Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments

Abstract

The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured in association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications.

Keywords: 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol); 1,2-dimyristoyl-sn-glycero-3-phosphocholine; 1,2-dioleoyl-sn-glycero-3-phosphocholine; 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]; 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Aso-PC; BTE; CECF; CF; CHS; CVs; Cell-free expression; DMPC; DMPG; DOPC; EPL; ETA; ETB; Escherichia coli polar lipids; FM; G-protein coupled receptor; GPCR; HTL; Human endothelin system; IMAC; LMPG; LPPG; LT-SDS-PAGE; Lipid screening; MSP; ND; Nanodisc; PBS; POPC; PR; RM; SDS-PAGE; SPR; Vasoactive peptide; brain total extract; cell-free; cholesteryl hemisuccinate; column volumes; continuous exchange cell-free; endothelin A receptor; endothelin B receptor; feeding mix; heart total lipids; immobilized metal affinity chromatography; low temperature SDS-PAGE; membrane scaffold protein; nanodisc; phosphate buffered saline; proteorhodopsin; reaction mix; red shifted green fluorescent protein; sGFP; sodium dodecyl sulfate polyacrylamide gel electrophoreses; soybean l-α-phosphatidylcholine; surface plasmon resonance.