Báo cáo khoa học: 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 pptx

Functional analysis of cell-free-produced human endothelin B receptor reveals transmembrane segment as an essential area for ET-1 binding and homodimer formation Christian Klammt1, Ankita Srivastava2, Nora Eifler3, Friederike Junge1, Michael Beyermann4, Daniel Schwarz1, Hartmut Michel2, Volker Doetsch1 and Frank Bernhard1 Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, University of Frankfurt ⁄ Main, Germany Max-Planck-Institute for Biophysics, Department of Molecular Membrane Biology, Frankfurt ⁄ Main, Germany M.E Mueller Institute for Microscopy, Biocentre, University of Basel, Switzerland Leibniz-Institute of Molecular Pharmacology, Department of Peptide Chemistry & Biochemistry, Berlin, Germany Keywords cell-free expression; detergent micelles; endothelin-1 ligand-binding site; G-protein coupled receptor; single-particle analysis Correspondence F Bernhard, Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, University of Frankfurt ⁄ Main, Max-von-Laue-Str 9, D-60438 Frankfurt ⁄ Main, Germany Fax: +49 69 798 29632 Tel: +49 69 798 29620 E-mail: fbern@bpc.uni-frankfurt.de (Received 27 March 2007, revised 26 April 2007, accepted 27 April 2007) doi:10.1111/j.1742-4658.2007.05854.x 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 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 Abbreviations bET-1, biotinylated endothelin-1; C, cytoplasmic; CECF, continuous exchange cell-free; cET-1, Cy3-labeled endothelin-1; CF, cell-free; CTD, C-terminal domain; E, extracellular; ETB, human endothelin B receptor; ET-1, endothelin-1; GPCR, G-protein-coupled receptor; LMPG, 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)]; NTD, N-terminal domain; RM, reaction mixture; SPR, surface plasmon resonance; TMS, transmembrane segment FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3257 Ligand binding of cell-free endothelin B receptor C Klammt et al G-protein-coupled receptors (GPCRs) form a large superfamily of membrane proteins, and their genes comprise an estimated 1–5% of vertebrate genomes They modulate the activity of specific targets such as ion channels or enzymes via G-protein coupling, and thus initiate intracellular signaling cascades in response to a broad range of external signals [1,2] GPCRs have a similar architecture, composed of seven transmembrane segments (TMS1–7) connected by three extracellular (E1–3) and three cytoplasmic (C1–3) loops In addition, GPCRs contain a more or less extended N-terminal domain and C-terminal domain, which are both often involved either in ligand binding or G-protein coupling Owing to their key role in signal transduction in eukaryotic cells, GPCRs are estimated to represent the targets for more than 50% of modern pharmaceutical drugs [3] Despite much investigation, the high-resolution structural evaluation of GPCRs as a prerequisite for directed drug design is so far still limited to the naturally abundant phototransducer rhodopsin [4] As for many other membrane proteins, the first bottleneck in structural and functional characterization of GPCRs is the production of sufficient amounts of protein sample Although considerable improvements have been made, the overproduction of GPCRs in cellular expression systems based on bacterial, yeast or insect cells is still complicated and often inefficient [5–11] Continuous exchange cell-free (CECF) expression systems based on Escherichia coli cell extracts have recently been demonstrated to provide a new and highly promising tool for the preparative-scale production of membrane proteins [12–14] Besides the elimination of toxic effects upon membrane protein overproduction, a unique advantage of CECF systems is the possibility of directly producing soluble membrane proteins in the presence of detergents [12,13,15,16] This completely new strategy provides an artificial hydrophobic environment that is able to interact with membrane proteins during translation Protein precipitation is prevented, and functional folding pathways can be facilitated [17,18] The protein–detergent association is initiated by hydrophobic interactions, and specific targeting or translocation systems are therefore not necessary The endothelin (ET) system is involved in many physiologic processes, such as control of vascular tone, neurotransmission, embryonic development, renal function, and regulation of cell proliferation, and it thus plays an important role in physiopathologic disorders such as congestive heart failure, diabetes, atherosclerosis, and primary pulmonary hypertension [19–21] The human ET receptor type B (ETB) is a prototypic 3258 GPCR distributed among multiple endothelial cell types as well as in smooth muscle cells, where it transmits vasoactive effects by binding the 21-mer isopeptides ET-1, ET-2, and ET-3 ETB has equally potent affinities for ET-1, ET-2 and ET-3, in contrast to the homologous ET receptor type A, which has a higher affinity for ET-1 and ET-2 We have established protocols for the high-level production of ETB and other GPCRs in an individual CECF system [22] The GPCRs can be synthesized as precipitates or in soluble form in micelles of selected detergents, and apart from small terminal peptide tags that facilitate detection and purification, no large fusion proteins are needed for expression and stabilization The functional folding of membrane proteins overproduced by the new cell-free (CF) approach is of primary interest, and we therefore further analyzed the quality of ETB samples obtained after CF production under different conditions Ligand-binding and oligomer formation studies demonstrated that CF-produced ETB is functionally folded when synthesized in the presence of Brij detergents, and single-particle analysis revealed nonaggregated proteins that predominantly form dimeric complexes On the basis of the functional in vitro analysis of rationally designed terminal ETB truncations, we specified a core domain responsible for ET-1 binding as well as for receptor dimerization in a relatively small region centered on TMS1 Results CF production of ETB CECF reaction protocols were essentially performed as previously described [22] Full-length ETBcHx and truncated derivatives were produced as translational fusions with the small 12 amino acid T7-tag at the N-terminus and a poly(His)10-tag at the C-terminus (Fig 1) To obtain the highest yields of the individual constructs, optimization of the Mg2+ and K+ concentrations in the ranges 12–16 mm and 250–340 mm was critical Under optimized conditions, ETBcHx could by synthesized in yields of