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Bradykinin recognizes different molecular forms of the B2 kinin receptor in the presence and absence of guanine nucleotides.

ABSTRACT: We have previously reported that [3H]bradykinin [( 3H]BK) identifies high- and low-affinity B2 kinin receptor sites in bovine myometrial membranes which are sensitive and insensitive respectively to guanine nucleotides. Here we show that these receptor-binding sites are solubilized by the detergent CHAPS. Equilibrium binding in soluble preparations revealed that [3H]BK identified a maximal number of binding sites (Bmax) of 1119 +/- 160 fmol/mg of protein, with an equilibrium dissociation constant (KD) of 314 +/- 70 pM and with a typical B2 kinin receptor specificity. Dissociation of equilibrium binding was biphasic. In the presence of the GTP analogue guanosine 5'[beta gamma-imido]triphosphate (Gpp[NH]p, [3H]BK bound to the soluble receptors with a KD of 929 +/- 129 pM and a Bmax. of 706 +/- 38 fmol/mg of protein. The Gpp(NH)p-promoted decrease in the apparent affinity and Bmax., which was half-maximal at 0.5 microM, was due at least in part to an increase in the dissociation rate of the slowly dissociating component of the equilibrium binding. Recoveries of guanine-nucleotide-sensitivity and of rapidly and slowly dissociating binding components were essentially identical, whether or not the receptor had been occupied by an agonist before solubilization. Sucrose-density-gradient sedimentation profiles revealed that [3H]BK recognized two different molecular forms of the receptor in the absence or presence of guanine nucleotides. These results provide for the first time direct evidence that guanine nucleotides promote a change in the structure of the B2 kinin-receptor complex. We propose that this structural change is due to dissociation of a guanine-nucleotide-regulatory (G-)protein.

SUBMITTER: Mathis SA

PROVIDER: S-EPMC1151156 | biostudies-other | 1991 May

REPOSITORIES: biostudies-other

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Project description:BackgroundKinins, with bradykinin and des-Arg(9)-bradykinin being the most important ones, are pro-inflammatory peptides released after tissue injury including stroke. Although the actions of bradykinin are in general well characterized; it remains controversial whether the effects of bradykinin are beneficial or not. Kinin-B2 receptor activation participates in various physiological processes including hypotension, neurotransmission and neuronal differentiation. The bradykinin metabolite des-Arg(9)-bradykinin as well as Lys-des-Arg(9)-bradykinin activates the kinin-B1 receptor known to be expressed under inflammatory conditions. We have investigated the effects of kinin-B1 and B2 receptor activation on N-methyl-D-aspartate (NMDA)-induced excitotoxicity measured as decreased capacity to produce synaptically evoked population spikes in the CA1 area of rat hippocampal slices.Principal findingsBradykinin at 10 nM and 1 µM concentrations triggered a neuroprotective cascade via kinin-B2 receptor activation which conferred protection against NMDA-induced excitotoxicity. Recovery of population spikes induced by 10 nM bradykinin was completely abolished when the peptide was co-applied with the selective kinin-B2 receptor antagonist HOE-140. Kinin-B2 receptor activation promoted survival of hippocampal neurons via phosphatidylinositol 3-kinase, while MEK/MAPK signaling was not involved in protection against NMDA-evoked excitotoxic effects. However, 100 nM Lys-des-Arg(9)-bradykinin, a potent kinin-B1 receptor agonist, reversed bradykinin-induced population spike recovery. The inhibition of population spikes recovery was reversed by PD98059, showing that MEK/MAPK was involved in the induction of apoptosis mediated by the B1 receptor.ConclusionsBradykinin exerted protection against NMDA-induced excitotoxicity which is reversed in the presence of a kinin-B1 receptor agonist. As bradykinin is converted to the kinin-B1 receptor metabolite des-Arg(9)-bradykinin by carboxypeptidases, present in different areas including in brain, our results provide a mechanism for the neuroprotective effect in vitro despite of the deleterious effect observed in vivo.

Project description:Binding affinity at the [3H]-BK binding site and activity as inositol phosphate (IP) production by the peptide bradykinin (BK) and the nonpeptide FR190997 were studied at wild-type or point-mutated human B2 receptors (hB2R) expressed in CHO cells. The effect of the following mutations were analyzed: E47A (TM1), W86A and T89A (TM2), I110A, L114A and S117A (TM3), T158A, M165T and L166F (TM4), T197A and S211A (TM5), F252A, W256A and F259A (TM6), S291A, F292A, Y295A and Y295F (TM7), and the double mutation W256A/Y295F. As the wild-type receptor-binding affinity of FR190997 was 40-fold lower than BK, whereas their agonist potency was comparable, both agonists produced similar maximal effects (Emax). Mutations were evaluated as affecting the affinity and/or efficacy of FR190997 compared with BK. Two mutations were found to impair the agonist affinity of both agonists drastically: W86A and F259A. BK agonist affinity (pEC50) was reduced by 1400- and 150-fold, and that of FR190997 was reduced by 400- and 25-fold, at the W86A and F259A mutant B2 receptors, respectively. Contrary to BK, the affinity of FR190997 was selectively decreased at I110A, Y295A, and Y295F mutants (>103-fold), and a different efficacy was measured at the Y295 mutants, FR190997 being devoid of the capability to trigger IP production at Y295A mutant. L114A, F252A, and W256A selectively impaired the efficacy of FR190997, whereas its binding affinity was not affected. As a consequence, FR190997 behaved as a high-affinity antagonist in blocking the IP production induced by BK. The lack of capability of FR190997 to activate or to bind the double mutant W256A/Y295F suggests that these residues are part of the same binding site, which is also important for receptor activation by the nonpeptide ligand. Overall, by means of mutational analysis, we indicate an hB2R recognition site for the nonpeptide agonist FR190997 (between TM3, 6, and 7), different from that of BK, and show that in the same binding crevice some mutations (L114, W256, and F252) are selectively responsible for the agonist properties of only FR190997.

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Bradykinin recognizes different molecular forms of the B2 kinin receptor in the presence and absence of guanine nucleotides.

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