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Bioluminescent Assay for GTPases Allows Measurement of GTPase, GAP and GEF Activities
Author(s) -
Mondal Subhanjan,
Hsiao Kevin,
Goueli Said
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.619.1
Subject(s) - gtpase , heterotrimeric g protein , microbiology and biotechnology , guanine nucleotide exchange factor , gtpase activating protein , biology , g protein coupled receptor , biochemistry , signal transduction , chemistry , g protein
Small‐GTPases and heterotrimeric GTPases, play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation and cell motility. Deregulation or mutation of these proteins resulting in serious pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays. To overcome the challenges in analyzing activities of GTPases and their regulatory proteins GTPase Activating Proteins (GAP) and Guanine Nucleotide Exchange Factors (GEF) we have developed a homogenous bioluminescent assay system to analyze these proteins in a simple, convenient “add‐mix‐read” format. The assay consists of optimized reaction buffers that allow continuous progression of the GTPase cycle and hydrolysis of GTP. The assay relies on enzymatic conversion of GTP remaining after the GTPase reaction to ATP and bioluminescent detection of the ATP. We show that the assay is sensitive, and robust when analyzing for analyzing intrinsic GTPase‐activity, GAP‐stimulated GTPase‐activity, GAP‐activity and GEF‐activity for various small GTPases. Heterotrimeric GTPases (Ga) are involved in the GPCR signaling pathway, where GPCRs acts as GEFs and RGS family proteins acts as GAPs for Ga. We also use the assay to monitor activities of Ga proteins. The assay has minimal false hits when tested for compound interference using the library of pharmacologically active compounds indicative of the robustness in identifying small molecule modulators of GTPase using high throughput screening.