The nonapeptide bradykinin (NH 2 -Arg-Pro-Pro-Gly-PheSer-Pro-Phe-Arg-COOH), a prototypic member of the kinin family, mediates important biological processes such as hypotension, edema formation, pain sensations, smooth muscle contraction, and cell growth (1). Kinins are locally released on the surface of target cells due to limited proteolysis of their parental molecules, kininogens, by the kallikreins (2). The broad spectrum of their (patho)physiological activities is mediated by cognate kinin receptors that classify pharmacologically as B1 and B2 subtypes (3). B1 receptors respond preferentially to des-Arg 10 -kallidin, whereas B2 receptors are stimulated by bradykinin and kallidin (lysylbradykinin). The multiplicity of biological effects elicited by the kinins is reflected by the complexity of their signaling pathways. B2 receptors couple to various G proteins such as G q , thereby triggering the inositol trisphosphate/Ca 2ϩ pathway via phospholipase C- and/or the arachidonic acid/prostaglandin pathway via phospholipase A 2 (4). Recent findings indicate that B2 receptors might also couple to the cAMP pathway via G s (5). In human foreskin fibroblasts, bradykinin induces, via phospholipase C, a transient rise in [Ca 2ϩ ] i that is counteracted by Ca 2ϩ extrusion (6). The increased [Ca 2ϩ ] i activates the nitric oxide/cGMP pathway and, together with diacylglycerol, drives the translocation and activation of protein kinase C, specifically of its isoforms ␣, ⑀, and (7).Given the remarkable pharmacological profile of these substances, it is evident that the activity of these peptides demands a careful control. Elaborate mechanisms exist that direct the kininogens to the surface of their target cells and allow kinin release at or next to its site of action (8). The liberated kinins are rapidly degraded in vivo by peptidases such as angiotensin-converting enzyme (kininase II), carboxypeptidase N (kininase I), and aminopeptidase P, which truncate and thereby inactivate the kinin peptides (9); the half-life of bradykinin in the plasma is Ͻ15 s (10). At the level of their receptors, the actions of kinins are restricted with respect to time and space by mechanisms involving receptor desensitization (11), internalization of the receptor-ligand complex (12, 13), loss of extracellular ligand-binding sites (14), and modulation of receptor affinity (11). Although tachyphylaxis and redistribution are well documented for the B2 receptor, the molecular mechanisms underlying these phenomena are not well understood. We hypothesized that reversible phosphorylation of kinin receptors might contribute to these phenomena.Here we have set out to investigate the agonist-induced phosphorylation of the bradykinin B2 receptor in a nontransformed human cell line (HF-15 fibroblasts) that endogenously expresses a high copy number of the B2 receptor. Using an anti-peptide antibody cross-reactive with the native receptor, we demonstrate the ligand-induced phosphorylation on Ser and Thr residues located in the carboxyl-terminal domain of th...
The kallikrein-kinin system is involved in the inflammatory process, in blood pressure regulation, and in renal homeostasis. The presence of kallikreins, kininogens, and kinins in renal tissues and fluids is well established however, the occurrence and distribution of the bradykinin (B2) receptor in the kidney are unknown. Using chemically cross-linked conjugates of bovine serum albumin and the B2 agonist bradykiuin or the potent B2 antagonist HOE140, followed by antibodies to the respective ligand and the peroxidase-anti-peroxidase system, we were able to detect the B2 receptor, The receptor has been found in straight portions of the proximal tubules, in distal straight tubules, in connecting tubules, and in collecting ducts of rat kidney. The staining patterns produced by the ligand conjugate-antiligand approach are in agreement with those obtained by conventional autoradiography using [1251]-Tyro-bradykinin.
Many of the physiological functions of bradykinin are mediated via the B2 receptor. Little is known about binding sites for bradykinin on the receptor. Therefore, antisera against peptides derived from the putative extracellular domains of the B2 receptor were raised. The antibodies strongly reacted with their corresponding antigens and cross-reacted both with the denatured and the native B2 receptor. Affinity-purified antibodies to the various extracellular domains were used to probe the contact sites between the receptor and its agonist, bradykinin or its antagonist HOE140. Antibodies to extracellular domain 3 (second loop) ]kallidin, whereas bradykinin is the agonist of B2 receptors. Molecular cloning has revealed the primary structures of the B1 (4) and the B2 receptors (5) and classified them as members of the G-protein-coupled receptor family that are thought to contain seven membrane spanning ␣-helices.The signaling pathways of the B2 receptors have been explored in some detail. The bradykinin B2 receptor is preferentially coupled to G proteins of the G␣ q subtype (6), which activate the phospholipase C-mediated cascade. This results in the hydrolysis of inositol-containing lipids, the generation of inositol phosphates, and the transient rise of the intracellular free Ca 2ϩ concentration (7). The initial increase of intracellular Ca 2ϩ is followed by Ca 2ϩ extrusion, which counteracts Ca 2ϩ influx, thereby regulating total cell calcium (8). B2-mediated release of diacylglycerol, another hydrolysis product of phospholipase C, results in the translocation of specific protein kinase C isoforms (9). The B2 receptor is also coupled to the phospholipase A2 pathway, which releases the prostaglandin precursor, arachidonic acid (10). Although the amino acid sequence of the B2 receptor has been deduced from its cDNA and its transmembrane topology has been predicted from the corresponding hydropathy plots, the specific role of the extracellular domains in ligand binding and in signal transduction is unknown. To address this question, we have raised antibodies against peptides derived from the ectodomains of the B2 receptor and used them to probe for the function(s) of the corresponding structures. Our data show that extracellular domain 3 is involved in ligand binding and may play an essential role in communicating the agonist signal through the receptor. EXPERIMENTAL PROCEDURES Materials-Na-[125 I] (17.4 Ci/mg) and the chemiluminescence detection kit (ECL) were from Amersham Corp.; [2,3-prolyl-3,4-3 H]bradykinin (specific activity 98 Ci/mmol) was from DuPont NEN; iodogen (1,3,4,6-tetrachloro-3␣-6␣-diphenyl-glycoluril) and 1,5-difluoro-2,4-dinitrobenzene were from Pierce; Sephadex-G50 was from Pharmacia Biotech Inc.; Dowex 1 (1 ϫ 8), wheat germ agglutinin (WGA) 1 from * This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (Mu 598/4 -2) and the Fonds der Chemischen Industrie (163323) (to W. M. E.).
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