up to mg per mL of reaction mixture (RM), and it was separated as a prominent band of c 46 kDa by SDS ⁄ PAGE (Fig 2) The fulllength synthesis of ETBcHx was verified by immunodetection with antibodies directed against the N-terminal T7-tag and the C-terminal poly(His)10-tag, respectively (data not shown) ETBcHx was CF produced either as a soluble protein in the presence of detergents or as a precipitate in the absence of detergents We analyzed the yield and sample quality of ETB synthesized in the presence of FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS C Klammt et al Ligand binding of cell-free endothelin B receptor Fig Proposed secondary structure of ETB The proposed seven TMSs are illustrated Various tags used for the modification of CF-produced ETBs are indicated Predicted sites for post-translational modifications are a glycosylation site at N59, a disulfide bridge connecting E1 and E2, and several palmitoylation sites at cysteine residues in the CTD The first (black) and last (gray) amino acid positions used for the construction of truncated ETB fragments are indicated Fig PAGE analysis of CF-expressed full-length ETB on a 12% SDS gel M, marker Lane 1: RM control Lane 2: supernatant of ETBcHx expression in the presence of Brij58 Lane 3: supernatant of ETBcHx expression in the presence of Brij78 Lane 4: ETBcHx after Ni–chelate acid chromatography Lane 5: supernatant of ETBStrep expression in the presence of Brij78 Lane 6: ETBStrep after Strep-Tactin purification Lane 7: ETBcHx precipitate after expression without detergent Lane 8: ETBcHx precipitate solubilized in 1% LMPG Lane 9: ETBcHx precipitate solubilized in 1% LMPG after Ni–chelate acid chromatography ETB is marked by arrows digitonin and long-chain Brij derivatives, as these detergents have been most effective for the soluble expression of GPCRs [15,22] In the presence of 1% Brij78 and 1.5% Brij58, completely soluble ETBcHx with final yields of c mg proteinỈ(mL RM))1 was produced With Brij35 (0.1%) and digitonin (0.4%), only 500 lg and 100 lg of soluble ETBcHx per mL of RM were obtained, respectively, in addition to ETBcHx precipitate The soluble ETBcHx produced was purified in one step by Ni-chelate chromatography, and, on average, c 60% of the synthesized ETBcHx in the RM was recovered ETBcHx precipitate CF produced in the absence of detergents was completely solubilized in 1% 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac(1-glycerol)] (LMPG) and purified by Ni-chelate chromatography Coelution of purified ETB with ET-1 The mode of CF expression, i.e expression as precipitate or as soluble protein, as well as the type of FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3259 Ligand binding of cell-free endothelin B receptor C Klammt et al detergent, could have a significant impact on the folding of ETBcHx into a functional conformation The affinity for its natural peptide ligand ET-1 was therefore analyzed with purified ETBcHx samples produced under different conditions Mixtures of the Cy3-dyelabeled derivative Cy3-labeled endothelin-1 (cET-1) with purified ETBcHx, either CF produced as precipitate and solubilized in 1% LMPG or directly produced as soluble protein in the presence of 1.5% Brij58, 1% Brij78 or 0.4% digitonin, respectively, were separated by gel filtration, and the elution fractions were analyzed by taking advantage of the different absorbancies of the two compounds (Fig 3) The 52 kDa ETBcHx elutes at a retention volume of 1.6 mL, whereas the 21-mer cET-1 starts to elute at a volume of 2.1 mL Coelution of cET-1 with ETBcHx therefore indicates complex forma- tion of the receptor with its ligand, giving evidence of a native protein conformation In contrast, CF-produced ETBcHx present in an unfolded or inactive conformation should result in the separation of the two compounds cET-1 was completely separated from ETBcHx samples that were CF produced as precipitate and solubilized in LMPG, indicating that, despite solubilization, the receptor might not have adopted its native conformation In contrast, significant amounts of cET-1 coeluted with ETBcHx synthesized in the soluble mode of CF expression in the presence of digitonin, Brij58 and Brij78 The highest apparent binding of cET-1 was obtained with protein CF expressed in the presence of Brij78 This expression condition was therefore chosen for further sample preparations of ETBcHx and its derivatives Fig Functional conformation of full-length ETBcHx ET-1 binding of ETBcHx analyzed by coelution Purified ETB samples produced CF at different conditions were incubated with cET-1 for h at 21 °C, and subsequently analyzed on a Superose PC 3.2 ⁄ 30 column The elution chromatograms show total protein absorption at 280 nm (solid line) and specific absorption of cET-1 at 550 nm (dashed line) The retention volume of ETBcHx is indicated by arrows (A) ETBcHx CF expressed as precipitate and resolubilized in 1% LMPG; (B) soluble ETBcHx expressed in the presence of 0.4% digitonin; (C) soluble ETBcHx expressed in the presence of 1.5% Brij58; (D) soluble ETBcHx expressed in the presence of 1% Brij78 3260 FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS C Klammt et al The percentage of ligand-binding receptor present in purified ETBcHx samples obtained after soluble CF expression in the presence of 1% Brij78 was determined by correlation of the molar ratio of complexed cET-1 with the amount of supplied ETBcHx After background subtraction, we estimated the amount of ligand-binding ETBcHx present in samples obtained under the described conditions as c 50% This value is similar to that for ETB samples obtained after conventional expression in insect cells Ligand binding of cell-free endothelin B receptor A Single-particle analysis of CF-produced ETBcHx The quality of CF-expressed and purified ETBcHx was analyzed by negative stain electron microscopy ETBcHx protein that was CF synthesized in the presence of Brij78 revealed evenly distributed particles with no detectable signs of aggregation (Fig 4A) ETBcHx synthesized under these conditions appears to be predominantly dimeric, and the good quality of the sample allowed further structural assessment using single-particle analysis Five hundred side views were reference-free aligned, classified, and averaged within the classes (Fig 4A) ETBcHx side view averages dis˚ play a pair of rods with a length of 63–68 A The distance between the centers of the rods corresponds to ˚ 35–38 A, and the rods are closely associated at one end These values are in excellent agreement with the dimensions observed for the rhodopsin dimer [23,24] Single