Bradykinin (BK) is a potent short-lived effector belonging to a class of peptides known as kinins. It participates in inflammatory and vascular regulation and processes including angioedema, tissue permeability, vascular dilation, and smooth muscle contraction. BK exerts its biological effects through the activation of the bradykinin B2 receptor (BKB2R) which is G-protein-coupled and is generally constitutively expressed. Upon binding, the receptor is activated and transduces signal cascades which have become paradigms for the actions of the Gai and Gaq G-protein subunits. Following activation the receptor is then desensitized, endocytosed, and resensitized. The bradykinin B1 (BKB1R) is a closely related receptor. It is activated by desArg 10 -kallidin or desArg 9 -BK, metabolites of kallidin and BK, respectively. This receptor is induced following tissue injury or after treatment with bacterial endotoxins such as lipopolysacharide or cytokines such as interleukin-1 or tumor necrosis factor-a. In this review we will summarize the BKB2R and BKB1R mediated signal transduction pathways. We will then emphasize the relevance of key residues and domains of the intracellular regions of the BKB2R as they relate to modulating its function (signal transduction) and selfmaintenance (desensitization, endocytosis, and resensitization). We will examine the features of the BKB1R gene promoter and its mRNA as these operate in the expression and self-maintenance of this inducible receptor. This communication will not cover areas discussed in earlier reviews pertaining to the actions of peptide analogs. For these we refer you to earlier reviews

Presently, little is known of the amino acid motif(s) participating in bradykinin B2 receptor-mediated signal transduction processes. In this report we investigate the potential role of the two existing tyrosine (Tyr) residues in the intracellular regions and the carboxyl terminus in the regulatory function of this receptor. Rat-1 cells, which do not contain detectable bradykinin B2 receptor, were transfected with wild type and mutant receptor cDNAs. Tyr-131 and Tyr-321 were each mutated to corresponding alanine-, serine-, and phenylalaninecontaining sequences. The last 34 amino acid residues of the carboxyl terminus were truncated. Rat-1 cells transfected with the mutant forms of the receptor cDNA including the truncated COOH-terminal cDNA all bound Bradykinin is a nine-amino acid peptide hormone exerting diverse biological action ranging from a role in the inflammatory process to regulatory effects on vascular permeability, blood pressure, generation of pain, and renal homeostasis (1, 2). Bradykinin expresses its physiological effect through activation of a B2 type receptor. The bradykinin B2 receptor cDNA has been isolated from several species such as mouse, rat, and human (3-5). The receptor is G-protein-coupled with a structure characterized by seven transmembrane regions (4). Upon binding, the receptor activates a number of enzyme systems involved in signal transduction including phospholipase C and cytosolic phospholipase A 2 (6). When stably transfected into such cells as hamster CCL39 the receptor retains its signal paths (7).Investigation of the regulatory regions or domains of the bradykinin B2 receptor which participate in the activation of second messenger responses has been hampered by the unavailability of immunoprecipitating antibodies against the receptor. Herein we target single amino acid and amino acid sequence moieties that could prove crucial to the function of this receptor in signal transduction. These include the two existing tyrosines at positions 131 and 321 and the carboxyl terminus consisting of 34 amino acid residues including four serines and two threonines. We analyze the ability of normal and mutant bradykinin B2 receptors to stimulate inositol phosphate (IP) 1 production and release of arachidonate. Our results show that tyrosine (Tyr) 131 and 321 and the carboxyl terminus participate variably in function of both paths as well as in receptor internalization. Cell Culture-Rat-1 cells were obtained from Dr. Robert Weinberg (Whitehead Institute, MIT) as a generous gift. Cells were grown at 37°C in a humidified atmosphere with 5% CO 2 in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum supplemented with 50 units/ml penicillin and 50 g/ml streptomycin. All tissue culture dishes were obtained from Fisher Scientific. EXPERIMENTAL PROCEDURES Materials-[Site-directed Mutagenesis-The overlap extension polymerase chain reaction method was used to generate mutants of the bradykinin B2 receptor utilizing as template the rat bradykinin B2 cDNA clone in the vector pRC/CMV (I...

