SARS-CoV-2 Spike Protein Destabilizes Microvascular Homeostasis.

SARS-CoV-2 infection can cause compromised respiratory function and thrombotic events. SARS-CoV-2 binds to and mediates downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. Theoretically, diminished enzymatic activity of ACE2 may result in increased concentrations of pro-inflammatory molecules, angiotensin II, and Bradykinin, contributing to SARS-CoV-2 pathology. Using immunofluorescence microscopy of lung tissues from uninfected, and SARS-CoV-2 infected individuals, we find evidence that ACE2 is highly expressed in human pulmonary alveolar epithelial cells and significantly reduced along the alveolar lining of SARS-CoV-2 infected lungs. Ex vivo analyses of primary human cells, indicated that ACE2 is readily detected in pulmonary alveolar epithelial and aortic endothelial cells. Exposure of these cells to spike protein of SARS-CoV-2 was sufficient to reduce ACE2 expression. Moreover, exposure of endothelial cells to spike protein-induced dysfunction, caspase activation, and apoptosis. Exposure of endothelial cells to bradykinin caused calcium signaling and endothelial dysfunction (increased expression of von Willibrand Factor and decreased expression of Krüppel-like Factor 2) but did not adversely affect viability in primary human aortic endothelial cells. Computer-assisted analyses of molecules with potential to bind bradykinin receptor B2 (BKRB2), suggested a potential role for aspirin as a BK antagonist. When tested in our in vitro model, we found evidence that aspirin can blunt cell signaling and endothelial dysfunction caused by bradykinin in these cells. Interference with interactions of spike protein or bradykinin with endothelial cells may serve as an important strategy to stabilize microvascular homeostasis in COVID-19 disease. IMPORTANCE SARS-CoV-2 causes complex effects on microvascular homeostasis that potentially contribute to organ dysfunction and coagulopathies. SARS-CoV-2 binds to, and causes downregulation of angiotensin converting enzyme 2 (ACE2) on cells that it infects. It is thought that reduced ACE2 enzymatic activity can contribute to inflammation and pathology in the lung. Our studies add to this understanding by providing evidence that spike protein alone can mediate adverse effects on vascular cells. Understanding these mechanisms of pathogenesis may provide rationale for interventions that could limit microvascular events associated with SARS-CoV-2 infection.

Homology Modeling of Class A G-Protein-Coupled Receptors in the Age of the Structure Boom.

With 700 members, G protein-coupled receptors (GPCRs) of the rhodopsin family (class A) form the largest membrane receptor family in humans and are the target of about 30% of presently available pharmaceutical drugs. The recent boom in GPCR structures led to the structural resolution of 57 unique receptors in different states (39 receptors in inactive state only, 2 receptors in active state only and 16 receptors in different activation states). In spite of these tremendous advances, most computational studies on GPCRs, including molecular dynamics simulations, virtual screening and drug design, rely on GPCR models obtained by homology modeling. In this protocol, we detail the different steps of homology modeling with the MODELLER software, from template selection to model evaluation. The present structure boom provides closely related templates for most receptors. If, in these templates, some of the loops are not resolved, in most cases, the numerous available structures enable to find loop templates with similar length for equivalent loops. However, simultaneously, the large number of putative templates leads to model ambiguities that may require additional information based on multiple sequence alignments or molecular dynamics simulations to be resolved. Using the modeling of the human bradykinin receptor B1 as a case study, we show how several templates are managed by MODELLER, and how the choice of template(s) and of template fragments can improve the quality of the models. We also give examples of how additional information and tools help the user to resolve ambiguities in GPCR modeling.

Various modifications of the amphipathic dynorphin A pharmacophore for rat brain bradykinin receptors.

As a unique endogenous opioid ligand, dynorphin A shows paradoxical neuroexcitatory effects at bradykinin receptors, and the effects are known to be amplified by the upregulation of dynorphin A under chronic pain and inflammatory conditions. In our earlier structure-activity relationship studies, the amphipathic dynorphin A fragment, [Des-Arg(7) ]-Dyn A-(4-11), was identified as a pharmacophore for the bradykinin receptors along with key structural features. Here, further modifications of the pharmacophore showed that the position of a Pro residue is also an important feature because of its role in making (or disrupting) a ß-turn or 310 helix structure which is crucial for receptor recognition.

