Journal of the Renin- Angiotensin- Aldosterone System (Including other peptidergic systems) September 2000 Volume 1 Number 3 283 Paper Lys 199 mutation of the human angiotensin type 1 receptor differentially affects the binding of surmountable and insurmountable non-peptide antagonists Frederik LP Fierens,* Patrick ML Vanderheyden,* Zsuzsanna Gáborik, † Tam Le Minh,* Jean-Paul De Backer,* László Hunyady, † Adriaan Ijzerman, § Georges Vauquelin* Keywords: CHO cells, angiotensin II, human AT 1 -receptor, mutation, insurmountable non-peptide antagonist * Department of Molecular and Biochemical Pharmacology, Institute of Molecular Biology and Biotechnology, Free University of Brussels (VUB), B-1640 Sint-Genesius Rode, Belgium † Department of Physiology, Semmelweis University Medical School, H-1444 Budapest, PO Box 259, Hungary § Division of Medicinal Chemistry, Leiden/Amsterdam Centre of Drug Research, Universiteit Leiden, PO Box 9502, 230 RA Leiden, The Netherlands Correspondence to: Frederik Fierens M.Sc., Department of Molecular and Biochemical Pharmacology, Free University of Brussels (VUB), Paardenstraat 65, B-1640 Sint-Genesius Rode, Belgium Tel: +32 2 3583139 Fax: +32 2 3590265 E-mail: ffierens@ vub.ac.be Accepted for publication 6th September 2000 JRAAS 2000;1:283-8 Abstract Many slow dissociating (insurmountable) non-peptide angiotensin type 1 receptor (AT 1 ) antagonists contain, besides the acidic biphenyltetrazole substructure of losartan, a second acidic group to stabilise antagonist- receptor complexes. To investigate the involved basic amino-acids of the human AT 1 -receptor, wild-type and mutant receptors were transiently transfected in CHO-K1 cells and characterised by [ 3 H]candesartan binding. Lys 199 Gln substitution decreased the affinity 45-fold for candesartan (95% insurmountable), 18-fold for EXP3174 (70% insurmountable), 10-fold for irbesartan (40% insurmountable) and 5-fold for losartan (surmountable). His 256 Ala substitution had only minor effects. This suggests that Lys 199 is important for the tight binding of non-peptide antagonists. Introduction There is much interest in the therapeutic use of selective non-peptide antagonists for the AT 1 - receptor, which plays an important role in cardio- vascular homeosatasis. When pre-equilibrated with rabbit aortic rings, surmountable antagonists (e.g. losartan) produce a dose-dependent right- ward shift of the angiotensin II (Ang II) dose-con- traction curve. Insurmountable antagonists also produce a partial (irbesartan, EXP3174) or almost complete (candesartan) depression of the maxi- mal response. 1-4 More recently, relevant informa- tion was also obtained by measuring Ang II- induced inositol phosphate production in stably transfected CHO-K1 cells expressing human AT 1 - receptors (CHO-hAT 1 cells). Antagonist pre-incu- bation of CHO-hAT 1 cells produced the same inhibitory effect on the concentration-response curves of subsequently added Ang II as in aortic ring contraction studies. 5 However, the same antagonists only produced a rightward shift of the Ang II dose-response curve under co-incubation conditions. 6 These findings suggest that the antag- onists inhibit the Ang II-mediated inositol phos- phate production in a competitive manner. Based on additional kinetic studies, it was proposed that the insurmountable behaviour of some AT 1 antag- onists in pre-incubation experiments is related to their slow dissociation from the receptor, thereby preventing the access of these receptors to subse- quently added Ang II. 7 Most non-peptide AT 1 -receptor antagonists (AIIAs) contain the same biphenyltetrazole sub- structure as losartan. Whereas losartan only pos- sesses an acidic tetrazole moiety, many insur- mountable AIIA are di-acidic molecules.This addi- tional acidic group could interact with basic amino acid residues of the receptor and stabilise slowly reversible antagonist-receptor complexes. Site-directed mutagenesis studies, involving the substitution of basic amino acid residues of the receptors, offer a way to evaluate this hypothesis. In the present study, we set out to evaluate the effect of mutation of Lys 199 on the binding of [ 3 H]candesartan and other non-peptide antago- nists and whether this is related to their order of insurmountability. Methods and materials Materials 2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4- yl)methyl]-1H-benzimidazoline-7-carboxylic acid (Candesartan), 3,8 2-n-butyl-4-chloro-1-[(2'-(1H-tetra- zol-5-yl)biphenyl-4-yl)methyl] imidazole-5-carboxylic acid (EXP3174), 9 2-n-butyl-4-chloro-5-hydrox- ymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4- yl)methyl] imidazole (losartan) 9 and 2-n-butyl-4- spirocyclopentane-1-[(2'-(1H-tetrazol-5-yl)biphenyl- 4-yl)methyl]2-imidazolin-5-one (irbesartan) 2 were obtained from AstraZeneca (Mölndal, Sweden). The chemical structures are shown in Figure 1. Ang II and [sar 1 -ile 8 ]Ang were obtained from Sigma. [ 3 H]candesartan (17 Ci/mmol) was kindly provided by AstraZeneca. Lipofectamine was from GibcoBRL, Life Technologies, Merelbeke, Belgium. All other chemicals were of the highest grade commercially available. Mutagenesis of human AT 1 -receptor DNA, cell culture and transient transfection The human AT 1 -receptor cDNA was subcloned in the mammalian expression vector pcDNA3 (Invitrogen, San Diego, USA) using the Hind III and Xba sites of this plasmid.Mutant human AT 1 -recep- tors were created using the Mutagene kit (Bio- Rad, Hercules, CA, USA). Each mutant contained a silent restriction site to facilitate the screening of the colonies. Oligonucleotides were from Creative Lab. Kft (Szeges, Hungary). All mutations were veri- fied by dideoxy sequencing using Sequenase II by guest on November 15, 2015 jra.sagepub.com Downloaded from