We describe a capillary electrophoresis (CE) assay todetect G protein-coupled receptor (GPCR)-sti
mulated Gprotein GTPase activity in cell
me
mbranes expressing
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2Aadrenoreceptor-G
mages/gifchars/alpha.gif" BORDER=0>o1 wild-type (wt) or C351I
mutantfusion proteins using a fluorescent, hydrolyzable GTPanalogue. As no change in total fluorescence is observedby conversion of substrate to product, CE is used toseparate the fluorescent substrate (*GTP) fro
m the fluorescent product (*GDP). Using the assay, the
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2a adrenoceptor agonist UK14,304 was shown to si
mulate specificproduction of *GDP in
me
mbranes fro
m HEK293T cellsexpressing receptor-G protein fusion to 525% of basallevels with an EC
50 of 0.48 ± 0.20
![](/i<font color=)
mages/entities/
mgr.gif">M. The EC
50increased to 9.4 ± 5
![](/i<font color=)
mages/entities/
mgr.gif">M with addition of the antagonistyohi
mbine. Nucleotide hydrolysis was increased furtherover agonist-sti
mulated levels with addition of the in vivo
modulator protein RGS (regulator of G protein signaling).It is envisioned that this technique could be used forscreening for novel GPCR ligands or other G proteinsignaling
modifiers.