Dynorphin A analogs for the treatment of chronic neuropathic pain.

Chronic pain is one of the most ubiquitous diseases in the world, but treatment is difficult with conventional methods, due to undesirable side effects of treatments and unknown mechanisms of pathological pain states. The endogenous peptide, dynorphin A has long been established as a target for the treatment of pain. Interestingly, this unique peptide has both inhibitory (opioid in nature) and excitatory activities (nonopioid) in the CNS. Both of these effects have been found to play a role in pain and much work has been done to develop therapeutics to enhance the inhibitory effects. Here we will review the dynorphin A compounds that have been designed for the modulation of pain and will discuss where the field stands today.

Short peptide constructs mimic agonist sites of AT(1)R and BK receptors.

Extracellular peptide ligand binding sites, which bind the N-termini of angiotensin II (AngII) and bradykinin (BK) peptides, are located on the N-terminal and extracellular loop 3 regions of the AT(1)R and BKRB(1) or BKRB(2) G-protein-coupled receptors (GPCRs). Here we synthesized peptides P15 and P13 corresponding to these receptor fragments and showed that only constructs in which these peptides were linked by S-S bond, and cyclized by closing the gap between them, could bind agonists. The formation of construct-agonist complexes was revealed by electron paramagnetic resonance spectra and fluorescence measurements of spin labeled biologically active analogs of AngII and BK (Toac(1)-AngII and Toac(0)-BK), where Toac is the amino acid-type paramagnetic and fluorescence quencher 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid. The inactive derivatives Toac(3)-AngII and Toac(3)-BK were used as controls. The interactions characterized by a significant immobilization of Toac and quenching of fluorescence in complexes between agonists and cyclic constructs were specific for each system of peptide-receptor construct assayed since no crossed reactions or reaction with inactive peptides could be detected. Similarities among AT, BKR, and chemokine receptors were identified, thus resulting in a configuration for AT(1)R and BKRB cyclic constructs based on the structure of the CXCR(4), an α-chemokine GPCR-type receptor.

N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes.

Peptide agonists and antagonists of both bradykinin (BK) B(1) and B(2) receptors (B(1)R, B(2)R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ε-aminocaproyl (ε-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B(1)R: B-10376: CF-ε-ACA-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; B(2)R: B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B(2)R agonists CF-ε-ACA-BK, AF350-ε-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.

Agonist-induced localization of Gq-coupled receptors and G protein-gated inwardly rectifying K+ (GIRK) channels to caveolae determines receptor specificity of phosphatidylinositol 4,5-bisphosphate signaling.

G protein-gated inwardly rectifying K(+) (GIRK) channels are parasympathetic effectors in cardiac myocytes that act as points of integration of signals from diverse pathways. Neurotransmitters and hormones acting on the Gq protein regulate GIRK channels by phosphatidylinositol 4,5-bisphosphate (PIP(2)) depletion. In previous studies, we found that endothelin-1, but not bradykinin, inhibited GIRK channels, even though both of them hydrolyze PIP(2) in cardiac myocytes, showing receptor specificity. The present study assessed whether the spatial organization of the PIP(2) signal into caveolar microdomains underlies the specificity of PIP(2)-mediated signaling. Using biochemical analysis, we examined the localization of GIRK and Gq protein-coupled receptors (GqPCRs) in mouse atrial myocytes. Agonist stimulation induced a transient co-localization of GIRK channels with endothelin receptors in the caveolae, excluding bradykinin receptors. Such redistribution was eliminated by caveolar disruption with methyl-ß-cyclodextrin (MßCD). Patch clamp studies showed that the specific response of GIRK channels to GqPCR agonists was abolished by MßCD, indicating the functional significance of the caveolae-dependent spatial organization. To assess whether low PIP(2) mobility is essential for PIP(2)-mediated signaling, we blocked the cytoskeletal restriction of PIP(2) diffusion by latrunculin B. This abolished the GIRK channel regulation by GqPCRs without affecting their targeting to caveolae. These data suggest that without the hindered diffusion of PIP(2) from microdomains, PIP(2) loses its signaling efficacy. Taken together, these data suggest that specific targeting combined with restricted diffusion of PIP(2) allows the PIP(2) signal to be compartmentalized to the targets localized closely to the GqPCRs, enabling cells to discriminate between identical PIP(2) signaling that is triggered by different receptors.

