Functional analysis of cell‐free‐produced human endothelin B receptor reveals transmembrane segment 1 as an essential area for ET‐1 binding and homodimer formation

Abstract

The functional and structural characterization of G-protein-coupled receptors (GPCRs) still suffers from tremendous difficulties during sample preparation. Cell-free expression has recently emerged as a promising alternative approach for the synthesis of polytopic integral membrane proteins and, in particular, for the production of G-protein-coupled receptors. We have now analyzed the quality and functional folding of cell-free produced human endothelin type B receptor samples as an example of the rhodopsin-type family of G-protein-coupled receptors in correlation with different cell-free expression modes. Human endothelin B receptor was cell-free produced as a precipitate and subsequently solubilized in detergent, or was directly synthesized in micelles of various supplied mild detergents. Purified cell-free-produced human endothelin B receptor samples were evaluated by single-particle analysis and by ligand-binding assays. The soluble human endothelin B receptor produced is predominantly present as dimeric complexes without detectable aggregation, and the quality of the sample is very similar to that of the related rhodopsin isolated from natural sources. The binding of human endothelin B receptor to its natural peptide ligand endothelin-1 is demonstrated by coelution, pull-down assays, and surface plasmon resonance assays. Systematic functional analysis of truncated human endothelin B receptor derivatives confined two key receptor functions to the membrane-localized part of human endothelin B receptor. A 39 amino acid fragment spanning residues 93-131 and including the proposed transmembrane segment 1 was identified as a central area involved in endothelin-1 binding as well as in human endothelin B receptor homo-oligomer formation. Our approach represents an efficient expression technique for G-protein-coupled receptors such as human endothelin B receptor, and might provide a valuable tool for fast structural and functional characterizations.

Breaking the bottleneck: Eukaryotic membrane protein expression for high-resolution structural studies

“…Another alternative involves bacterial expression of fusion proteins with the membrane-integrating protein mistic: preliminary data have suggested that it may facilitate the folding and insertion of eukaryotic proteins in bacterial hosts (Roosild et al, 2005). Finally, cell-free expression systems have been shown to support the production of milligram quantities of solubilized, functional IMPs (Liguori et al, 2007), particularly for GPCRs, in which ligand binding and structural homogeneity could readily be demonstrated (Ishihara et al, 2005;Klammt et al, 2007).…”

Biosynthesis and NMR-studies of a double transmembrane domain from the Y4 receptor, a human GPCR

“…Recent advances in the expression and purification of membrane proteins have been described for various expression hosts, for example: Escherichia coli (Drew et al 2003(Drew et al , 2005Grisshammer et al 2005), yeast (Wedekind et al 2006;Lee et al 2007), insect cells (Massotte 2003), mammalian cells (Yin et al 2005;Werner et al 2008) and cell-free systems (Klammt et al 2007). However, from approximately 1000 known GPCRs, only five high-resolution 3-D structures of two distinct receptor types have been reported: bovine rhodopsin (Palczewski et al 2000) and opsin (Park et al 2008), squid rhodopsin (Murakami and Kouyama 2008), the human b2-adrenergic receptor Rosenbaum et al 2007) and the turkey b1-adrenergic receptor (Warne et al 2008).…”

Validation of histological diagnostic methods for detecting endothelin B receptor expression

“…It has been reported that ETBR can form a homodimer (20,21). In this case, the homodimer shows a size of 100 kDa, and polymerization of the homodimer or a combination of the homodimer and other membrane proteins can show a size exceeding 200 kDa.…”