rods, which presumably represent side views of dimers but could also be ETBcHx monomers, represent less than 10% of all particles In contrast, and in agreement with the observed inability to bind cET-1, ETBcHx produced as a precipitate and solubilized in LMPG was found to be aggregated, and is therefore most likely unfolded (Fig 4B) Localization of the ET-1-binding site In order to determine the ETB region that is essential for binding of ET-1, a series of eight plasmids coding for terminally truncated ETB fragments and containing different secondary structural elements were constructed (Table 1, Fig 1) All fragments could be overproduced in amounts of at least mgỈ(mL RM))1 in our CF system as soluble proteins in the presence of 1% Brij78 (Fig 5A) The fragments were purified after expression in one step by Ni-chelate chromatography, and the purity was evaluated by SDS ⁄ PAGE analysis (Fig 5B) The ligand binding of ETBcHx and truncated derivatives was characterized by pull-down assays of purified proteins with immobilized biotinylated ET-1 (bET-1), B Fig Single-particle analysis of CF-produced ETB Representative views of electronmicrographs of the negatively stained ETB fulllength construct (A) ETB produced as soluble protein in the presence of Brij78 The ETB sample appears to be nonaggregated, and particles are predominantly dimeric (black arrow); ETB monomers can be seen occasionally (white arrow) Side view class averages of reference-free aligned ETB dimers are displayed in the gallery on the right (B) ETB produced as precipitate and solubilized in LMPG The sample is no longer monodisperse, but rather forms aggregates as described in Experimental procedures Fractions containing complexes of bET-1 with ETB derivatives were separated by SDS ⁄ PAGE and blotted, and the proteins were identified by immunodetection with antibody to T7-tag (Fig 6) Only fragments containing TMS1-like full-length ETBcHx, ETB131 [N-terminal domain (NTD)-TMS1], ETB168 (NTD-TMS2), ETB203 (NTD-TMS3) and also the NTD-deleted fragment ETB93 (TMS1-TMS3) were detected in the eluted fractions, and formed complexes with bET-1 Accord- FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3261 Ligand binding of cell-free endothelin B receptor C Klammt et al Table Structural characteristics of CF-produced ETB derivatives With the exception of ETBDT7, all proteins contain additionally an N-terminal T7-tag x, included; –, deleted; ⁄ , partially truncated Included domains Fragment Region kDa NTD T1 C1 T2 E1 T3 C2 T4 E2 T5 C3 T6 E3 T7 CTD C-terminal tag ETBDT7 ETBcHx ETBStrep ETB131 ETB168 ETB203 ETB306 ETB132 ETB204 ETB307 ETB93 M1–S443 Q2–S443 E27–S443 E27–C131 E27–P168 E27–V203 E27–G306 M132–S443 A204–S443 M307–S443 P93–V203 50.7 52.5 49.5 14.4 18.5 22.2 34.1 38.6 30.8 18.8 15.3 x x x x x x x – – – ⁄ x x x x x x x – – – x x x x ⁄ x x x ⁄ – – x x x x – x x x x – – x x x x – ⁄ x x x – – x x x x – – x x x – – x x x x – – ⁄ x x ⁄ – ⁄ x x x – – – x x x – – x x x – – – x x x – – x x x – – – x x x – – x x x – – – ⁄ x x ⁄ – x x x – – – – x x x – x x x – – – – x x x – x x x – – – – x x x – x x x – – – – x x x – cH6 cHx Strep cHx cHx cHx cHx cHx cHx cHx cHx A B Fig PAGE analysis of CF-expressed ETB fragments on SDS gels (A) CF expression of ETB fragments in the presence of Brij78; 0.7 lL of supernatant (S) and lL of eluate (E) after Ni–chelate chromatography were analyzed The overproduced ETB truncations are indicated by arrows (B) Soluble CF-expressed, purified and reconstituted ETB fragments Nine microliters of each sample was analyzed on a 16.5% SDS gel 1, ETB93; 2, ETB131; 3, ETB168; 4, ETB203, 5, ETB306; 6, ETB132; 7, ETB204; 8, ETB307; 9, ETBcHx Arrows indicate the ETB derivative monomers Putative oligomeric forms are also visible ingly, proteins devoid of TMS1-like ETB132 (TMS2CTD) and ETB204 (TMS4-CTD) did not interact with bET-1 Analysis of ETBcHx–ligand interaction by surface plasmon resonance (SPR) Although the coelution approach gives good evidence for a ligand-binding activity of CF-produced ETB 3262 Fig Ligand binding of ETB derivatives Binding of ETB derivatives to bET-1 was analyzed by pull-down assays Bound proteins eluted from avidin matrix were separated on 16.5% SDS gels and detected by immunoblotting with antibody to T7-tag Arrows indicate detected bET-1-interacting ETB fragments, and asterisks indicate expected positions of noninteracting ETB derivatives M, marker samples, it is primarily not a quantitative assay SPR allows the sensitive detection and quantification of molecular interactions in real time We immobilized bET-1 on the streptavidin surface of the biosensor chip and analyzed the direct binding of functionally active ETBcHx ETBcHx solutions with increasing concentrations from 10 nm to 250 nm were loaded on the bET-1 chip, and binding kinetics were evaluated using biaevaluation 3.1 software In general, signals obtained from the Biacore assay were lower than expected for loading of ETBcHx as a relatively large analyte, and this effect is probably due to ligand occlusion by the detergent micelles Binding constants were therefore not determined by steady-state kinetics, but FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS C Klammt et al rather by association and dissociation rates, which were fitted by using : Langmuir models The determined kd was used for calculation of ka, and the binding constants KD were determined from kd ⁄ ka We determined the binding constant KD for binding of ETBcHx to bET-1 as 6.2 ± 1.7 · 10)9 (Fig 7) Similar assays with the C-terminal-truncated derivatives ETB131 and ETB93 revealed KD values of (2.7 ± 1.9) · 10)8 and (1.7 ± 0.5) · 10)8, respectively Ligand binding of cell-free endothelin B receptor A Identification of TMS1 as an essential element for ETB dimerization Several GPCRs are known to form dimers that remain stable even after SDS ⁄ PAGE analysis Protein bands corresponding to dimers or even higher oligomers of full-length ETBcHx and of most of the truncated derivatives are visible after separation of purified protein samples by SDS ⁄ PAGE (Fig 5A,B) In addition, our single-particle studies provided strong evidence of ETBcHx dimer formation We therefore attempted to identify the structural elements responsible for ETB oligomerization by analyzing heterodimer formation between full-length ETBStrep and the various truncated ETB fragments in two different pull-down assays First, purified ETB fragments and full-length ETBStrep were incubated at equimolar concentrations and then loaded on Strep-Tactin columns In a second assay, the full-length ETBStrep receptor was coexpressed with