ArticleAssay and drug development technologies2015
A Homogenous Bioluminescent System for Measuring GTPase, GTPase Activating Protein, and Guanine Nucleotide Exchange Factor Activities.
Article in Assay and drug development technologies, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 34 papers.
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Who cites it
34 citing papers in PubMed, 60 citations in OpenAlex.
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- Balancing G protein selectivity and efficacy in the adenosine ANature chemical biology · 2025Article
- Structural basis of antimicrobial membrane coat assembly by human GBP1.Nature structural & molecular biology · 2025Article
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- Oscillatory dynamics of Rac1 activity in Dictyostelium discoideum amoebae.PLoS computational biology · 2024Article
- Tumor-derived RHOA mutants interact with effectors in the GDP-bound state.Nature communications · 2024Article
- K128 ubiquitination constrains RAS activity by expanding its binding interface with GAP proteins.The EMBO journal · 2024Article
- Structure and function of a ligand-free GPCR-Gαβγ intermediate complex.Research square · 2024Article
- From bench to bedside: current development and emerging trend of KRAS-targeted therapy.Acta pharmacologica Sinica · 2024Review
- Measurement of KRAS-GTPase Activity.Methods in molecular biology (Clifton, N.J.) · 2024Article
- Genomic analysis reveals HDAC1 regulates clinically relevant transcriptional programs in Pancreatic cancer.BMC cancer · 2023Article
- The Role ofInternational journal of molecular sciences · 2023Article
- Dual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition.Structure (London, England : 1993) · 2023Article
- Direct Observation of Membrane-Associated H-Ras in the Native Cellular Environment by In-CellJACS Au · 2023Article
- A missense variant at the RAC1-PAK1 binding site of RAC1 inactivates downstream signaling in VACTERL association.Scientific reports · 2023Article
- Cholesterol Biases the Conformational Landscape of the Chemokine Receptor CCR3: A MAS SSNMR-Filtered Molecular Dynamics Study.Journal of chemical information and modeling · 2023Article
- Mapping the conformational landscape of the stimulatory heterotrimeric G protein.Nature structural & molecular biology · 2023Article
- Intermediate-state-trapped mutants pinpoint G protein-coupled receptor conformational allostery.Nature communications · 2023Article
Corrections and comments
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Authors and funding
3 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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 has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators has been challenging due to paucity of convenient assays. In this study, we describe a homogenous bioluminescent assay for monitoring the activities of GTPase and its immediate regulators: GTPase activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). Since Mg(2+) plays a critical role in influencing the affinity of GTPases with guanosine triphosphate/guanosine diphosphate (GTP/GDP) and the process of nucleotide exchange, manipulating Mg(2+) concentrations in the GTPase reaction buffer allows continuous progression of the GTPase cycle and faster hydrolysis of GTP. The assay relies on enzymatic conversion of GTP that remains after the GTPase reaction to ATP and detection of the generated ATP using the luciferin/luciferase combination. The GTPase/GAP/GEF-Glo assay system enables monitoring of GTPase, GAP-stimulated GTPase, GAP, and GEF activities. The system can also be used to analyze these proteins when expressed in cells as fusion proteins by performing the assay in a pulldown format. The assays showed minimal false hits upon testing for compound interference using the library of pharmacologically active compounds and its robustness was demonstrated by a high Z'-factor of 0.93 and CV of 2.2%. The assay system has a high dynamic range, formatted in a convenient add-mix-read, and applicable to high-throughput screening.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.