, PA, USA.
authors.forename: Preston S
authors.initials: PS
authors.lastname: Donover
authors.affiliation: Lankenau Institute for Medical Research Chemical Genomics Center, Wynnewood, PA, USA.
authors.forename: Lisa D
authors.initials: LD
authors.lastname: Laury-Kleintop
authors.affiliation: BellBrook Labs, Madison, WI, USA bob.lowery@bellbrooklabs.com.
authors.forename: Robert G
authors.initials: RG
authors.lastname: Lowery
mesh_terms: D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007118:Immunoassay / Q000379:methods*; D064067:Rho Guanine Nucleotide Exchange Factors / Q000032:analysis*
chemical_list: D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors
keywords: GDP; GEF; GTPase; Rho; Transcreener