A novel bioassay for detecting GPCR heterodimerization: transactivation of beta 2 adrenergic receptor by bradykinin receptor.

Many G-protein-coupled receptors (GPCRs) have been shown to form heteromeric complexes primarily by biochemical methods, including competitive radioligand binding assays or measurements of changes in second-messenger concentration in lysed cells. These results are often cell line specific, and the expression of other cell surface proteins makes it difficult to detect potential functional consequences of GPCR interaction. Here, 2-electrode voltage clamping in Xenopus oocytes was used as a bioassay to explore heterodimerization of bradykinin type 2 receptor (Bk2R) and beta 2 adrenergic receptor (beta(2)AR), using chloride channels as outputs for receptor activation. The data show for the first time that these 2 receptors heterodimerize with functional consequences. Stimulation with bradykinin induced activation of Galphaq- and transactivation of Galphas-coupled pathways in oocytes expressing Bk2R and beta(2)AR. To corroborate these data, potential receptor interaction was examined in PC12 cells, a cell line that endogenously expresses both receptors, and confirmed that stimulation with bradykinin transactivates beta(2)AR. In both oocytes and PC12 cells, transactivation was ablated by Bk2R or beta(2)AR inverse agonists, suggesting that transactivation occurred directly through both receptors. This is the first evidence of Bk2R/beta(2)AR physical interaction, forming a functional heterodimer. The oocyte system may prove highly useful for exploration of GPCR heterodimerization and the functional consequences thereof.

Bradykinin receptors in the zebrafish (Danio rerio).

Ligand interactions of a zebrafish bradykinin (BK) receptor expressed in vitro were characterized by measuring inositol phosphate accumulation. The ligands were analogues of zebrafish BK. Substitutions of Arg1, Gly4, Ser6, Pro7, Leu8, and Arg9 caused greatly reduced potency and maximum response. The Pro3 to Ala analogue had higher potency, but lower maximum response. These and other differences show that the zebrafish BK receptor has a ligand-interaction profile that is distinct from mammalian B1 and B2 receptors and from the previously characterized BK receptor in trout stomach. The results increase our understanding of the evolution of BK receptors and their ligands.

Solution-phase combinatorial synthesis of nonpeptide bradykinin antagonists.

We describe the solution-phase combinatorial synthesis and pharmacological effect of fifty N,N(')-substituted-N"-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid triamide derivatives as nonpeptide B2 antagonists. The synthesized compounds were tested for their antibradykinin activity by utilizing guinea-pig ileum smooth muscle. Most of the compounds showed antagonistic effects on bradykinin induced contraction. N-acetyl-N(')-(4-methylbenzyl)-N"-1-(4-chlorobenzhydryl)piperazine iminodiacetic acid triamide (A3B1C1) showed the 46% inhibition at 100nM.

Identification and characterisation of GPR100 as a novel human G-protein-coupled bradykinin receptor.

G-protein-coupled receptor 100 (GPR100) was discovered by searching the human genome database for novel G-protein-coupled peptide receptors. Full-length GPR100 was amplified from a cDNA library of the neuroendocrine cell line BON, which is derived from a human pancreas carcinoid. The open-reading frame, present on a single exon, coded for a protein of 374 amino acids with highest sequence identity (43%) to the human orphan somatostatin- and angiotensin-like peptide receptor. The analysis of chromosomal localisation mapped the GPR100 gene to chromosome 1q21.2-q21.3. The stable expression of GPR100 in Chinese hamster ovary cells together with aequorin as calcium sensor and the promiscuous G-protein subunit alpha16 as signal transducer revealed bradykinin and kallidin as effectors to elicit a calcium response. Dose-response curves yielded EC50 values for both ligands in the low nanomolar range, while the respective analogues without arginine at the carboxy-terminus were inactive. Calcium mobilisation was inhibited by the phospholipase C blocker U73122, but not by pertussis toxin, suggesting the involvement of the G-protein subunit alphaq and not alphai or alphao in signal transduction. In line with the main function of kinins as peripheral hormones, we found that GPR100 was expressed predominantly in tissues like pancreas, heart, skeletal muscle, salivary gland, bladder, kidney, liver, placenta, stomach, jejunum, thyroid gland, ovary, and bone marrow, but smaller amounts were also detected in the brain and in cell lines derived from tumours of various origins.

