Live cell PNA labelling enables erasable fluorescence imaging of membrane proteins.

DNA nanotechnology is an emerging field that promises fascinating opportunities for the manipulation and imaging of proteins on a cell surface. The key to progress is the ability to create a nucleic acid-protein junction in the context of living cells. Here we report a covalent labelling reaction that installs a biostable peptide nucleic acid (PNA) tag. The reaction proceeds within minutes and is specific for proteins carrying a 2 kDa coiled-coil peptide tag. Once installed, the PNA label serves as a generic landing platform that enables the recruitment of fluorescent dyes via nucleic acid hybridization. We demonstrate the versatility of this approach by recruiting different fluorophores, assembling multiple fluorophores for increased brightness and achieving reversible labelling by way of toehold-mediated strand displacement. Additionally, we show that labelling can be carried out using two different coiled-coil systems, with epidermal growth factor receptor and endothelin receptor type B, on both HEK293 and CHO cells. Finally, we apply the method to monitor internalization of epidermal growth factor receptor on CHO cells.

Crystal structures of human ETB receptor provide mechanistic insight into receptor activation and partial activation.

Endothelin receptors (ETA and ETB) are class A GPCRs activated by vasoactive peptide endothelins, and are involved in blood pressure regulation. ETB-selective signalling induces vasorelaxation, and thus selective ETB agonists are expected to be utilized for improved anti-tumour drug delivery and neuroprotection. Here, we report the crystal structures of human ETB receptor in complex with ETB-selective agonist, endothelin-3 and an ETB-selective endothelin analogue IRL1620. The structure of the endothelin-3-bound receptor reveals that the disruption of water-mediated interactions between W6.48 and D2.50 is critical for receptor activation, while these hydrogen-bonding interactions are partially preserved in the IRL1620-bound structure. Consistently, functional analysis reveals the partial agonistic effect of IRL1620. The current findings clarify the detailed molecular mechanism for the coupling between the orthosteric pocket and the G-protein binding, and the partial agonistic effect of IRL1620, thus paving the way for the design of improved agonistic drugs targeting ETB.

Reduction of Uterine Perfusion Pressure Induced Redistribution of Endothelin Receptor Type-B Between the Intima and Media Contributes to the Pathogenesis of Pregnancy-Induced Hypertension.

BACKGROUND/AIMS: Studies have shown that a change in endothelin receptor expression in the artery is related to pregnancy-induced hypertension (PIH). However, the mechanism underlying this change remains unclear. METHODS: To test whether the distribution of endothelin receptor type-A (ETAR) and type-B (ETBR) plays an important role in PIH, a reduction of uterine perfusion pressure (RUPP) rat model was used to mimic some of the features of PIH; the resulting variable endothelin receptor expression was investigated in the media and intima of the aorta. Single vascular smooth muscle cells (VSMCs) were isolated from RUPP and normal pregnant (NP) rats to study the effect of ETAR and ETBR in smooth muscle cells. RESULTS: Compared with NP rats, RUPP rats had a significant redistribution of ETBR expression in the intima and media, while there was no significant difference in ETAR expression between the two groups. ETBR upregulation in VSMCs enhanced cellular contraction and contributed to PIH. The TNF-α plasma levels in RUPP rats were two-fold higher than those of NP rats, which upregulated the expression of ETBR in VSMCS through the NF-κB pathways in RUPP rats. CONCLUSION: Redistribution of ETBR between the media and intima played an important role in the pathogenesis of PIH.

X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog.