In the search for the structural elements participating in signal transduction, internalization, and resensitization of the bradykinin B2 receptor, we identified two critical motifs, one in the second intracellular loop (IC2), the other in the proximal C terminus. We previously described the contribution of tyrosines within each of the two motifs (Tyr interact cooperatively in conjunction with at least two separate signaling functions. Given these results, a molecular model of the receptor was generated with the IC2 and the proximal C terminus in close spatial proximity. Conformations were identified to provide structural explanation for these observations. The conserved Thr 137 in the IC2 was next substituted with proline (T137P) to prevent phosphorylation at this position or with aspartate (T137D) to emulate phosphorylation. The T137P mutant demonstrated no change from WT with respect to either BK-activated PI turnover or arachidonic acid release. However, the mutant exhibited a markedly reduced capacity to internalize. It also resensitized poorly. The T137D mutant lacked both BK responsive activities. However, it internalized and resensitized normally, as did WT. These final results suggest that Thr 137 is functioning as a switch in termination of signal transduction and the initiation of internalization.The bradykinin B2 receptor (BKB2R) 1 cDNA has been successfully transfected into such cell types as hamster lung fibroblasts, Rat-1 fibroblasts and CHO cells with the expected binding properties (1-3). The BKB2R is a typical G protein-coupled receptor (GPCR) that has been reported to be associated with Gq (4 -7), Gi, and G 12 (8 -10). Binding of a GPCR to more than one G␣ subunit is not unprecedented. For example, the angiotensin II type 1 (AT1) receptor couples to both Gi and Gq (11). Whereas Gq has been linked to phospholipase C activation (6, 7), the release of arachidonate, via phospholipase A2 activation, has been linked to .The cellular response of GPCRs to agonists is regulated under a tightly controlled process of desensitization and resensitization (15, 16). Desensitization prevents cells from uncontrolled stimulation, and resensitization allows cells to recover and maintain responsiveness (17). Both processes appear to be regulated at the receptor level. Recent studies by Haasemann et al. (18) showed that stimulation of BKB2R results in redistribution of the receptor and its internalization in caveolae (18). On/off regulatory elements have been identified in other receptors (19 -22). However, to date, no clear consensus motifs have been identified to determine either internalization or resensitization. The DRYXXI/VXXP motif located in the second intracellular loop (IC2) of the human muscarinic cholinergic receptor has been proposed as important for receptor internalization (23). Another important motif is the tyrosine-containing motif located in the proximal BKB2R C terminus. The motifs in this region have been implicated in receptor internalization in other GPCRs, such as neurokinin 1 receptor (1...

Interleukin-8 (IL-8), a member of the chemokine superfamily, exists as both monomers and dimers, and mediates its function by binding to neutrophil CXCR1 and CXCR2 receptors that belong to the G protein-coupled receptor class. It is now well established that the monomer functions as a high-affinity ligand, but the binding affinity of the dimer remains controversial. The approximately 1000-fold difference between monomer-dimer equilibrium constant (microM) and receptor binding constant (nM) of IL-8 does not allow receptor-binding affinity measurements of the native IL-8 dimer. In this study, we overcame this roadblock by creating a "trapped" nondissociating dimer that contains a disulfide bond across the dimer interface at the 2-fold symmetry point. The NMR studies show that the structure of this trapped dimer is indistinguishable from the native dimer. The trapped dimer, compared to a trapped monomer, bound CXCR1 with approximately 70-fold and CXCR2 with approximately 20-fold lower affinities. Receptor binding involves two interactions, between the IL-8 N-loop and receptor N-domain residues, and between IL-8 N-terminal and receptor extracellular loop residues. In contrast to a trapped monomer that bound an isolated CXCR1 N-domain peptide with microM affinity, the trapped dimer failed to show any binding, indicating that dimerization predominantly perturbs the binding of only the N-loop residues. These results demonstrate that only the monomer is a high-affinity ligand for both receptors, and also provide a structural basis for the lower binding affinity of the dimer.

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