the various truncated fragments in CF reactions, and the RMs were then loaded on Strep-Tactin columns In both assays, the interacting protein fragments were identified after washing, elution and SDS ⁄ PAGE separation by immunoblotting with antibody to T7-tag (Fig 8) In the coexpression assays, the synthesis of fulllength ETBStrep and that of the corresponding ETB fragment was always visible by immunoblotting (Fig 8A) After loading of the RMs on Strep-Tactin columns, the fragments ETB93 (TMS1-TMS3), ETB131 (NTD-TMS1), ETB168 (NTD-TMS2), ETB203 (NTDTMS3) and ETB306 (NTD-TMS5) were coeluted together with ETBStrep, indicating an interaction of the proteins However, fragment ETB132 (TMS2-CTD) lacking the TMS1 region was not detectable in the eluted fraction, and therefore seems not to interact with ETBStrep After mixing of purified proteins, again the fragments ETB131 (NTD-TMS1), ETB168 (NTD-TMS2), ETB203 (NTD-TMS3) and ETB306 (NTD-TMS5) were found to interact with ETBStrep, whereas fragments lacking TMS1, such as ETB132 (TMS2-CTD) and ETB204 (TMS4-CTD), could not be B C Fig SPR response curves for the interaction of immobilized bET-1 with full-length ETB, ETB131 and ETB93 (A) Interaction of ETB between 10 and 250 nM (B) Interaction of ETB131 between 200 and 1600 nM (C) Interaction of ETB93 between 10 and 200 nM coeluted with full-length ETBStrep and were localized only in the flow-through of the Strep-Tactin column (Fig 8B) FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3263 Ligand binding of cell-free endothelin B receptor C Klammt et al A B Fig Analysis of dimerization of truncated ETB fragments with full-length ETBStrep containing a StrepII-tag Interacting proteins eluted from Strep-Tactin spin columns were separated on 16.5% SDS gels and immunoblotted with an antibody against the T7-tag (A) Interaction of ETBStrep and truncated ETB fragments after coexpression in the CF system in the presence of 1% Brij78 S, supernatant of the RM; E, corresponding eluted fractions from the Strep-Tactin columns (B) In vitro interaction of purified ETB fragments with purified ETBStrep Bound fractions or flow-throughs (F) were analyzed 1, ETB131–ETBStrep; 2, ETB168–ETBStrep; 3, ETB203– ETBStrep; 4, ETB306–ETBStrep; 5, ETB132–ETBStrep, flow-through; 6, ETB132–ETBStrep; 7, ETB204–ETBStrep, flow-through; 8, ETB204–ETBStrep M, marker; dotted arrow, full-length ETBstrep; solid arrow, truncated ETB fragments; gray arrow, putative ETBStrep–ETBxx heterodimers Discussion The high-level production of GPCRs in conventional in vivo systems such as E coli or Pichia pastoris cells can be very difficult and inefficient Successful approaches require the construction of large fusion proteins or intensive optimization [11,25] In addition, a variety of steps in conventional expression and purification protocols, such as kinetics of membrane insertion, saturation of biosynthetic translocation machinery, control of proteolysis, growth conditions, and extraction of recombinant membrane proteins from cellular membranes, are highly critical and need intensive optimization We have established a fast and efficient protocol for the high-level production of functionally folded human ETB and other GPCRs that eliminates most of the critical steps of conventional expression systems, as membranes and living cells are no longer involved 3264 Furthermore, the proteolysis of synthesized membrane proteins can easily be prevented by protease inhibitors N-terminal digestion of ETB, which is frequently observed upon in vivo expression, was not detectable by CF expression [26] Also, terminal truncated derivatives of ETB, which are often very difficult to express in vivo, due to proteolysis or translocation problems, can be produced at high levels in the CF system [27] A unique advantage of CF expression systems is the possibility of inserting membrane proteins directly into detergent micelles upon translation The efficiency of this solubilization mode was nearly 100% in the case of ETB, as no residual precipitate was detectable and expression levels were similar to those obtained in the absence of detergent The CF approach is very straightforward, and purified ETB protein in sufficient amounts for structural analysis can now be obtained in less than days It should also be mentioned that the production of labeled membrane proteins, even with complicated label combinations, is easily feasible by CF expression without the need for extensive optimization screens and without any loss of productivity [28–30] The CF expression technique might therefore become applicable also for the production of other GPCRs In this regard, we have already produced the porcine vasopressin type receptor and the rat corticotropin-releasing factor precursor receptor at high levels of several milligrams per milliliter of RM by using protocols very similar to that for ETB [15,17,22] In addition, larger thioredoxin fusions of the human M2 muscarinic acetylcholine receptor, of the human b2-adrenergic receptor and of the rat neurotensin receptor have been produced in CF systems in yields approaching mg of protein [16] As observed for production of the porcine vasopressin type receptor [15,22], only the steroid detergent digitonin and several long-chain polyoxyethylene derivatives such as Bri35, Brij58 and Brij78 were suitable for the CF synthesis of soluble ETB in milligram amounts Detergents of the Brij family are extremely mild detergents, being unable to disintegrate membranes, and they are tolerated by the CF transcription ⁄ translation machinery in amounts far exceeding 100 · critical micellar concentration [15] The quality of synthesized receptor can vary with the type of detergent, and the highest apparent ET-1 binding was obtained with Brij78, with some lower activity being seen in digitonin and other Brij derivatives In cellular systems, it is also known that the binding activity and structural integrity of GPCRs can be sensitive to the supplied detergents during solubilization [31] The extraction of active ligand-free ETB from cell membranes was only possible with digitonin [27] Accordingly, specific detergents FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS C Klammt et al were required for the functional folding of other membrane proteins, such as the nucleoside transporter Tsx, during CF expression [15] CF-produced precipitates of Tsx as well as of ETB did not adopt functional conformations upon solubilization An initial screen for suitable detergents is therefore most important for the production of functionally folded membrane proteins during CF expression in the