Capillary Electrophoresis Assay for G Protein-Coupled Receptor-Mediated GTPase Activity

Abstract

We describe a capillary electrophoresis (CE) assay to detect G protein-coupled receptor (GPCR)-stimulated G protein GTPase activity in cell membranes expressing alpha2A adrenoreceptor-Galphao1 wild-type (wt) or C351I mutant fusion proteins using a fluorescent, hydrolyzable GTP analogue. As no change in total fluorescence is observed by conversion of substrate to product, CE is used to separate the fluorescent substrate (*GTP) from the fluorescent product (*GDP). Using the assay, the alpha2a adrenoceptor agonist UK14,304 was shown to simulate specific production of *GDP in membranes from HEK293T cells expressing receptor-G protein fusion to 525% of basal levels with an EC50 of 0.48 +/- 0.20 microM. The EC50 increased to 9.4 +/- 5 muM with addition of the antagonist yohimbine. Nucleotide hydrolysis was increased further over agonist-stimulated levels with addition of the in vivo modulator protein RGS (regulator of G protein signaling). It is envisioned that this technique could be used for screening for novel GPCR ligands or other G protein signaling modifiers.

Capillary Electrophoresis in Bioanalysis

“…CE was used to monitor this activity, which results in the transformation of BODIPY FL GTP (substrate) to BODIPY FL GDP (product) (187). The approach was used to determine the EC 50 of the antagonist yohimbine and UK14,304.…”

“…GTPase activity is important in cell signaling and can be used to monitor compounds that modulate G protein-coupled receptors. CE was used to monitor this activity, which results in the transformation of BODIPY FL GTP (substrate) to BODIPY FL GDP (product) (187). The approach was used to determine the EC 50 of the antagonist yohimbine and UK14,304.…”

Antibodies that identify only the active conformation of Gifamily G protein α subunits

“…However, both in efforts to identify activated G proteins in a cellular setting and in the context of ligand screening in the pharmaceutical and biotechnology industries, such assays present major challenges. First, although the greatest amount of information can be gathered from measures of ligand regulation of high‐affinity GTPase activity, because they require the use of radiolabeled or fluorescent forms of GTP and are not easily adapted to a homogenous format (7, 8), such enzyme assays have not found favor outside academic laboratories. Second, although assays based on the binding of the poorly hydrolyzed analog of GTP, guanosine 5'‐ O ‐(3‐thio)‐triphosphate ([ 35 S]GTPγS), have been used widely (9–11), the desire to move away from the use of radioactivity and to develop “label‐free” assays is strong (12).…”

Microfabricated Channel Array Electrophoresis for Characterization and Screening of Enzymes Using RGS−G Protein Interactions as a Model System

“…We used Z ′ value to evaluate the reproducibility of the assay. The Z ′ is by definition = 1.0 − (3.0( S neg + S pos )/ R ), where S neg and S pos are the standard deviation of the signal of negative and positive controls, respectively, and R is the difference between the mean of positive and negative controls . We determined a Z ′ = 0.7 for the assay, which is well above the 0.5 considered necessary for high-throughput screening.…”