Cloning, structural characterization and functional expression of a zebrafish bradykinin B2-related receptor.
Ontology highlight
ABSTRACT:
SUBMITTER: Duner T
PROVIDER: S-EPMC1222631 | biostudies-other | 2002 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:A cDNA encoding a functional bradykinin receptor was isolated from a rat uterus library by a clonal selection strategy using Xenopus laevis oocytes to assay for expression of bradykinin responses. The predicted protein is homologous to the seven transmembrane G protein-coupled superfamily of receptors. Bradykinin and its analogs stimulate a Cl- current oocytes expressing the receptor with the rank order of potency: bradykinin approximately Lys-bradykinin greater than [Tyr8]-bradykinin much greater than [Phe6]bradykinin. This is the rank order of potency observed for these compounds in competitive binding assays on soluble receptor from rat uterus. Des-Arg9-bradykinin (10 microM) elicits no response when applied to oocytes expressing the receptor; thus, the cDNA encodes a B2 type bradykinin receptor. [Thi5,8,DPhe7]bradykinin, where Thi is beta-(2-thienyl)-alanine, is a very weak partial agonist and inhibits the bradykinin-mediated ion flux, suggesting the cDNA encodes a smooth muscle, rather than a neuronal, B2 receptor subtype. Receptor message has a distribution consistent with previous reports of bradykinin function and/or binding in several tissues and is found in rat uterus, vas deferens, kidney, lung, heart, ileum, testis, and brain. Receptor subtypes are a possibility because several tissues contain two or three message species (4.0, 5.7, and 6.5 kilobases). Southern blot high-stringency analysis demonstrated that the rat, guinea pig, and human genomes contain a single gene. As bradykinin is a key mediator of pain, knowledge of the primary structure of this receptor will allow a molecular understanding of the receptor and aid the design of antagonists for pain relief.
| S-EPMC52375 | biostudies-other
Project description:In the present study, pharmacological properties of a bradykinin B(2) receptor amplified either from guinea-pig ileum or lung and homologous to the previously reported sequence except two amino-acid changes L(124)-->P and N(227)-->Y in the receptor protein were characterized. Tritiated bradykinin ([(3)H]-BK) specifically bound to the cloned guinea-pig B(2) bradykinin receptor stably expressed in Chinese hamster ovary cells (CHO-K1) with a K(D) value of 0.29+/-0.07 nM. In competition experiments, bradykinin (BK) affinity constant value was 0.21+/-0.05 nM while the two specific kinin B(1) ligands, des-Arg(9)-bradykinin (DBK) and des-Arg(9)-Leu(8)-bradykinin (DLBK) were unable to compete with [(3)H]-BK. As the specific peptide antagonist D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]-bradykinin (HOE140), (E)-3-(6-acetamido-3-pyridil)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide (FR173657) and 1-[[3-[2,4-dimethylquinolin-8-yl)oxymethyl] - 2,4 - dichloro - phenyl]sulfonyl] - 2(S) - [[4-[4-(aminoiminomethyl)-phenylcarbonyl]piperazin-1-yl]carbonyl]pyrrolidine (LF16-0335C) exhibited a high affinity for this receptor with K(i) values of 7.34+/-2.45 nM and 8.54+/-1.55 nM respectively. BK and kallidin (KD) increased inositol phosphates (IPs) levels with EC(50) values of 0.44+/-0.12 nM and 6.88+/-0.28 nM, respectively. Neither DLBK nor DBK (0.01 nM to 10 microM) stimulated or inhibited IPs turnover and as expected HOE140 did not raise IPs production. HOE140 (0.1 microM) and LF 16-0335c (1 microM) right shifted the BK response curve with pK(B) values of 9.2+/-0.4 and 8.4+/-0.3, respectively. The results indicate that this cloned guinea-pig receptor displayed typical pharmacological properties of a bradykinin B(2) receptor and support the existence of a single B(2) receptor in this species.