Endothelin receptors (ETRs) have crucial roles in vascular control and are targets for drugs designed to treat circulatory-system diseases and cancer progression. The nonpeptide dual-ETR antagonist bosentan is the first oral drug approved to treat pulmonary arterial hypertension. Here we report crystal structures of human endothelin ETB receptor bound to bosentan and to the ETB-selective analog K-8794, at 3.6-Å and 2.2-Å resolution, respectively. The K-8794-bound structure reveals the detailed water-mediated hydrogen-bonding network at the transmembrane core, which could account for the weak negative allosteric modulation of ETB by Na+ ions. The bosentan-bound structure reveals detailed interactions with ETB, which are probably conserved in the ETA receptor. A comparison of the two structures shows unexpected similarity between antagonist and agonist binding. Despite this similarity, bosentan sterically prevents the inward movement of transmembrane helix 6 (TM6), and thus exerts its antagonistic activity. These structural insights will facilitate the rational design of new ETR-targeting drugs.

TNFα-induced airway smooth muscle cell proliferation depends on endothelin receptor signaling, GM-CSF and IL-6.

UNLABELLED: Pathological proliferation of human airway smooth muscle cells (HASMCs) causes hyperplasia in chronic lung diseases. Signaling pathways that link airway inflammation to HASMC proliferation might provide therapeutic targets for the prevention of airway remodeling and chronic lung diseases. Endothelin-1 (ET-1) signals via endothelin-A- and B-receptors (ETAR, ETBR) to perpetuate HASMC-associated and TNFα-dependent inflammatory processes. HYPOTHESIS: endothelin receptor antagonists (ERAs) suppress HASMC proliferation induced by inflammatory cytokines. HASMCs were stimulated ex vivo with cytokines in the presence or absence of ERAs (ETAR-specific/selective: BQ123, ambrisentan; ETBR-specific: BQ788; non-selective: bosentan, macitentan, ACT-132577) or cytokine-blocking antibodies. Cell counts, DNA-synthesis (BrdU-incorporation assay), cytokine production (ELISA) and ETBR expression (whole-genome microarray data, western blot) were analyzed. ET-1-induced HASMC proliferation and DNA-synthesis were reduced by protein kinase inhibitors and ETAR-specific/selective ERAs but not by BQ788. TNFα-induced HASMC proliferation and DNA-synthesis were reduced by all ERAs. TNFα induced ET-1 and ETBR expression. TNFα- and ET-1-induced GM-CSF releases were both reduced by BQ123 and BQ788. TNFα- and ET-1-induced IL-6 releases were both reduced by BQ123 but not by BQ788. Combined but not single blockade of GM-CSF-receptor-α-chain and IL-6 reduced TNFα- and ET-1-induced HASMC proliferation and DNA-synthesis. Combined but not single treatment with GM-CSF and IL-6 induced HASMC proliferation and DNA-synthesis in the presence of ET-1. In conclusion, TNFα induces HASMC proliferation via ET-1/GM-CSF/IL-6. ETBR requires up-regulation by TNFα to mediate ET-1 effects on HASMC proliferation. This signaling cascade links airway inflammation to HASMC-associated remodeling processes and is sensitive to ERAs. Therefore, ERAs could prevent inflammation-induced airway smooth muscle hyperplasia.

Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ETA and ETB.

The molecular basis for recognition of peptide ligands endothelin-1, -2 and -3 in endothelin receptors is poorly understood. Especially the origin of ligand selectivity for ET(A) or ET(B) is not clearly resolved. We derived sequence-structure-function relationships of peptides and receptors from mutational data and homology modeling. Our major findings are the dissection of peptide ligands into four epitopes and the delineation of four complementary structural portions on receptor side explaining ligand recognition in both endothelin receptor subtypes. In addition, structural determinants for ligand selectivity could be described. As a result, we could improve the selectivity of BQ3020 about 10-fold by a single amino acid substitution, validating our hypothesis for ligand selectivity caused by different entrances to the receptors' transmembrane binding sites. A narrow tunnel shape in ET(A) is restrictive for a selected group of peptide ligands' N-termini, whereas a broad funnel-shaped entrance in ET(B) accepts a variety of different shapes and properties of ligands.

Identification of ETA and ETB binding domains using ET-derived photoprobes.

