Capillary Electrophoresis Assay for G Protein-Coupled Receptor-Mediated GTPase Activity
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- 作者：Emily E. Jameson ; Jian Pei ; Susan M. Wade ; Richard R. Neubig ; Graeme Milligan ; Robert T. Kennedy
- 刊名：Analytical Chemistry
- 出版年：2007
- 出版时间：February 1, 2007
- 年：2007
- 卷：79
- 期：3
- 页码：1158 - 1163
- 全文大小：198K
- 年卷期：v.79,no.3(February 1, 2007)
- ISSN：1520-6882
文摘
We describe a capillary electrophoresis (CE) assay todetect G protein-coupled receptor (GPCR)-stimulated Gprotein GTPase activity in cell membranes expressing 
2Aadrenoreceptor-Go1 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) from the fluorescent product (*GDP). Using the assay, the 2a adrenoceptor agonist UK14,304 was shown to simulate specificproduction of *GDP in membranes from HEK293T cellsexpressing receptor-G protein fusion to 525% of basallevels with an EC50 of 0.48 ± 0.20 
M. The EC50increased to 9.4 ± 5 M with addition of the antagonistyohimbine. Nucleotide hydrolysis was increased furtherover agonist-stimulated levels with addition of the in vivomodulator 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.
2Aadrenoreceptor-Go1 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) from the fluorescent product (*GDP). Using the assay, the 2a adrenoceptor agonist UK14,304 was shown to simulate specificproduction of *GDP in membranes from HEK293T cellsexpressing receptor-G protein fusion to 525% of basallevels with an EC50 of 0.48 ± 0.20 M. The EC50increased to 9.4 ± 5 M with addition of the antagonistyohimbine. Nucleotide hydrolysis was increased furtherover agonist-stimulated levels with addition of the in vivomodulator 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.