| S-EPMC1573155 | biostudies-literature
Project description:Bradykinin B2 receptor (B2R) and angiotensin-(1-7) Mas receptor (MasR)-mediated effects are physiologically interconnected. The molecular basis for such cross talk is unknown. It is hypothesized that the cross talk occurs at the receptor level. We investigated B2R-MasR heteromerization and the functional consequences of such interaction. B2R fused to the cyan fluorescent protein and MasR fused to the yellow fluorescent protein were transiently coexpressed in human embryonic kidney293T cells. Fluorescence resonance energy transfer analysis showed that B2R and MasR formed a constitutive heteromer, which was not modified by their agonists. B2R or MasR antagonists decreased fluorescence resonance energy transfer efficiency, suggesting that the antagonist promoted heteromer dissociation. B2R-MasR heteromerization induced an 8-fold increase in the MasR ligand-binding affinity. On agonist stimulation, the heteromer was internalized into early endosomes with a slower sequestration rate from the plasma membrane, compared with single receptors. B2R-MasR heteromerization induced a greater increase in arachidonic acid release and extracellular signal-regulated kinase phosphorylation after angiotensin-(1-7) stimulation, and this effect was blocked by the B2R antagonist. Concerning serine/threonine kinase Akt activity, a significant bradykinin-promoted activation was detected in B2R-MasR but not in B2R-expressing cells. Angiotensin-(1-7) and bradykinin elicited antiproliferative effects only in cells expressing B2R-MasR heteromers, but not in cells expressing each receptor alone. Proximity ligation assay confirmed B2R-MasR interaction in human glomerular endothelial cells supporting the interaction between both receptors in vivo. Our findings provide an explanation for the cross talk between bradykinin B2R and angiotensin-(1-7) MasR-mediated effects. B2R-MasR heteromerization induces functional changes in the receptor that may lead to long-lasting protective properties.
| S-EPMC5016258 | biostudies-literature
Project description:Progestagenic sex steroid hormones play critical roles in reproduction across vertebrates, including teleost fish. To further our understanding of how progesterone modulates testis functions in fish, we set out to clone a progesterone receptor (pgr) cDNA exhibiting nuclear hormone receptor features from zebrafish testis. The open reading frame of pgr consists of 1854 bp, coding for a 617-amino acid-long protein showing the highest similarity with other piscine Pgr proteins. Functional characterization of the receptor expressed in mammalian cells revealed that zebrafish Pgr exhibited progesterone-specific, dose-dependent induction of reporter gene expression, with 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), a typical piscine progesterone, showing the highest potency. Expression of pgr mRNA: 1) appeared in embryos at 8 h after fertilization; 2) was significantly higher in developing ovary than in early transforming testis at 4 wk of age but vice versa in young adults at 12 wk of age, and thus resembling the expression pattern of the germ cell marker piwil1; and, 3) was restricted to Leydig and Sertoli cells in adult testis. Zebrafish testicular explants released DHP concentration dependently in response to high concentrations of recombinant zebrafish gonadotropins. In addition, DHP stimulated 11-ketotestosterone release from zebrafish testicular explants, but only in the presence of its immediate precursor, 11 beta-hydroxytestosterone. This stimulatory activity was blocked by a Pgr antagonist (RU486), suggesting that 11 beta-hydroxysteroid dehydrogenase activity was stimulated by DHP via Pgr. Our data suggest that DHP contributes to the regulation of Leydig cell steroidogenesis, and potentially--via Sertoli cells--also to germ cell differentiation in zebrafish testis.
| S-EPMC2802120 | biostudies-literature
Project description:Recent functional and proteomic studies in eukaryotes (www.openprot.org) predict the translation of alternative open reading frames (AltORFs) in mature G-protein-coupled receptor (GPCR) mRNAs, including that of bradykinin B2 receptor (B2R). Our main objective was to determine the implication of a newly discovered AltORF resulting protein, termed AltB2R, in the known signaling properties of B2R using complementary methodological approaches. When ectopically expressed in HeLa cells, AltB2R presented predominant punctate cytoplasmic/perinuclear distribution and apparent cointeraction with B2R at plasma and endosomal/vesicular membranes. The presence of AltB2R increases intracellular [Ca2+] and ERK1/2-MAPK activation (via phosphorylation) following B2R stimulation. Moreover, HEK293A cells expressing mutant B2R lacking concomitant expression of AltB2R displayed significantly decreased maximal responses in agonist-stimulated Gαq-Gαi2/3-protein coupling, IP3 generation, and ERK1/2-MAPK activation as compared with wild-type controls. Conversely, there was no difference in cell-surface density as well as ligand-binding properties of B2R and in efficiencies of cognate agonists at promoting B2R internalization and β-arrestin 2 recruitment. Importantly, both AltB2R and B2R proteins were overexpressed in prostate and breast cancers, compared with their normal counterparts suggesting new associative roles of AltB2R in these diseases. Our study shows that BDKRB2 is a dual-coding gene and identifies AltB2R as a novel positive modulator of some B2R signaling pathways. More broadly, it also supports a new, unexpected alternative proteome for GPCRs, which opens new frontiers in fields of GPCR biology, diseases, and drug discovery.