Discovery of a potent, non-peptide bradykinin B1 receptor antagonist.

Bradykinin (BK) plays an important role in the pathophysiological processes accompanying pain and inflammation. Selective bradykinin B1 receptor antagonists have been shown to be anti-nociceptive in animal models and could be novel therapeutic agents for the treatment of pain and inflammation. We have explored chemical modifications in a series of dihydroquinoxalinone sulfonamides to evaluate the effects of various structural changes on biological activity. The optimization of a screening lead compound, facilitated by a homology model of the BK B1 receptor, culminated in the discovery of a potent human BK B1 receptor antagonist. Results from site-directed mutagenesis studies and experiments in an animal pain model are presented.

Chimeric exchanges within the bradykinin B2 receptor intracellular face with the prostaglandin EP2 receptor as the donor: importance of the second intracellular loop for cAMP synthesis.

The prostaglandin E2 (PGE(2)) EP2 receptor (EP2R) type is G protein coupled (GPCR) and links to Galphas. Through this receptor PGE(2) activates cAMP production. The bradykinin (BK) B2 receptor (BKB2R) is also a GPCR but links to Galphaq and Galphai and does not activate cAMP production in response to bradykinin. In an attempt to convert the BKB2R into a Galphas-linked adenylate cyclase-activating receptor we proceeded to make global and discrete motif replacements of the intracellular (IC) face of the BKB2R with the corresponding regions of the human EP2R. With this approach we produced hybrid receptors which, when stably transfected into wild type (WT) Rat-1 cells, bound BK but produced cAMP. Replacement of the second loop (IC2), third loop (IC3), the entire C terminus, and the distal C terminus resulted in receptors which bound BK. However, only the IC2 and IC3 exchanges resulted in cAMP-producing receptors. Of these two regions, the IC2 exchange was by far the better cAMP-generating receptor, producing cAMP at approximately 6.6-fold above WT BKB2R or approximately one fourth the amount produced by WT EP2R-transfected Rat-1 cells. Both human and rat EP2R and human beta2-adrenergic receptor exchanges of the IC2 produced equal quantities of cAMP. Focusing on the rBKB2R/hEP2R IC2 chimeras, the region consisting of residues 136-147 (BKB2R residue numbering) proved to contain a cAMP-generating motif. Within this region, the proximal six amino acids from the EP2R (HPYFYQ) at position 136-141 proved crucial for cAMP production (10-fold over WT BKB2R). The distal part of this region, the six residues at 142-147, played no role in cAMP production. On the other hand, the ALV motif of the BKB2R IC2, residues 133-135, proved important with respect to phosphatydilinositol (PI) turnover. Replacing the entire IC2 of BKB2R resulted in poor PI turnover, while including the AVL of BKB2R retained approximately half of the WT PI turnover. With respect to receptor uptake, all the IC2 mutants endocytosed as WT BKB2R (60% in 1h). However, the exchange of the distal and the whole C termini resulted in a marked drop in endocytosis (30% in 1h). These results demonstrate that the construction of a cAMP-producing BKB2/EP2 receptor hybrid is possible, with the IC2 region distal to DRYLALV proving important to Galphas linkage and the LALV motif within the IC2 of BKB2R and the region proximal to it proving important for Galphaq and Galphai linkage. Additionally, our results confirm the importance of the distal C terminus in determining receptor uptake.

The therapeutic potential of bradykinin B2 receptor agonists in the treatment of cardiovascular disease.

