title: AT1 receptor blocker-insensitive mutant AT1A angiotensin receptors reveal the presence of G protein-independent signaling in C9 cells.
abstract: Although mutant receptors are highly useful to dissect the signal transduction pathways of receptors, they are difficult to study in physiological target tissues, due to the presence of endogenous receptors. To study AT(1) angiotensin receptors in their physiological environment, we constructed a mutant receptor, which differs only from the AT(1A) receptor in its reduced affinity for candesartan, a biphenylimidazole antagonist. We have determined that the conserved S109Y substitution of the rat AT(1A) receptor eliminates its candesartan binding, without exerting any major effect on its angiotensin II and peptide angiotensin receptor antagonist binding, internalization kinetics, beta-arrestin binding, and potency or efficacy of the inositol phosphate response. To demonstrate the usefulness of this mutant receptor in signal transduction studies, we combined it with substitution of the highly conserved DRY sequence with AAY, which abolishes G protein activation. In rat C9 hepatocytes the S109Y receptor caused ERK activation with the same mechanism as the endogenous AT(1) receptor. After combination with the DRY/AAY mutation G protein-independent ERK activation was detected demonstrating that this approach can be used to study the angiotensin II-stimulated signaling pathways in cells endogenously expressing AT(1) receptors.
authors.affiliation: Department of Physiology, Semmelweis University, Budapest, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Szidonya
authors.forename: Katinka
authors.initials: K
authors.lastname: Süpeki
authors.forename: Eszter
authors.initials: E
authors.lastname: Karip
authors.forename: Gábor
authors.initials: G
authors.lastname: Turu
authors.forename: Péter
authors.initials: P
authors.lastname: Várnai
authors.forename: Adrian J L
authors.initials: AJ
authors.lastname: Clark
authors.forename: László
authors.initials: L
authors.lastname: Hunyady
mesh_terms: D019943:Amino Acid Substitution; D047228:Angiotensin II Type 1 Receptor Blockers / Q000494:pharmacology*; D000818:Animals; D019390:Arrestins / Q000494:pharmacology; D001562:Benzimidazoles / Q000494:pharmacology*; D001713:Biphenyl Compounds; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D009132:Muscles / Q000166:cytology; D009154:Mutation; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics*; D013777:Tetrazoles / Q000494:pharmacology*; D014443:Tyrosine / Q000235:genetics; D000071557:beta-Arrestins
chemical_list: D047228:Angiotensin II Type 1 Receptor Blockers; D019390:Arrestins; D001562:Benzimidazoles; D001713:Biphenyl Compounds; D044140:Receptor, Angiotensin, Type 1; D013777:Tetrazoles; D000071557:beta-Arrestins; D014443:Tyrosine; D012694:Serine; D019204:GTP-Binding Proteins; C081643:candesartan