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1.  Two Gαi1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity 
Journal of biomolecular screening  2009;14(10):1195-1206.
RGS proteins are critical modulators of G protein-coupled receptor (GPCR) signaling given their ability to deactivate Gα subunits via “GTPase-accelerating protein” (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow GDP release from Gα and lack of solution-phase assays for detecting free GDP in the presence of excess GTP. To overcome these hurdles, we developed a Gαi1 mutant with increased GDP dissociation and decreased GTP hydrolysis, enabling detection of GAP activity using steady-state GTP hydrolysis. Gαi1(R178M/A326S) GTPase activity was stimulated 6~12 fold by RGS proteins known to act on Gαi subunits, and not affected by those unable to act on Gαi, demonstrating that the Gα/RGS domain interaction selectivity was not altered by mutation. Gαi1(R178M/A326S) interacted with RGS proteins with expected binding specificity and affinities. To enable non-radioactive, homogenous detection of RGS protein effects on Gαi1(R178M/A326S), we developed a Transcreener® fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining Gαi1(R178M/A326S) with a homogenous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling.
doi:10.1177/1087057109347473
PMCID: PMC2795102  PMID: 19820068
Fluorescence polarization; GDP detection; regulators of G-protein signaling; surface plasmon resonance
2.  Evaluating PI3 Kinase Isoforms Using Transcreener™ ADP Assays 
Journal of biomolecular screening  2008;13(6):476-485.
Development of drugs targeting lipid kinases has been delayed by the lack of robust screening assays. Methods are needed that can accommodate the presentation of different acceptor substrates in the optimal lipid environment. The Trancreener™ ADP Assay relies on homogenous immunodetection of ADP, using either fluorescence polarization (FP) or time-resolved fluorescence resonance energy transfer (TR-FRET) as a signal output. Detection of ADP - the invariant product of all kinase reactions - provides complete flexibility for varying lipid substrate parameters. We used this assay to optimize dispersal methods for C8 and C16 phosphatidylinositol 4,5 bisphosphate substrates and to assess the effects of chain length on the activity and inhibition of phosphoinositide-3-kinase (PI3K) isoforms. The non-physiological C8 substrate supported the highest activity. Known inhibitors were profiled using both the FP and TR-FRET based assays and there was excellent concordance (r2 = 0.93) in the IC50 values. The overall rank order of inhibitors was the same using the C8 and C16 substrates, except for minor deviations. ATP hydrolysis in the absence of substrate was detected with the PI3Kα isoform, and inhibitors affected PI3Kα intrinsic ATP hydrolysis activity similarly to lipid phosphorylation.
doi:10.1177/1087057108319864
PMCID: PMC2787408  PMID: 18566477
ADP detection; phosphoinositide 3-kinase; fluorescence polarization; time-resolved fluorescence resonance energy transfer; intrinsic ATPase activity

Results 1-2 (2)