| S-EPMC7949122 | biostudies-literature
Project description:The role of bradykinin (BK) receptors in activating and sensitizing peripheral nociceptors is well known. Recently, we showed that spinal dynorphin was pronociceptive through direct or indirect BK receptor activation. Here, we explored the potential role of BK receptors in pain associated with persistent pancreatitis in rats.Experimental pancreatitis and abdominal hypersensitivity were induced by intravenous administrations of dibutyltin dichloride (DBTC). [des-Arg-Leu]BK (B1 antagonist) and HOE 140 (B2 antagonist) were given by intraperitoneal or intrathecal injection. Dynorphin antiserum was given intrathecally. Reverse transcription-polymerase chain reaction was used to detect spinal mRNA for BK receptors.Dibutyltin dichloride-induced pancreatitis upregulated B1 and B2 mRNA in the thoracic dorsal root ganglion and B2, but not B1, in the pancreas. No changes in spinal B1 or B2 mRNA were observed. Intraperitoneal or intrathecal administration of HOE 140 dose dependently abolished DBTC-induced abdominal hypersensitivity, whereas [des-Arg-Leu]BK was without effect by either route of administration. Antiserum to dynorphin (intrathecal) abolished DBTC-induced hypersensitivity.These results suggest that blockade of peripheral or spinal BK B2 receptors may be an effective approach for diminishing pain associated with pancreatitis. Moreover, it is suggested that spinal dynorphin may maintain pancreatitis pain through direct or indirect activation of BK B2 receptors in the spinal cord.
| S-EPMC5690478 | biostudies-literature
Project description:Type I human diabetics and streptozotocin-induced diabetic mice with higher genetically determined levels of angiotensin-converting enzyme have an increased risk of developing nephropathy. However, previous experiments in mice and computer simulations indicate that modest increases in angiotensin-converting enzyme have minimal effects on blood pressure and angiotensin II levels, although bradykinin decreases significantly, inferring that bradykinin is critical for protecting the kidney in diabetics. Here, we confirm this inference by demonstrating that Akita diabetic mice lacking the bradykinin B2 receptor develop overt albuminuria, excreting the equivalent of >550 mg/day albumin in humans, which contrasts with the microalbuminuria (equivalent to <150 mg/day) seen in their simply diabetic littermates. The overt albuminuria is accompanied by a marked increase in glomerular mesangial sclerosis. The importance of bradykinin demonstrated here bears strongly on how current drugs reduce diabetic nephropathy and suggests that B2 receptor-specific agonists merit consideration in this context.
| S-EPMC516527 | biostudies-literature
Project description:1. A zebrafish M2 muscarinic acetylcholine receptor (mAChR) gene was cloned. It encodes 495 amino acids in a single exon. The derived amino acid sequence is 73.5% identical to its human homologue. 2. Competitive binding studies of the zebrafish M2 receptor and [(3)H]-NMS gave negative log dissociation constants (pK(i)) for each antagonist as follows: atropine (9.16)>himbacine (8.05)>/=4-DAMP (7.83)>AF-DX 116 (7.26)>/=pirenzepine (7.18)>/=tropicamide (6.97)>/=methoctramine (6.82)>/=p-F-HHSiD (6.67)>carbachol (5.20). The antagonist affinity profile correlated with the profile of the human M2 receptor, except for pirenzepine. 3. Reverse transcription polymerase chain reaction and Southern blotting analysis demonstrated that the M2 mAChR mRNA levels increased during the segmentation period (12 h post-fertilization; h.p.f.) in zebrafish. By whole-mount in situ hybridization, the M2 mAChR was first detectable in the heart, vagus motor ganglion, and vagus sensory ganglion at 30, 48 and 60 h.p.f., respectively. 4. The muscarinic receptor that mediates carbachol (CCh)-induced bradycardia was functionally mature at 72 h.p.f. The effect of CCh-induced bradycardia was antagonized by several muscarinic receptor antagonists with the order of potency (pIC(50) values): atropine (6.76)>methoctramine (6.47)>himbacine (6.10)>4-DAMP (5.72)>AF-DX 116 (4.77), however, not by pirenzepine, p-F-HHSiD, or tropicamide (<10 micro M). 5. The effect of CCh-induced bradycardia was abolished completely before 56 h.p.f. by M2 RNA interference, and the bradycardia effect gradually recovered after 72 h.p.f. The basal heart rate was increased in embryos injected with M2 mAChR morpholino antisense oligonucleotide (M2 MO) and the effect of CCh-induced bradycardia was abolished by M2 MO in a dose-dependent manner. In conclusion, the results suggest that the M2 mAChR inhibit basal heart rate in zebrafish embryo and the M2 mAChR mediates the CCh-induced bradycardia.
| S-EPMC1573553 | biostudies-literature
Project description:The zebrafish genome encodes homologs for most of the proteins involved in inflammatory pathways; however, the molecular components and activation mechanisms of fish inflammasomes are largely unknown. ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)] is the only adaptor involved in the formation of multiple types of inflammasomes. Here, we demonstrate that zASC is also involved in inflammasome activation in zebrafish. When overexpressed in vitro and in vivo in zebrafish, both the zASC and zASC pyrin domain (PYD) proteins form speck and filament structures. Importantly, the crystal structures of the N-terminal PYD and C-terminal CARD of zebrafish ASC were determined independently as two separate entities fused to maltose-binding protein. Structure-guided mutagenesis revealed the functional relevance of the PYD hydrophilic surface found in the crystal lattice. Finally, the fish caspase-1 homolog Caspy, but not the caspase-4/11 homolog Caspy2, interacts with zASC through homotypic PYD-PYD interactions, which differ from those in mammals. These observations establish the conserved and unique structural/functional features of the zASC-dependent inflammasome pathway.DatabaseStructural data are available in the PDB under accession numbers 5GPP and 5GPQ.
| S-EPMC6105367 | biostudies-literature
Project description:We here report the discovery and early characterization of Compound 3, a representative of a novel class of small molecule bradykinin (BK) B2 receptor antagonists, and its superior profile to the prior art B2 receptor antagonists Compound 1 and Compound 2. Compound 3, Compound 2, and Compound 1 are highly potent antagonists of the human recombinant B2 receptor (Kb values 0.24, 0.95, and 1.24 nM, respectively, calcium mobilization assay). Compound 3 is more potent than the prior art compounds and icatibant in this assay (Kb icatibant 2.81 nM). The compounds also potently inhibit BK-induced contraction of endogenous B2 receptors in a human isolated umbilical vein bioassay. The potencies of Compound 3, Compound 2, Compound 1, and icatibant are (pA2 values) 9.67, 9.02, 8.58, and 8.06 (i.e. 0.21, 0.95, 2.63, and 8.71 nM), respectively. Compound 3 and Compound 2 were further characterized. They inhibit BK-induced c-Fos signaling and internalization of recombinant human B2 receptors in HEK293 cells, and do not antagonize the venous effects mediated by other G protein-coupled receptors in the umbilical vein model, including the bradykinin B1 receptor. Antagonist potency of Compound 3 at cloned cynomolgus monkey, dog, rat, and mouse B2 receptors revealed species selectivity, with a high antagonist potency for human and monkey B2 receptors, but several hundred-fold lower potency for the other B2 receptors. The in vitro off-target profile of Compound 3 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B1 receptor. Compound 3 showed a lower intrinsic clearance in the microsomal stability assay than the prior art compounds. With an efflux ratio of 1.0 in the Caco-2 permeability assay Compound 3 is predicted to be not a substrate of efflux pumps. In conclusion, we discovered a novel chemical class of highly selective and very potent B2 receptor antagonists, as exemplified by Compound 3. The compound showed excellent absorption in the Caco-2 assay, predictive of good oral bioavailability, and favourable metabolic stability in liver microsomes. Compound 3 has provided a significant stepping stone towards the discovery of the orally bioavailable B2 antagonist PHA-022121, currently in phase 1 clinical development.
| S-EPMC7316994 | biostudies-literature