title: Purification and characterization of recombinant human endothelin receptor type A.
abstract: Human endothelin receptor type A (ET(A)) is a G-protein coupled receptor that mediates vasoconstriction of blood vessels. To determine the structural characteristics and signaling mechanism of ET(A), we have expressed recombinant ET(A) as a fusion protein with p9 envelope protein from phi6 bacteriophage. The His-tag-labeled p9-ET(A) fusion protein was highly expressed in the membrane fraction of Escherichia coli and purified to homogeneity by single affinity chromatography after solubilization with detergents. Purified p9-ET(A) appeared as an oligomer and presented mainly as an α-helical structure. The protein also showed specific binding to endothelin-1 (ET-1) and the alpha subunit of G(q) protein with apparent K(D) values of 17 and 20 nM, respectively. An antagonist of ET(A), bosentan, prevented the interaction between p9-ET(A) and ET-1 in a concentration-dependent manner. These results indicate that recombinant p9-ET(A) has a competent conformation for interactions with ET-1 and the alpha subunit of G(q) protein.
authors.affiliation: Department of Chemistry, Kookmin University, 861-1 Jeongneung-dong, Seongbuk-gu, Seoul 136-702, Republic of Korea.
authors.forename: Kwangkyu
authors.initials: K
authors.lastname: Lee
authors.forename: Yuna
authors.initials: Y
authors.lastname: Jung
authors.forename: Jae Youl
authors.initials: JY
authors.lastname: Lee
authors.forename: Won-Kyu
authors.initials: WK
authors.lastname: Lee
authors.forename: Dongbin
authors.initials: D
authors.lastname: Lim
authors.forename: Yeon Gyu
authors.initials: YG
authors.lastname: Yu
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D019332:Endothelin-1 / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011487:Protein Conformation; D044022:Receptor, Endothelin A / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D019332:Endothelin-1; D044022:Receptor, Endothelin A; D011993:Recombinant Fusion Proteins; D014763:Viral Matrix Proteins; C088019:protein P9, bacteriophage phi6; C033223:polyhistidine; D006639:Histidine; D043802:GTP-Binding Protein alpha Subunits, Gq-G11