soluble mode ETB is known to become post-translationally modified by palmitoylation, phosphorylation, and glycosylation However, these modifications not play a role in the ligand-binding capacities of ETB [32], and they are most likely absent in CF-produced ETB, resulting in more homogeneous sample preparations that might be even more suitable for crystallization studies On the other hand, disulfide bridge formation is very likely to occur in CF systems as long as no specific chaperones are required [33] SPR studies of GPCRs are generally difficult to perform, due to the intrinsic properties of these proteins Hydrophobic environments are necessary, and the SPR sensitivity level requires high receptor concentrations on the biosensor surface in order to detect the binding of low molecular weight ligands Therefore, only a few SPR measurements with GPCRs have been successful so far [31,34], but these reports have shown that ligands can bind solubilized GPCRs even in lipid-free environments and without the need for membrane reconstitution Most recently, a modified assay that employs the detergent-solubilized neurotensin receptor as the analyte has been described [35], and we successfully applied this approach to the characterization of ETB Interestingly, for both GPCRs, the amplitude of the observed response was lower than might be expected if the relatively high mass of the receptor used as analyte is considered Ligand occlusion by immobilization on the sensor chip surface, as well as limited access to the ligand-binding site of the receptor due to the presence of detergent molecules, might account for this effect Although there is still some potential for the optimization of this technique, e.g by systematic evaluation of sensor chip surfaces or of linker structures, the good correlation of the findings presented here with the published results obtained with neurotensin receptor indicate that the SPR technique could become a promising tool for the optimization of GPCR expression conditions, for the localization of ligand-binding sites, and for the identification of compounds with new properties that could be important for the pharmaceutical industry Human ETB forms a very tight complex with ET-1 that remains stable even in 2% SDS [36] ET-1 binds with high affinity to purified ETB in Brij78 micelles, as Ligand binding of cell-free endothelin B receptor indicated by the determined KD of nm, which is even lower than the value of 29 nm previously determined by total internal reflection fluorescence spectrometry with linear fluorescent labeled ET-1 [22] ETB ⁄ ET-1 dissociation constants determined in vivo in various cellular environments range between 40 pm and 300 pm [37–41] It is known that the ligand-binding kinetics of ETB in intact cells are different from those in corresponding membrane preparations [42] In addition, the interaction of ETB in vivo with other proteins, such as G-proteins or receptor activity-modifying proteins, might dramatically increase the affinity for distinct ligands [43] In this work, we determined the dissociation constant of pure ETB in the environment of detergent micelles, and this is also the first analysis of ETB by SPR measurement The different assay conditions, in addition to the use of a modified biotinylated ET-1 derivative as a ligand, have therefore most likely resulted in modified binding kinetics The localization of ligand-binding sites in ETB is still a subject of controversy Labeling of ETB with radioactive ET-1, followed by chemical crosslinking and trypsin-digest analysis, located the ET-1-binding domain between residues I85 in the NTD and Y200 in the second cytoplasmic loop C2 [44] In addition, deletions, mutations and the lack of glycosylation in the NTD were found to have no effect on ET-1 binding to ETB [27] Our direct in vitro analysis of purified N-terminally and C-terminally truncated ETB derivatives confined the ET-1-binding site to a 39 amino acid area between P93 in the NTD and C131 in the first cytoplasmic loop C1 These data are in agreement with the above-mentioned findings, and they further define TMS1 as a central determinant for ET-1 binding On the basis of chimeric ETB derivatives and binding of antagonists, Wada et al proposed a 60 amino acid area spanning I138-I197, and thus covering TMS2 and TMS3, as the ET-1-binding site [44] In addition, other regions, such as TMS5, have been proposed to be involved in ligand binding as evaluated by photoaffinity labeling with ETB-specific agonists [45] This result might have been caused by side-effects of the crosslink approaches, different binding sites of the supplied antagonists, or conformational changes of the analyzed chimeric ETB derivatives We showed that ETB truncations devoid of TMS1 but still retaining TMS2 and TMS3 are not able to bind ET-1 in detectable amounts Nevertheless, the affinity of ETB93 and ETB131 for ET-1 was reduced by approximately one order of magnitude, indicating that other regions of ETB still might contribute to the ligand binding Evidence for several and partially overlapping binding sites of ETB for different ligands has been documented [46] FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3265 Ligand binding of cell-free endothelin B receptor C Klammt et al Homo-oligomerization of rhodopsin-like GPCRs is an increasingly recognized mechanism, and might represent an important platform for the modulation of GPCR activities such as ligand binding, signaling or trafficking [24,47–49] Even SDS-resistant dimerization of b2-adrenergic receptor and vasopressin type receptor has been reported [47], and SDS-resistant dimers of CF-produced porcine vasopressin type receptor have also been detected [15] The ETB dimer bands observed during our SDS ⁄ PAGE analysis indicate a similar stable association The first evidence of the formation of ETB homodimer and also of its homolog human endothelin A in vivo was recently obtained by fluorescence resonance energy transfer analysis in HEK293 cells [50] Interestingly, ETB dimer formation in vivo did not depend on the presence of ET-1 This is in accordance with our observed oligomerization of CF-produced ETB in the absence of any ligand Furthermore, ETB dimer formation is strongly supported by single-particle analysis, and the bilobed structures described are almost identical to that of rhodopsin [24] and to those of the vasopressin type receptor and corticotropin-releasing factor receptor type [22] By analyzing truncated ETB derivatives, we confined the site that was essential for dimer formation to the TMS1 fragment, which was also identified as