Method for identifying ligands activating either excitatory or inhibitory G-protein-coupled receptors by functional coexpression in Xenopus oocytes.
Xenopus oocytes devoid of their follicular enclosure provide a frequently used expression system for investigating receptors that transduce through activation of adenylyl cyclase following injection of the appropriate mRNA. However, due to a low basal activity of the cyclase they cannot be utilized to investigate receptor-mediated reductions in endogenous cAMP levels. In order to overcome this limitation, a model was designed in which test clones for such inhibitory receptors were co-expressed with a beta 2-adrenoceptor, which elevated cAMP upon exposure to isoproterenol. Following injection of mRNA to express the alpha 2 test receptor in the oocytes, marked reduction in cAMP could be measured after exposure to clonidine. Attenuation of cAMP levels was also seen following co-expression of the dopamine D2 receptor along with dopamine administration. Thus, after inducing a receptor-mediated tone in adenylyl cyclase activity, Xenopus oocytes can be conveniently used to study also ligands that bind to inhibitory G-protein coupled receptors.
Owman
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Department of Physiology and Neuroscience, Wallenberg Neuroscience Center, University of Lund, Sweden. christer.owman@mphy.lu.se
Mahan
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LC
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D005260:Female; D019204:GTP-Binding Proteins / Q000378:metabolism*; D015870:Gene Expression; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008024:Ligands; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018343:Receptors, Adrenergic, beta-2 / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
D008024:Ligands; D012333:RNA, Messenger; D018343:Receptors, Adrenergic, beta-2; D011956:Receptors, Cell Surface; D017448:Receptors, Dopamine D2; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007545:Isoproterenol