The nonapeptide bradykinin (BK) is a Janus-faced hormone, which exerts pathophysiological as well as pronounced beneficial physiological effects, mainly by stimulation of BK B(2) receptors. In various animal models and in humans it has been shown that the stimulation of BK B(2) receptors is not only implicated in the pathogenesis of inflammation, pain and tissue injury but also in powerful cardioprotective mechanisms. Either exogenous administration of BK or locally increased BK concentrations as a consequence of the inhibition of its metabolic breakdown by angiotensin-converting enzyme inhibitors, reveal the significant contribution of BK in powerful cardioprotective mechanisms. These are mainly triggered by the synthesis and release of the vasorelaxant, anti-hypertrophic and anti-atherosclerotic endothelial mediators nitric oxide, prostaglandins and tissue-type plasminogen activator, by ischaemic preconditioning and by an increase in insulin sensitivity. Consequently, BK B(2) receptor agonists may have important clinical value in the treatment and prevention of various cardiovascular disorders such as hypertension, ischaemic heart disease, left ventricular hypertrophy, ventricular remodelling and congestive heart failure as well as diabetic disorders by mimicking the reported beneficial effects of BK. However, none of the currently known potent and selective peptide and non-peptide agonists of BK B(2) receptors--RMP-7 (lobradamil, Cereport; Alkermes), JMV-1116 (Fournier), FR-190997 (Fujisawa) and FR-191413 (Fujisawa)--have been selected for a clinical assessment in cardiovascular indications. One major challenge of this approach is the still unanswered question of whether there is a sufficient safe therapeutic window between potential cardioprotective and pro-inflammatory effects following BK B(2) receptor agonism.

Tissue kallikrein actions at the rabbit natural or recombinant kinin B2 receptors.

We have examined whether exogenous human tissue kallikrein exerts pharmacological actions via the bradykinin B2 receptor; specifically, whether the protease can bind to, cleave, internalize, and/or activate a fusion protein composed of the rabbit B2 receptor conjugated to the green fluorescent protein (B2R-GFP). The enzyme partially digested the fusion protein at 1 micromol/L, but not 100 nmol/L, and promoted B2R-GFP endocytosis in HEK 293 cells (> or =50 nmol/L). Trypsin and endoproteinase Lys-C, but not plasma kallikrein, also cleaved B2R-GFP. Phospholipase A2 was activated by 50 nmol/L tissue kallikrein in HEK 293 cells expressing B2R-GFP, and this was mediated by the receptor, as shown by the effect of a B2 receptor antagonist and by the lack of response in untransfected cells. However, 500 nmol/L kallikrein elicited a strong receptor-independent activation of phospholipase A2. Tissue kallikrein competed for [3H]bradykinin binding to B2R-GFP only at 1 micromol/L. A simulation involving kallikrein treatment of HEK 293 cells, pretreated or not with human plasma, evidenced the formation of immunoreactive bradykinin. The enzyme (50 nmol/L) contracted the rabbit isolated jugular vein via its endogenous B2 receptors, but the effect was tachyphylactic, and there was no cross-desensitization with bradykinin effects. Aprotinin prevented all pharmacological responses to tissue kallikrein, indicating that the enzyme activity is required for its effect. The local generation of kinins is a plausible mechanism for the pharmacological effects of lower concentrations of tissue kallikrein (50 to 100 nmol/L); higher levels (0.5 to 1 micromol/L) can not only initiate the degradation of rabbit B2 receptors but also exert nonreceptor-mediated effects.

Structure and signalling pathways of kinin receptors.

Kinins are peptide hormones that transmit their biological effects via G protein-coupled receptors. They are generated by kallikrein-mediated proteolysis of their precursors, the kininogens. Kinins have been implicated in the regulation of blood pressure, pain sensation and cell growth. Interestingly, all components of the kallikrein-kinin system have also been localized in testis. Effects of kallikrein and bradykinin on pre-spermatogonial cell proliferation and on sperm motility suggest a regulatory function of kinins and their cognate receptors in the male reproductive system. This review is dedicated to summarize the current knowledge about structure, signal transduction and regulation of kinin receptors. Particular emphasis will be given to the kinin-induced activation of the mitogen-activated protein kinase cascade which might represent an important signalling pathway involved in regulation of spermatogenesis and sperm function.

Changes in amino-terminal portion of human B2 receptor selectively increase efficacy of synthetic ligand HOE 140 but not of cognate ligand bradykinin.

