Capillary electrophoresis assay for G protein-coupled receptor-mediated GTPase activity.
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.
Jameson
Emily E
EE
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Pei
Jian
J
Wade
Susan M
SM
Neubig
Richard R
RR
Milligan
Graeme
G
Kennedy
Robert T
RT
D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D020558:GTP Phosphohydrolases / Q000032:analysis* / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000032:analysis / Q000096:biosynthesis; D006160:Guanosine Triphosphate / Q000032:analysis / Q000378:metabolism; D006801:Humans; D008024:Ligands; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; D014162:Transfection
C547768:ADRA2A protein, human; D005456:Fluorescent Dyes; D008024:Ligands; D018341:Receptors, Adrenergic, alpha-2; D043562:Receptors, G-Protein-Coupled; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases