Co-solubilization of bradykinin B2 receptors and angiotensin-converting enzyme from guinea pig lung membranes.

Bradykinin B2 receptor-like binding activity was solubilized from guinea pig lung using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (Chaps). The binding of [3H]bradykinin to the soluble fraction was time-dependent and saturable. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of binding sites with a Kd of 696 pM and a Bmax of 57 fmol/mg protein. Unlabelled bradykinin and B2 antagonists inhibited the binding of [3H]bradykinin to Chaps-solubilized extracts with relative potencies similar to those observed with the low-affinity membrane-bound binding sites. Following partial purification of the soluble preparation, using anion exchange (DEAE-Sephacel) and gel filtration (Aca 34) column chromatography steps, two peaks eluted off the column were able to bind [3H]bradykinin and have molecular masses of 168 and 98.5 kDa. The former seems to represent binding of bradykinin to angiotensin converting enzyme (ACE, EC 3.4.15.1) and the latter binding to bradykinin receptor. Using purified commercial ACE, we show that the binding of [3H]bradykinin to ACE can easily be distinguished from that of the bradykinin receptor, since both B1 and B2 ligands were able to inhibit bradykinin binding with affinities clearly different from that expected for a bradykinin receptor.

Purification and characterization of a human bradykinin binding protein from inflammatory cells.

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.

[125I]PIP HOE 140, a high affinity radioligand for bradykinin B2 receptors.

A high affinity radioligand for bradykinin B2 receptors was prepared by coupling an activated ester of [125I]4-iodobenzoic acid to the amino terminus nitrogen of the potent B2 antagonist HOE 140. The ligand, [125I]para-iodophenyl HOE 140 ([125I]PIP HOE 140), bound to a homogeneous set of sites in guinea pig ileal membranes with an equilibrium dissociation constant of 15 pM and a maximal binding density of 193 fmole/mg protein. Competition studies with a number of BK-related peptides indicated that the ligand specifically labeled B2 receptors in the preparation. The results suggest that [125I]PIP HOE 140 will be a useful tool for future studies of B2 receptors.

Purification and characterisation of B2 bradykinin receptor from rat uterus.

A B2 bradykinin (BK) receptor was solubilised and partially purified from rat uterine membranes by a combination of ammonium sulphate precipitation, desalting on Sephadex G-50, and hydroxyapatite and wheat germ agglutinin affinity chromatography. The partially purified BK receptor, enriched 1,500-fold, was then cross-linked to 125I-Tyr0-BK using disuccinimidyl suberate and purified to homogeneity as a single protein species on two-dimensional gel electrophoresis with a molecular mass of 81 kDa. This molecular size was in agreement with the value of 80-120 kDa estimated from Sephacryl 300 size exclusion column chromatography of the B2 receptor. The partially purified and the crude solubilised B2 BK receptor from rat uterus showed similar affinities for BK and the BK analogues iodo-Tyr0-Bk, D-Phe7-BK, and des-Arg9-BK, indicating that the ligand binding specificity of the receptor had been retained during the purification procedures. The biochemical properties of the solubilised B2 BK receptor correspond to those of a hydrophobic acidic glycoprotein (isoelectric focusing gave a value of 4.5-4.7) that binds specifically to wheat germ agglutinin but has no affinity for either concanavalin A or lentil lectin, suggesting the absence of terminal mannose or glucose residues.

Kinin B1 and B2 receptors in the mouse.

A systematic study has been performed in various segments of the intestine and in the urinary bladder of the mouse to identify tissues that respond to kinins and possess B1 and (or) B2 receptors. The stomach was found to contain B1 and B2 functional sites that show pharmacological profiles compatible with B1 and B2 receptors, whereas the urinary bladder possesses only B2 sites. Myotropic responses mediated by B1 receptors show slow onset and reversibility compared with responses evoked by the activation of B2 receptors. The order of potency of agonists is bradykinin (BK) > or = [Hyp3]BK > [Aib7]BK on the B2 of both the stomach and urinary bladder, while desArg9-BK is inactive. The order of potency of agonists on the B1 receptor is [Lys]desArg9BK < or = desArg9BK, while BK and the other B2 agonists are inactive. B2 antagonists of the first generation, such as DArg[Hyp3,DPhe7]BK, act as partial agonists and show residual agonistic activities higher than 0.5, while HOE-140 shows high affinity and very little residual agonistic activity; WIN 64338 is almost inactive. On the B1 receptor, classical antagonists, such as [Leu8]desArg9BK and Lys[Leu8]desArg9BK, act as partial agonists. A modification of their structures has led to a new compound (R-715) that shows fairly high affinity (pA2 7.0) and little residual agonistic effect. This compound has been used for B1 receptor characterization in the stomach. Residual agonistic activities of both B2 and B1 antagonists appear to be mediated by B2 and B1 receptors, respectively. Data presented in this paper provide the pharmacological basis for sensitive and selective preparations to be used for studying B1 and B2 receptors in the mouse.

Structure of the bradykinin B2 receptors' amino terminus.

The peptide hormone bradykinin exerts important biological functions by binding to and activating bradykinin B2 receptors. B2 receptors belong to the seven transmembrane domain (7TM) receptor family. Cloning of the cDNA sequences for the rat, human, and mouse bradykinin B2 receptor revealed several in-frame AUG triplets as potential initiation sites for translation. Due to "Kozak-like" consensus nucleotides, the AUG codon closest to transmembrane domain 1 was assumed the preferred initiation site for translation, but the real amino terminus of the B2 receptor protein was unknown. The amino terminus of several 7TM receptors has been shown to be essentially involved in receptor activation and/or ligand binding. Therefore we determined the amino terminus of the human and of the rat B2 receptor using domain-specific antipeptide antibodies, amino acid sequence analysis, and in vitro transcription/translation. We report that the human and rat B2 receptor protein start with the methionine which is translated from the first in-frame AUG. This start site extends the known amino terminus of the human and rat B2 receptors by 27 or 30 amino acid residues, respectively. Antibodies raised against a peptide of the initial 27 amino acids of the human B2 receptor stained B2 receptors on intact cells. This finding excludes the existence of a signal sequence for this receptor.

The N-terminal amino group of [Tyr8]bradykinin is bound adjacent to analogous amino acids of the human and rat B2 receptor.

To obtain data of the bradykinin B2 receptor's agonist binding site, we used a combined approach of affinity labeling and "immunoidentification" of receptor fragments generated by cyanogen bromide cleavage. Domain-specific antibodies to the various extracellular receptor domains were applied to detect receptor fragments with covalently attached [125I-Tyr8]bradykinin. As a cross-linker we used the homobifunctional reagent disuccinimidyl tartarate (DST), which reacts preferentially with primary amines. With this technique a [125I-Tyr8]bradykinin-labeled receptor fragment derived from the third extracellular domain was identified. The epsilon-amino group of lysine (Lys172) of the human B2 receptor provides the only primary amino group within this receptor fragment. This strongly suggests that DST attached the N-terminal amino group of [Tyr8]bradykinin to Lys172 of the human B2 receptor. Next we asked whether DST attaches [Tyr8]bradykinin to the analogous residue, Lys174 of the rat B2 receptor, which is 81% identical to the human B2 receptor, and we attempted to label the wild-type rat B2 receptor and a rat B2 receptor mutant where Lys174 had been exchanged for alanine. Affinity labeling of the wild-type rat B2 receptor worked efficiently, whereas DST did not attach detectable amounts of [125I-Tyr8]bradykinin to the K174A rat B2 receptor mutant. Taken together these observations indicate that the N-terminal amino group of [Tyr8]bradykinin is bound to analogous positions of the rat and of the human B2 receptor, i.e. [Tyr8]bradykinin's N terminus is bound adjacent to Lys172 of the human and Lys174 of the rat B2 receptor.

Human bradykinin B2 receptors isolated by receptor-specific monoclonal antibodies are tyrosine phosphorylated.

We report the immunoaffinity isolation of bradykinin B2 receptors in a tyrosine-phosphorylated state from WI-38 human lung fibroblasts. We generated six monoclonal antibodies directed against B2 bradykinin receptor biologic activity mediating prostaglandin E2 production in WI-38. These cells express a repertoire of bradykinin receptor affinity forms with closely correlated biologic activity and [3H]bradykinin binding. Some of the monoclonal antibodies selectively recognize intermediate-affinity (Kd = 5.6 nM) or low-affinity (Kd = 42 nM) receptor forms, whereas others recognize epitopes common to both. The monoclonal antibodies block bradykinin binding and biologic activity. Immunoaffinity chromatography on an immobilized monoclonal antibody of intermediate- plus low-affinity specificity yields WI-38 B2 receptors with intact [3H]bradykinin binding activity and a molecular mass of 78 kDa. The same band is immunoblotted by all the monoclonal antibodies, indicating a similar molecular mass for receptor forms of different affinity. Anti-phosphotyrosine antibodies demonstrate that the receptors are tyrosine phosphorylated, with implications for receptor function and regulation. Genistein completely inhibits bradykinin-mediated prostaglandin E2 production with an IC50 of 8 microM, indicating that tyrosine kinase activity is critical for the signal transduction leading to arachidonic acid release.

Inhibitory interactions of the bradykinin B2 receptor with endothelial nitric-oxide synthase.

It has been shown previously that the endothelial nitric-oxide synthase (eNOS) interacts reversibly with the plasmalemmal caveolae structural protein, caveolin-1. The eNOS-caveolin-1 interaction inhibits eNOS catalytic activity. In the present study, we show that eNOS also participates in reversible inhibitory interactions with the G protein-coupled bradykinin B2 receptor. eNOS and the B2 receptor are coimmunoprecipitated from endothelial cell lysates by antibodies directed against either of the two proteins. A glutathione S-transferase fusion protein containing intracellular domain 4 of the receptor is bound by purified recombinant eNOS in in vitro binding assays. The fusion protein selectively inhibits the activity of purified eNOS. A synthetic peptide corresponding to membrane-proximal residues 310-334 in intracellular domain 4 also potently inhibits eNOS activity (IC50 < 1 microM). Treatment of cultured endothelial cells with bradykinin or Ca2+ ionophore promotes a rapid dissociation of the eNOS.B2 receptor complex. These data demonstrate that the bradykinin B2 receptor physically associates with eNOS in a ligand- and Ca2+-dependent manner. Reversible and inhibitory membrane-docking interactions of eNOS, therefore, are not restricted to those with caveolin-1 but also occur with the bradykinin B2 receptor.