Recently, we have shown that a widely used antagonist of the human bradykinin B(2) receptor (B(2)R) HOE 140 acts as a full agonist of the chicken ornithokinin receptor (B(o)R). To understand the molecular mechanisms underlying differential efficacy of HOE 140 for the various kinin receptors, we have constructed chimeric kinin receptors (CKR) in which the amino-terminal portion including the first two transmembrane regions and the first extracellular loop (CKR-2) or only the second transmembrane region and the first extracellular loop (CKR-1) of B(2)R were substituted with the corresponding segments of B(o)R. Ligand efficacy of synthetic ligand HOE 140 decreased in the order B(o)R > CKR-2 > CKR-1 > B(2)R, whereas the efficacy of the endogenous kinin ligand was unchanged. Enhanced HOE 140 efficacy was not due to a structural change in the ligand binding site or to an enhanced receptor expression level. Rather, heterologous binding competition studies indicated that structural change(s) introduced into the engineered receptors caused a selective reduction in apparent affinity of HOE 140 for the uncoupled inactive receptor state R but not for the active G protein-coupled state R*, thereby increasing the ratio of R* over R for a given ligand concentration. Our results may help explain the unusually broad efficacy spectrum of HOE 140, which varies from inverse to full agonism, depending on kinin receptor subtype, tissue origin, or species.

Negative and positive regulatory epitopes in the C-terminal domains of the human B1 and B2 bradykinin receptor subtypes determine receptor coupling efficacy to G(q/11)-mediated [correction of G(9/11)-mediated] phospholipase Cbeta activity.

The human B1 bradykinin (BK) receptor (B1R) is more efficacious than the human B2 BK receptor (B2R) in both ligand-independent and agonist-dependent coupling to G(q/11)-mediated phospholipase Cbeta activity. In fact, B1R is constitutively active, whereas B2R exhibits little if any constitutive activity. To evaluate the role of the C-terminal domain in receptor G(q/11) coupling, we constructed chimeric and C-terminally truncated receptors. The slopes of the increase in basal and agonist-dependent cellular phosphoinositide hydrolysis as a function of receptor density in transiently transfected human embryonic kidney 293 cells provided parameters of receptor coupling. Exchanging the C-terminal domains between the two receptors revealed that these domains are largely responsible for the difference in coupling. B1R truncation showed that this receptor does not directly depend on the C-terminal domain for efficient coupling, although coupling is dramatically augmented by residues in the membrane-distal portion of the domain downstream from Tyr(327). On the other hand, coupling of B2R is absolutely dependent on a membrane-proximal epitope in the C-terminal domain upstream from Lys(315). This epitope is adjacent to a basic residue, Arg(311), which exerts an inhibitory effect on coupling. Arg(311) is not conserved in B1R, and complementary mutations in B2R and B1R showed that this residue, together with previously identified serines and threonines, acts to attenuate the coupling efficacy of B2R. Therefore, the C-terminal domain participates intimately in the efficacy of B1R and B2R G(q/11) coupling by contributing both positive and negative regulatory epitopes.

Cloning, structural characterization and functional expression of a zebrafish bradykinin B2-related receptor.

The actions of bradykinin (BK) in mammals are mediated through the activation of the B1 and B2 BK receptors. The only BK receptor that has been cloned from a non-mammalian species is a B2-like receptor from the chicken (termed the ornithokinin receptor). Pharmacological studies have demonstrated the presence of BK receptors in tissues of teleost fishes, such as trout and cod, but the ligand-binding properties of these receptors differ appreciably from those of the mammalian and chicken receptors. We report here the cloning of a B2-like receptor in zebrafish that shares 35% identity with human B2 and 30% identity with human B1. Phylogenetic analyses confirm a closer relationship with B2 than B1. The receptor gene was mapped to linkage group 17, which is syntenic to the human B2-B1 gene region. After functional expression of the zebrafish B2 receptor in mammalian cells, nanomolar concentrations of trout BK ([Arg0,Trp5,Leu8]-BK) and the derivative [des-Arg0,Trp5,Leu8]-BK (where 'des' indicates a missing amino acid) induced a significant transient rise in intracellular free Ca2+. The B1-selective analogue [Arg0,Trp5,Leu8,des-Arg9]-BK was inactive at nanomolar concentrations. Taken together, these results strongly support the gene's identity as a piscine orthologue of the mammalian B2 receptor.