covering the ET-1-binding site The two fragments ETB131 and ETB93, which overlapped in that region, did still form homodimers as well as heterodimers with full-length ETB Our results therefore indicate that TMS1 is a key area for two main functions of ETB: the binding of ET-1 as one of the main natural peptide ligands, and ETB dimerization This close colocalization raises the question of whether dimer formation could modulate the ligand-binding activity of ETB In summary, the presented work provides an interesting alternative approach for the generation of high-quality samples for the functional and structural characterization of ETB and similar GPCRs Further analysis of the identified ETB131 and ETB93 fragments will help to identify residues involved in ligand binding and dimerization, and they might even represent suitable targets for structural studies by high-resolution NMR analysis Experimental procedures CA, USA) with a molecular mass cut-off of 25 kDa in an RM volume of 70 lL with an RM ⁄ feeding mixture ratio of : 14 Preparative-scale reactions were carried out in dispodialyzers (Spectrum Laboratories) in an RM volume of mL with an RM ⁄ feeding mixture ratio of : 17 The reaction was optimized for the concentrations of the ions Mg2+ (15 mm) and K+ (290 mm) For soluble expression, detergent was supplied during the reaction at the following final concentrations: Brij35, 0.1%; Brij58, 1.5%; Brij78, 1%; and digitonin, 0.4% Cloning procedures and protein analysis Coding regions of full-length ETB and its derivatives were amplified from cDNA by standard PCR techniques, and the fragments were inserted into the expression vector pET21a(+) (Merck Biosciences, Darmstadt, Germany) Additional codons for extended poly(His)10-tags or for StrepII-tags were inserted by the Quickchange procedure (Stratagene, La Jolla, CA, USA) Protein separated on 12% or 16.5% (w ⁄ v) Tris ⁄ glycine ⁄ SDS gels were transferred to 0.45 lm Immobilon-P poly(vinylidene difluoride) membranes (Millipore, Eschborn, Germany) blocked for h in blocking buffer containing · Tris-buffered saline, 7% skim milk powder (Fluka, Buchs, Switzerland), and 0.1% (w ⁄ v) Triton X-100 Horseradish peroxidase-conjugated T7-tag antibody (Merck Biosciences) was diluted : 5000 and incubated for h with the membrane Washed blots were analyzed by chemiluminescence in a Lumi-Imager F1 (Roche Diagnostics, Penzberg, Germany) Protein concentrations were determined by the bicinchoninic acid assay (Sigma, Taufkirchen, Germany) Soluble fractions diluted : 10 in column buffer (20 mm Tris, pH 8.0, 500 mm NaCl) were applied to mL HistrapHP columns (GE Healthcare, Freiburg, Germany) equilibrated in column buffer with 0.1% Brij78 Chromatography was performed at a flow rate of mLỈmin)1 with washing steps of six column volumes of column buffer supplemented with 10 mm, 20 mm and 50 mm imidazole, respectively, and bound protein was eluted with 375 mm imidazole ETBStrep was purified on Strep-Tactin Spin columns (IBA, Gottingen, ă Germany) according to the manufacturers recommendations Precipitates produced CF in the absence of detergent were suspended in 1% LMPG in 20 mm phosphate buffer (pH 7.0), in volumes equal to the RM volume Suspensions were incubated for h at room temperature with gentle shaking, and this was followed by centrifugation for 10 at 20 000 g (using an Eppendorf table top centrifuge 5810) in order to remove residual precipitate CF expression Proteins were produced in CECF systems essentially as previously described [14,22] Analytical-scale reactions for the optimization of reaction conditions were performed in microdialyzers (Spectrum Laboratories, Rancho Dominguez, 3266 Ligand-binding analysis The Cy3 dye was attached at Lys9 of cET-1 Biotin was covalently attached to Cys1 of ET-1 and Lys-9, resulting in bET-1 For coelution studies of ET-1 with ETB, FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS C Klammt et al receptor samples produced CF under different conditions were bound on HistrapHP columns equilibrated in 20 mm Hepes (pH 7.4), 150 mm NaCl and 0.02% dodecyl maltoside, and eluted as described above Ten to thirty micrograms of the purified receptor was mixed with cET-1 dissolved in 20% dimethylsulfoxide The mixtures were incubated for h at 21 °C, filtered, and then separated on a pre-equilibrated Superose PC column (3.2 mm ⁄ 30 cm) (GE Healthcare) at a flow rate of 0.05 mLỈmin)1 on a SMART chromatography station (GE Healthcare) The cET-1 ligand was detected by specific absorption at 550 nm Peak area values were calculated by smart manager software and plotted with kaleidagraph 3.52 software Nonspecific binding of cET-1 was monitored by saturation of the ETB sample with unlabeled ET-1 for h at 21 °C, followed by incubation with cET-1 for an additional h and subsequent gel filtration The nonspecific binding of ET-1 was determined to be below 10% of the total binding For pull-down assays, bET-1 was mixed with ETB derivatives in 20 mm Tris (pH 8.0), 500 mm NaCl and 0.1% Brij78 at a molar ratio of : 1, and incubated for h at 25 °C Mixtures were added to 100 lL of presaturated monomeric avidin matrix (Pierce, Rockford, IL, USA), and incubated for h at °C with gentle mixing The matrix was subsequently packed in mL gravity flow columns, washed with five column volumes of 20 mm Hepes (pH 7.4), 150 mm NaCl and 0.02% dodecyl maltoside, and eluted with mm biotin in a total volume of mL The eluate was mixed with 25 lL of 1% sodium deoxycholate, and incubated for 15 at 25 °C Then, mL of 12% icecold trichloroacetic acid was added, and the mixture was centrifuged at 10 000 g for 20 at °C (Eppendorf table top centrifuge 5810) The resulting pellet was dried, suspended in 50 lL of 0.1% SDS, and analyzed by SDS ⁄ PAGE Ligand binding of cell-free endothelin B receptor Parlodion grids (SPI-Supplies, West Chester, PA, USA) and negatively stained with 2% (w ⁄ v) uranyl acetate For this, a dilution series of the protein at constant detergent concentration was generated to obtain an optimal protein concentration on the grid Images were recorded at a magnification of · 50 000 on Kodak SO163 film (Rochester, NY, USA), using a Hitachi H-8.000 microscope (Tokyo, Japan) operating at an acceleration voltage of 200 kV For image processing, negatives were digitized on a Heidelberg PrimescanD 7100 (Heidelberg, Germany) ˚ drum scanner at a resolution of A ⁄ pixel at the specimen level The eman boxer program [51] was used to select a total of approximately 500 particles from electron micrographs Particle projections were subjected to reference-free alignment [52] and classification by