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Role of basic amino acids of the human angiotensin type 1 receptor in the binding of the non-peptide antagonist candesartan

Department of Molecular and Biochemical Pharmacology, Institute for Molecular Biology and Biotechnology, Free University of Brussels (VUB), B-1640 Sint-Genesius Rode, Belgium, gvauquel@ vub.ac.be

Frederik LP Fierens

Department of Molecular and Biochemical Pharmacology, Institute for Molecular Biology and Biotechnology, Free University of Brussels (VUB), B-1640 Sint-Genesius Rode, Belgium

Zsuzsanna Gáborik

Department of Physiology, Semmelweis University Medical School, H-1444 Budapest, PO Box 259, Hungary

Tam Le Minh

Department of Molecular and Biochemical Pharmacology, Institute for Molecular Biology and Biotechnology, Free University of Brussels (VUB), B-1640 Sint-Genesius Rode, Belgium

Jean-Paul De Backer

Department of Molecular and Biochemical Pharmacology, Institute for Molecular Biology and Biotechnology, Free University of Brussels (VUB), B-1640 Sint-Genesius Rode, Belgium

László Hunyady

Department of Physiology, Semmelweis University Medical School, H-1444 Budapest, PO Box 259, Hungary

Patrick ML Vanderheyden

Department of Molecular and Biochemical Pharmacology, Institute for Molecular Biology and Biotechnology, Free University of Brussels (VUB), B-1640 Sint-Genesius Rode, Belgium

To explain the insurmountable/long-lasting binding of biphenyltetrazole-containing AT₁-receptor antagonists such as candesartan, to the human angiotensin II type 1-receptor, a model is proposed in which the basic amino acids Lys¹⁹⁹ and Arg ¹⁶⁷ of the receptor interact respectively with the carboxylate and the tetrazole group of the antagonists. To validate this model, we have investigated the impact of substitution of Lys¹⁹⁹ by Ala or Gln and of Arg¹⁶⁷ by Ala on the binding properties of [³H]candesartan and on competition binding by candesartan, EXP3174, irbesartan, losartan, angiotensin II (Ang II) and [Sar¹-Ile⁸]angiotensin. Our results indicate that both amino acids play an important role in the AT₁-receptor ligand binding. Whereas the negative charge of Lys ¹⁹⁹ is involved in an ionic bond with the end-standing carboxylate group of the peptide ligands, its polarity also contributes to the non-peptide antagonist binding. Substitution of Arg¹⁶⁷ by Ala completely abolished [³H]Ang II, as well as [³H] candesartan, binding. Whereas these results are in line with the proposed model, it cannot be excluded that both amino acid residues are important for the structural integrity of the AT₁-receptor with respect to its ligand binding properties.

Key Words: CHO cells • angiotensin II • human AT1-receptor • mutation • candesartan • insurmountable • non-peptide antagonist

Journal of Renin-Angiotensin-Aldosterone System, Vol. 2, No. 1 suppl, S32-S36 (2001)
DOI: 10.1177/14703203010020010501


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