Purification and characterization of recombinant human endothelin receptor type A

Human endothelin receptor type A (ETA) is a G-protein coupled receptor that mediates vasoconstriction of blood vessels. To determine the structural characteristics and signaling mechanism of ETA, we have expressed recombinant ETA as a fusion protein with p9 envelope protein from phi6 bacteriophage. The His-tag-labeled p9-ETA 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-ETA 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 Gq protein with apparent KD values of 17 and 20 nM, respectively. An antagonist of ETA, bosentan, prevented the interaction between p9-ETA and ET-1 in a concentration-dependent manner. These results indicate that recombinant p9-ETA has a competent conformation for interactions with ET-1 and the alpha subunit of Gq protein.

► An overexpression system of human endothelin receptor (ETA) was established.
► Bacterially expressed recombinant ETA was purified to homogeneity.
► Recombinant ETA composed of mainly α-helices.
► Recombinant ETA interacts specifically with ET-1 and Gαq.