multivariate statistical analysis [53], employing the spider package [54] Protein interaction studies Soluble fractions containing ETBStrep and the various Histagged ETB fragments were obtained from analytical CF reactions in the presence of Brij78 after centrifugation of the RM at 20 000 g for 10 (Eppendorf table top centrifuge 5810) After dilution : 20 with 100 mm Tris ⁄ HCl (pH 8.0) and 150 mm NaCl to a final volume of 1.4 mL, the solution was split into · 0.7 mL volumes and loaded on pre-equilibrated Strep-Tactin Spin columns (IBA) Washing and elution was performed essentially according to the manufacturer’s recommendations with the exception that all buffers were adjusted to 0.05% Brij78 For interaction of purified proteins, approximately equimolar concentrations of ETBStrep and ETB fragments were combined in 20 mm Tris ⁄ HCl (pH 8.0), 150 mm NaCl, and 0.05% Brij78, incubated for h at 25 °C, and purified on StrepTactin columns as described above Biacore measurements Kinetic measurements were done with a Biacore T100 (Uppsala, Sweden) in 20 mm Hepes ⁄ NaOH (pH 7.4), 500 mm NaCl and 0.1% Brij78 at 25 °C The ligand bET1 was loaded at 400–450 resonance units on Biacore Sensor Chips streptavidin, with 60 s contact time, a flow rate of 10 lLỈmin)1, and a stabilization time of 1.500 s Responses obtained for the reference flow cell were directly subtracted from the curves, and revealed negligible nonspecific binding to the control surface ETB binding was analyzed at a flow rate of 30 lLỈmin)1, with 480 s contact time and 1500 s dissociation time Data were processed with biaevaluation 3.1 software Single-particle analysis Different concentrations of purified ETB particles were adsorbed to glow-discharged 400 mesh carbon-coated Acknowledgements We are grateful to Clemens Glaubitz and Andreas Engel for valuable discussions, and we thank Walter Rosenthal for the cDNA of human ETB We further ´ thank Robert Tampe and Katrin Schulze for their help with SPR analysis The work was financially supported by SFB 628 ‘Functional Membrane Proteomics’ References Bockaert J & Pin JP (1999) Molecular tinkering of G protein-coupled receptors: an evolutionary success EMBO J 18, 1723–1729 Kristiansen K (2004) Molecular mechanisms of ligand binding, signaling, and regulation within the superfamily of G-protein-coupled receptors: molecular modeling and FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3267 Ligand binding of cell-free endothelin B receptor 10 11 12 13 14 C Klammt et al mutagenesis approaches to receptor structure and function Pharmacol Ther 103, 21–80 Klabunde T & Hessler G (2002) Drug design strategies for targeting G-protein-coupled receptors Chembiochem 3, 928–944 Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, LeTrong I, Teller DC, Okada T, Stenkamp RE et al (2000) Crystal structure of rhodopsin: a G protein-coupled receptor Science 289, 739–745 Massotte D (2003) G protein-coupled receptor overexpression with the baculovirus–insect cell system: a tool for structural and functional studies Biochim Biophys Acta 1610, 77–89 Sarramegna V, Talmont F, Demange P & Milon A (2003) Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification Cell Mol Life Sci 60, 1529–1546 Tate CG, Haase J, Baker C, Boorsma M, Magnani F, Vallis Y & Williams DC (2003) Comparison of seven different heterologous protein expression systems for the production of the serotonin transporter Biochim Biophys Acta 1610, 141–153 Akermoun M, Koglin M, Zvalova-Iooss D, Folschweiller N, Dowell SJ & Gearing KL (2005) Characterization of 16 human G protein-coupled receptors expressed in baculovirus-infected insect cells Protein Express Purif 44, 65–74 Lundstrom K (2005) Structural biology of G proteincoupled receptors Bioorg Med Chem Lett 15, 3654– 3657 ´ Andre N, Cherouati N, Prual C, Steffan T, Zeder-Lutz G, Magnin T, Pattuı` F, Michel H, Wagner R & Reinhart C (2006) Enhancing functional production of G protein-coupled receptors in Pichia pastoris to levels required for structural studies via a single expression screen Protein Sci 15, 1115–1126 Lundstrom K, Wagner R, Reinhart C, Desmyter A, Cherouati N, Magnin T, Zeder-Lutz G, Courtot M, Prual C, Andre N et al (2006) Structural genomics on membrane proteins: comparison of more than 100 GPCRs in expression systems J Struct Funct Genomics 7, 77–91 Berrier C, Park KH, Abes S, Bibonne A, Betton JM & Ghazi A (2004) Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent Biochemistry 43, 12585–12591 Elbaz Y, Steiner-Mordoch S, Danieli T & Schuldiner S (2004) In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state Proc Natl Acad Sci USA 101, 1519–1524 Klammt C, Lohr F, Schafer B, Haase W, Dotsch V, ă ă ă Ruterjans H, Glaubitz C & Bernhard F (2004) High ă level cell-free expression and specific labeling of integral membrane proteins Eur J Biochem 271, 568–580 3268 15 Klammt C, Schwarz D, Fendler K, Haase W, Dotsch V ă & Bernhard F (2005) Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system FEBS J 272, 6024–6038 16 Ishihara G, Goto M, Saeki M, Ito K, Hori T, Kigawa T, Shirouzu M & Yokoyama S (2005) Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors Protein Express Purif 41, 27–37 17 Klammt C, Schwarz D, Lohr F, Schneider B, Dotsch V ă ă & Bernhard F (2006) Cell-free expression as an emerging technique for the large scale production of integral membrane proteins FEBS J 273, 4141–4153 18 Schwarz D, Klammt C, Koglin A, Lohr F, Schneider B, ă Dotsch V & Bernhard F (2006) Preparative scale cellă free expression systems: new tools for the large scale preparation of integral membrane proteins for functional and structural studies Methods 41, 355–369 19 Sokolowsky M (1995) Endothelin receptor subtypes and their role in transmembrane signalling mechanisms Pharmacol Ther 68, 435–471 20 Miyauchi T & Masaki T (1999) Pathophysiology of endothelin in the cardiovascular system Annu Rev Physiol 61, 391–415 ´ ´ 21 D’Orleans-Juste P, Labonte J, Bkaily G, Choufani S, ´ Plante M & Honore JC (2002) Function of the endothelin B receptor in cardiovascular physiology and pathophysiology Pharmacol Ther 95, 221–238 22 Klammt C, Schwarz D, Eifler N, Engel A, Piehler J, Haase W, Hahn S, Dotsch V & Bernhard F (2007) Cellă free production of G protein-coupled receptors for functional and structural studies J Struct Biol XX, doi: 10.1016/j.jsb.2007.01.006 23 Fotiadis D, Liang Y, Filipek S, Saperstein DA, Engel A & Palczewski K (2003) Atomic-force microscopy: rhodopsin dimers in native disc membranes Nature 421, 127–128 24 Fotiadis D, Jastrzebska B, Philippsen A, Muller DJ, ă Palczewski K & Engel A (2006) Structure