Extracellular Domains of the Bradykinin B2 Receptor Involved in Ligand Binding and Agonist Sensing Defined by Anti-peptide Antibodies

Abstract

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.).

International Union of Pharmacology. XLV. Classification of the Kinin Receptor Family: from Molecular Mechanisms to Pathophysiological Consequences

“…Indeed, the close proximity of the BK N terminus (3 Å) to both Cys 277 in EL-3 and Cys 20 in the receptor N terminus provided direct support for the helical bundle GPCR structure (Herzig et al, 1996). Inhibition of BK binding by antipeptide antibodies specific for the N-terminal halves of EL-3 and EL-2 provided further evidence for this orientation (AbdAlla et al, 1996c). The sensitivity of the human B 1 receptor to the presence and absence of L-Lys at the N-terminal end of the peptide ligand was also used to orient the bound peptide agonists in the human B 2 receptor (Fathy et al, 2000).…”

Differential Distribution of Bradykinin B 2 Receptors in the Rat and Human Cardiovascular System

“…37 Thoracic aortas were prepared as described and, cut into rings Ϸ10 mm long, and the sections were incubated with 5 nmol/L [ Table). After incubation for 15 minutes at 4°C, the radioactivity present in each ring was measured in a ␥-counter (Packard).…”

“…This antibody docks to a ligand binding of the receptor, thereby preventing bradykinin attachment to its cognate receptor. 37 (Figure 4, bottom and middle). Similarly, 100 mol/L bradykinin or HOE140 displaced the radioligand, whereas control antibody to an intracellular domain ID4 of B 2 receptor or des [Arg 9 ]bradykinin had no effect (Figure 4, bottom).…”

Palmitoylation of Endothelin Receptor A

“…To investigate the involvement of PLC in the transient increase in [Ca 2ϩ ] i , Sf9 cells were incubated with 4 M U73122, a specific PLC inhibitor, or with 10 M LaCl 3 , an inhibitor of Ca 2ϩ channel opening, prior to stimulation with 100 nM ET-1. Fluorescence monitoring, calibration procedures, and all other experimental details were as described (23,24).…”