title: Thermostabilization of the Human Endothelin Type B Receptor.
abstract: The peptide hormone endothelin, produced by the vascular endothelium, is involved in several physiological functions, including maintenance of vascular tone and humoral homeostasis. Endothelin transmits signals through the endothelin receptor, a G-protein-coupled receptor. Structural studies of the endothelin type B receptor (ETBR) have been unsuccessful due to its structural flexibility and instability in detergent-solubilized solution. To overcome these problems, we explored thermostabilization of human ETBR by establishing an ETBR expression system in Escherichia coli, followed by systematic alanine scanning mutagenesis. Among 297 point mutations, 11 thermostabilizing residues were selected and further mutated to other amino acids. The thermostability indices of these residues, represented by the ratios of endothelin-1 (ET-1) binding activities with or without heat treatment at 27°C for 30min in a ligand-free form, were compared. The ligand affinity and apparent melting temperature (Tm) of the five most thermostable mutants, R124Y, D154A, K270A, S342A, and I381A, were then examined. The apparent Tm of three single mutants, R124Y, D154A, and K270A, was approximately 7°C higher than that of the wild type. The apparent Tm value of a combination of the five residues, named the Y5 ETBR mutant, was 17°C higher than that of the wild type. The Y5 ETBR mutant exhibited an affinity for ET-1 and activated Gq similar to the wild type. Further investigation of the pharmacological properties affected by combinatorial mutations of ET-1, ET-3, TxET-1, and K8794 suggested that Y5 ETBR is highly suitable for representing a ligand-free form of ETBR and is potentially applicable for studying an ET-1-bound form.
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan; Cellular and Structural Physiology Institute, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Okuta
authors.affiliation: Cellular and Structural Physiology Institute, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Tani
authors.affiliation: Cellular and Structural Physiology Institute, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
authors.forename: Shoko
authors.initials: S
authors.lastname: Nishimura
authors.affiliation: Cellular and Structural Physiology Institute, Nagoya University, Chikusa, Nagoya 464-8601, Japan; Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Fujiyoshi
authors.affiliation: Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan. Electronic address: doi@mb.biophys.kyoto-u.ac.jp.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Doi
mesh_terms: D019332:Endothelin-1 / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000502:physiology; D017466:Receptors, Endothelin / Q000235:genetics / Q000378:metabolism*; D044366:Transition Temperature
chemical_list: D019332:Endothelin-1; D008024:Ligands; D017466:Receptors, Endothelin
keywords: G-protein-coupled receptor; alanine scanning mutagenesis; homology model; thermostabilization