Using the structure of ET-1 as a template, a series of photosensitive analogs were developed to investigate the binding domain of ETA and ETB receptors. Accordingly, a p-benzoyl-l-phenylalanine (Bpa) residue was introduced into the peptide chain following a pattern aiming at scanning N- to C-terminal portions of the molecule. Among the analogs, those containing a Bpa amino acid in position 7 ([L-Bpa7, Tyr(125I)13]hET-1) or 12 ([Nle7, L-Bpa12, Tyr(125I)13]hET-1) exhibited the capacity to activate both receptors, thus showing that residues Met-7 and Val-12 of ET-1 do not play a key role in the activation process. The binding capacity of the probes was also evaluated on transfected CHO cells overexpressing either ETA or ETB receptors. Subsequently, these photoprobes were used to label ETA and ETB receptors overexpressed in transfected CHO cells. Enzymatic digestions and chemical cleavages were then performed on ligand-receptor complexes and fragments produced by the lysis were analyzed to point out putative interaction areas on the receptors. Results showed that Phe147-Lys166, covering the second segment of EC I and the top part of TM III, contains a contact point for [Nle7, L-Bpa12, Tyr(125I)13]hET-1 on ETA receptors whereas Ile292-Trp319, spanning from the second half of the intracellular loop III up to the middle turns of TM VI, includes a residue that can interact with [L-Bpa7, Tyr(125I)13]hET-1. Moreover, upon binding of [Nle7, L-Bpa12, Tyr(125I)13]hET-1, it was observed that Thr263-Met266 (EC II) of the ETB receptor would come close with the ligand.

N-terminal proteolysis of the endothelin B receptor abolishes its ability to induce EGF receptor transactivation and contractile protein expression in vascular smooth muscle cells.

OBJECTIVE: The extracellular N terminus of the endothelin B (ETB) receptor is cleaved by a metalloprotease in an agonist-dependent manner, but the physiological role of this N-terminal proteolysis is not known. In this study, we aimed to determine the functional role of the ETB receptor and of its N-terminal cleavage in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: VSMCs expressing either the full-length ETB receptor or an N-terminally truncated ETB receptor (corresponding to the N-terminally cleaved receptor) were analyzed for ligand-induced mitogen-activated protein kinase activation and expression of contractile proteins. In VSMCs expressing the full-length ETB receptor, IRL1620 (an ETB-selective agonist) induced a biphasic extracellular signal-regulated kinase 1/2 (ERK1/2) activation and increased expression of contractile proteins (smooth muscle myosin-1 [SM-1]/SM-2, SM22alpha, and alpha-actin). Interestingly, the second phase of ERK1/2 activation required metalloprotease activity, epidermal growth factor (EGF) receptor transactivation, and predominantly activation of Gi proteins. In contrast, in VSMCs expressing N-terminally truncated ETB receptors, IRL1620 did not elicit EGF transactivation and failed to increase contractile protein expression. CONCLUSIONS: This study is the first to show that stimulation of full-length ETB receptors promotes expression of contractile proteins and may thus participate in the differentiation of VSMCs.

Characterization and application of monoclonal antibodies against human endothelin B receptor expressed in insect cells.

Endothelin B receptor (ET(B)R) is a G protein-coupled receptor that mediates a variety of signals by binding to vasoconstrictive peptides, endothelins. Monoclonal antibodies were prepared against human ET(B)R using the full-length protein expressed in Sf9 cells. Five typical monoclonal antibodies were characterized further for their recognition. The epitopes for the 2A5, 9A3 and 21A1 antibodies were mapped within the N-terminal extracellular sequences, V71-I85 and E27-Q41, respectively, which differ between the human and mouse ET(B)Rs. All of these antibodies labeled cell surface ET(B)R expressed in COS cells, suggesting that their recognition sites exist in the extracellular domain. In addition, the immobilized antibodies could purify ET(B)R expressed in Sf9 cells to the majority under mild conditions. Thus, immunization with the recombinant full-length membrane protein provides a strategy to produce monoclonal antibodies recognizing the native protein.