title: Constitutive activity of endogenous receptors by inducible Gq overexpression.
abstract: We have developed an inducible cell line that transiently expresses Gq alpha G protein subunits in response to doxycycline. HEK293/Tet-On pBI(Gq alpha) cells worked consistently, achieving high and tightly regulated levels of Gq alpha overexpression (38-fold increase compared with non-induced cells). We investigated the possibility of using an inducible system to increase the proportion of constitutively active endogenously expressed G protein-coupled receptors (GPCRs) by overexpressing Gq alpha. Not only did we observe an increase in basal activity following doxycycline treatment, but also increased intrinsic activity of agonists such as carbachol, endothelin, lysophosphatidic acid (LPA), and bradykinin. Furthermore, carbachol and LPA potency increased following Gq alpha overexpression, as did the intrinsic activity of the partial agonist pilocarpine, observations indicative of constitutive activity. An inducible cell line, transiently expressing G proteins, can therefore be employed to induce constitutive activity of endogenously expressed GPCRs. This model system could be used to identify clinically important inverse agonists.
authors.affiliation: Cardiovascular Research Institute at Leeds, School of Medicine, University of Leeds, Leeds LS2 9JT, UK. J.Scragg@leeds.ac.uk
authors.forename: Jason L
authors.initials: JL
authors.lastname: Scragg
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Ball
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Balmforth
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001920:Bradykinin / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D006224:Cricetinae; D004318:Doxycycline / Q000494:pharmacology; D016232:Endothelins / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism*; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*
chemical_list: D016232:Endothelins; D008246:Lysophospholipids; D043562:Receptors, G-Protein-Coupled; D002217:Carbachol; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D004318:Doxycycline; C032881:lysophosphatidic acid; D001920:Bradykinin