of the rhodopsin dimer: a working model for G-protein-coupled receptors Curr Opin Struct Biol 16, 252–259 25 Grisshammer R, White JF, Trinh LB & Shiloach J (2005) Large-scale expression and purification of a G-protein-coupled receptor for structure determination ) an overview J Struct Funct Genom 6, 159–163 26 Satoh M, Miyamoto C, Terashima H, Tachibana Y, Wada K, Watanabe T, Hayes AE, Gentz R & Furuichi Y (1997) Human endothelin receptors ETa and ETB expressed in baculovirus-infected insect cells Eur J Biochem 249, 803–811 27 Doi T, Hiroaki Y, Arimoto I, Fujiyoshi Y, Okamoto T, Satoh M & Furuichi Y (1997) Characterization of human endothelin B receptor and mutant receptors expressed in insect cells Eur J Biochem 248, 139–148 FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS C Klammt et al 28 Ozawa K, Headlam MJ, Schaeffer PM, Henderson BR, Dixon NE & Otting G (2004) Optimization of an Escherichia coli system for cell-free synthesis of selectively 15N-labelled proteins for rapid analysis by NMR spectroscopy Eur J Biochem 271, 4084–4093 29 Torizawa T, Shimizu M, Taoka M, Miyano H & Kainosho M (2004) Efficient protocol of isotopically labelled proteins by cell-free synthesis: a practical protocol J Biomol NMR 30, 311–325 30 Trbovic N, Klammt C, Koglin A, Lohr F, Bernhard F ă & Dotsch V (2005) Efcient strategy for the rapid backă bone assignment of membrane proteins J Am Chem Soc 127, 13504–13505 31 Stenlund P, Babcock GJ, Sodrowski J & Myszka DG (2003) Capture and reconstitution of G protein-coupled receptors on a biosensor surface Anal Biochem 316, 243–250 32 Okamoto Y, Ninomiya H & Masaki T (1998) Posttranslational modifications of endothelin receptor type B Trends Cardiovasc Med 8, 327–329 33 Galeffi P, Lombardi A, Pietraforte I, Novelli F, Donato MD, Sperandei M, Tornambe A, Fraioli R, Martayan A, Natali PG et al (2006) Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems J Transl Med 4, 39–52 34 Sen S, Jaakola VP, Pirila P, Finel M & Goldman A (2005) Functional studies with membrane-bound and detergent-solubilized a2-adrenergic receptors expressed in Sf9 cells Biochim Biophys Acta 1712, 62–70 35 Harding PJ, Hadingham TC, McDonnell JM & Watts A (2006) Direct analysis of a GPCR–agonist interaction by surface plasmon resonance Eur Biophys J 35, 709– 712 36 Takasuka T, Sakurai T, Goto K, Furuichi Y & Watanabe T (1994) Human endothelin receptor ETB Amino acid sequence requirements for super stable complex formation with its ligand J Biol Chem 269, 7509–7513 ´ 37 De Leon H & Garcia R (1995) Characterization of endothelin receptor subtypes in isolated rat renal preglomerular microvessels Reg Peptides 60, 1–8 38 Elshourbagy NA, Korman DR, Wu HL, Sylvester DR, Lee JA, Nuthalaganti P, Bergsma DJ, Kumar CS & Nambi P (1993) Molecular characterization and regulation of the human endothelin receptors J Biol Chem 268, 3873–3879 39 Nakamichi K, Ihara M, Kobayashi M, Saeki T, Ishikawa K & Yano M (1992) Different distribution of endothelin receptor subtypes in pulmonary tissues revealed by the novel selective ligands BQ-123 and [Ala1,3,11,15]ET-1 Biochem Biophys Res Commun 182, 144–150 40 Saeki T, Ihara M, Fukuroda T, Yamagiwa M & Yano M (1991) [Ala1,3,11,15]Endothelin-1 analogs with ETB agonistic activity Biochem Biophys Res Commun 179, 286–292 Ligand binding of cell-free endothelin B receptor 41 Schiller H, Haase W, Molsberger E, Janssen P, Michel H & Reilander H (2000) The human ET (B) endothelin receptor heterologously produced in the methylotrophic yeast Pichia pastoris shows high-affinity binding and induction of stacked membranes Receptors Channels 7, 93–107 42 Hara M, Tozawa F, Itazaki K, Mihara S & Fujimoto M (1998) Endothelin ETB receptors show different binding profiles in intact cells and cell membrane preparations Eur J Pharmacol 345, 339–342 43 Hay DL, Poyner DR & Sexton PM (2006) GPCR modulation by RAMPs Pharmacol Ther 109, 173–197 44 Wada K, Hashido K, Terashima H, Adachi M, Fujii Y, Hiraoka O, Furuichi Y & Miyamoto C (1995) Ligand binding domain of the human endothelin-B subtype receptor Protein Express Purif 6, 228–236 45 Boivin S, Tessier S, Aubin J, Lampron P, Detheux M & Fournier A (2004) Identification of a binding domain of the endothelin-B receptor using a selective IRL-1620derived photoprobe Biochemistry 43, 11516–11525 46 Lee JA, Brinkmann JA, Longton ED, Peishoff CE, Lago MA, Leber JD, Cousins RD, Gao A, Stadel JM, Kumar CS et al (1994) Lysine 182 of endothelin B receptor modulates agonist selectivity and antagonist affinity: evidence for the overlap of peptide and nonpeptide ligand binding sites Biochemistry 48, 14543– 14549 47 Hebert TE & Bouvier M (1998) Structural and functional aspects of G protein-coupled receptor oligomerization Biochem Cell Biol 76, 1–10 48 Schoneberg T, Schultz G & Gudermann T (1999) Strucă tural basis of G protein-coupled receptor function Mol Cell Endocrinol 151, 181–193 49 Rios CD, Jordan BA, Gomes I & Devi LA (2001) G-protein-coupled receptor dimerization: modulation of receptor function Pharmacol Ther 92, 71–87 50 Gregan B, Schaefer M, Rosenthal W & Oksche A (2004) Fluorescence resonance energy transfer analysis reveals the existence of endothelin-A and endothelin-B receptor homodimers J Cardiovasc Pharmacol 44, S30– S33 51 Ludtke SJ, Baldwin PR & Chiu W (1999) EMAN: semiautomated software for high-resolution single-particle reconstructions J Struct Biol 128, 82–97 52 Penczek P, Radermacher M & Frank J (1992) Threedimensional reconstruction of single particles embedded in ice Ultramicroscopy 40, 33–53 53 Frank J, Verschoor A & Boublik M (1982) Multivariate statistical analysis of ribosome electron micrographs L and R lateral views of the 40 S subunit from HeLa cells J Mol Biol 161, 107–133 54 Frank J, Radermacher M, Pencek P, Zhu J, Li Y, Ladjadj M & Leith A (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields J Struct Biol 116, 190–199 FEBS Journal 274 (2007) 3257–3269 ª 2007 The Authors Journal compilation ª 2007 FEBS 3269 ... selective ligands BQ -12 3 and [Ala1,3 ,11 ,15 ]ET -1 Biochem Biophys Res Commun 18 2, 14 4? ?15 0 40 Saeki T, Ihara M, Fukuroda T, Yamagiwa M & Yano M (19 91) [Ala1,3 ,11 ,15 ]Endothelin- 1 analogs with ETB agonistic... 3266 Ligand -binding analysis The Cy3 dye was attached at Lys9 of cET -1 Biotin was covalently attached to Cys1 of ET -1 and Lys-9, resulting in bET -1 For coelution studies of ET -1 with ETB, FEBS Journal... gives good evidence for a ligand -binding activity of CF-produced ETB 3262 Fig Ligand binding of ETB derivatives Binding of ETB derivatives to bET -1 was analyzed by pull-down assays Bound proteins