A novel expression system for Gs-coupled receptors.
Renal epithelial cells LLC-PK1 secrete high amounts of urokinase-type plasminogen activator (uPA) when stimulated through the cAMP-signaling pathway. Here we show that this characteristic can be used to identify rapidly LLC-PK1-derived cell lines expressing heterologous receptors that couple to Gs-proteins. A genomic clone encoding the mouse beta 2 adrenergic receptor (beta 2AR) was transfected stably into LLC-PK1 cells. Drug-resistant colonies were subsequently stimulated with isoproterenol and screened for the secretion uPA activity. Positive clones displayed dose-dependent uPA activity in response to isoproterenol, specifically bound the beta 2AR-agonist iodocyanopindolol and expressed beta 2AR-mRNA. The functional coupling of beta 2AR expression to the inducible uPA gene establishes that LLC-PK1 cells can be used to assay for beta 2AR (and beta 2AR-agonist) function. Our data suggest that this system can be used for functional expression of other heterologous receptors that couple to Gs-proteins.
Catanzariti
L
L
Friedrich Miescher-Institut, Basel, Switzerland.
Allen
J M
JM
Hemmings
B A
BA
D000818:Animals; D015152:Blotting, Northern; D002116:Calcitonin / Q000494:pharmacology; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004848:Epithelium; D019204:GTP-Binding Proteins / Q000502:physiology*; D015870:Gene Expression; D007545:Isoproterenol / Q000008:administration & dosage / Q000494:pharmacology; D007668:Kidney; D051379:Mice; D011943:Receptors, Adrenergic, beta / Q000235:genetics*; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism; D014667:Vasopressins / Q000494:pharmacology
D011943:Receptors, Adrenergic, beta; D014667:Vasopressins; D002116:Calcitonin; D014568:Urokinase-Type Plasminogen Activator; D019204:GTP-Binding Proteins; D007545:Isoproterenol