Detection of adenylate cyclase-coupled receptors in Xenopus oocytes by coexpression with cystic fibrosis transmembrane conductance regulator.
To detect heterologous expression of receptors coupled via G proteins to the stimulation of adenylate cyclase in Xenopus laevis oocytes, the receptor of interest is coexpressed with the cystic fibrosis transmembrane conductance regulator (CFTR)--a cAMP-dependent Cl- channel. The binding of an agonist to the expressed receptor stimulates adenylate cyclase resulting in intracellular cAMP elevation, which in turn activates the CFTR. The CFTR-mediated Cl- current response is then measured using the standard two-electrode voltage-clamp technique. This method has allowed us to detect functional expression in oocytes of the human EP2 and IP prostanoid receptors. This method should prove valuable for expression and identification of putative G protein-coupled receptors signaling through stimulation of adenylate cyclase, for structure/function studies, and for analysis of receptor antagonists and agonists.
Grygorczyk
R
R
Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Pointe Claire-Dorval, Quebec, Canada.
Abramovitz
M
M
Boie
Y
Y
Bastien
L
L
Adam
M
M
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism*; D009865:Oocytes / Q000737:chemistry*; D011982:Receptors, Prostaglandin / Q000032:analysis* / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors; D014982:Xenopus laevis
C505032:CFTR protein, human; D015220:Calcium Channels; D008565:Membrane Proteins; D011982:Receptors, Prostaglandin; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D016285:Iloprost; D015232:Dinoprostone; D015056:1-Methyl-3-isobutylxanthine