Mutation of Aspartate Residues in the Third Extracellular Loop of the Rat B2 Bradykinin Receptor Decreases Affinity for Bradykinin

Novotny, Elizabeth A.; Bednář, David; Connolly, Maureen A.; Connor, Judy; Stormann, Thomas M.

doi:10.1006/bbrc.1994.1733

Cited by 37 publications

(24 citation statements)

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“…Our results in combination with those of Novotny et al (54) and Maradone and Hogan (55) provide mutations of all of the acidic residues in extracellular domains 3 and 4 and the extracellular half of TM-4, TM-5, TM-6, and TM-7 of the B 2 receptor. Only residues Glu 199 (4.7-fold reduced in bradykinin affinity (54)), Asp 268 (3.5-fold reduced in affinity, Table I) and Asp…”

Section: Discussionsupporting

confidence: 83%

Mutations in the B2 Bradykinin Receptor Reveal a Different Pattern of Contacts for Peptidic Agonists and Peptidic Antagonists

Jarnagin¹,

Bhakta²,

Zuppan³

et al. 1996

Journal of Biological Chemistry

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The B 2 bradykinin receptor, a seven-helix transmembrane receptor, binds the inflammatory mediator bradykinin (BK) and the structurally related peptide antagonist HOE-140. The binding of HOE-140 and the binding of bradykinin are mutually exclusive and competitive. Fifty-four site-specific receptor mutations were made. BK's affinity is reduced 2200-fold by F261A, 490-fold by T265A, 60-fold by D286A, and 3-10-fold by N200A, D268A, and Q290A. In contrast, HOE-140 affinity is reduced less than 7-fold by F254A, F261A, Y297A, and Q262A. Bradykinin is a central initiator of acute and chronic inflammation and the associated pain and edema. Most of the acute and many of the chronic responses to bradykinin are mediated by B 2 bradykinin receptors (1, 2), while some of the chronic responses to bradykinin are mediated by B 1 bradykinin receptors (3). B 1 and B 2 bradykinin receptors are members of the G-protein-coupled seven-transmembrane (GPC-7TM) 1 receptor superfamily (4 -7). For B 1 receptors, [des-Arg 9 ]bradykinin is a more potent agonist than bradykinin, whereas B 2 receptors bind and respond to bradykinin about 10,000-fold more effectively than [des-Arg 9 ]bradykinin. Several peptidic B 2 bradykinin antagonists have been identified; these compounds reduce pain and inflammation (8 -12). Peptidic antagonists also reduce death from experimental shock (13-15). Bradykinin receptor antagonists are potentially useful in the treatment of pain, acute and chronic inflammation, shock, allergic or infectious rhinitis, and asthma. The peptidic antagonists are useful tools; but, to date, these compounds have made poor human therapeutic agents because of their poor bioavailability and formulation difficulties (16,17). The discovery of a nonpeptide antagonist of bradykinin would improve the prospects of treating bradykinin-instigated inflammation, pain, or edema. Thus, we have focused on a molecular understanding of the bradykinin receptor ligand binding site, believing that this information may help in the discovery and design of nonpeptidic antagonists.We used the results from molecular modeling studies of B 2 bradykinin receptors and NMR studies of peptidic agonists and antagonists to generate a number of models for agonist binding to the B 2 BKR. These models were tested by site-directed mutagenesis of the receptor and by making single amino acid changes in agonist and antagonist peptides. The data reveal a disparity between the way peptidic agonists and antagonists bind to the BKR. We attempt to reconcile the disparities by proposing new models of the BKR-ligand complex. -140 (18, 19). The product was HPLC-purified and then characterized by HPLC and mass spectrometry as to its chemical purity (Ն96%), identity, and specific activity (56.5 Ci/mmol). Media and other cell culture additives were from Life Technologies, Inc. Biochemicals and enzymes were from Boehringer Mannheim. Common reagents were from Sigma. EXPERIMENTAL PROCEDURES Materials MethodsStandard molecular biological and cell culture methods were used except as speci...

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Section: Discussionsupporting

confidence: 83%

Mutations in the B2 Bradykinin Receptor Reveal a Different Pattern of Contacts for Peptidic Agonists and Peptidic Antagonists

Jarnagin¹,

Bhakta²,

Zuppan³

et al. 1996

Journal of Biological Chemistry

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“…It was proposed that these aspartates interact electrostatically with the N-terminal amino group, the guanidinyl side chain, or both on Arg 1 in BK, a residue absolutely critical for function (Regoli and Barabé, 1980). Alanine mutation of these aspartate residues in the rat receptor confirmed their critical role in BK binding (Novotny et al, 1994. Conclusive evidence for this orientation of the receptorbound BK was gained from chemical cross-linking of the N termini of bound [ in EL-3 (Herzig and Leeb-Lundberg, 1995;Herzig et al, 1996) and Lys 172 near TM-4 (AbdAlla et al, 1996b), respectively, in the human B 2 receptor (Fig.…”

Section: Agonist and Antagonist Binding Sites In The Receptorsmentioning

confidence: 82%

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

Marceau²,

et al. 2005

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“…However, this region is not involved in forming the agonist-binding site, since we did not detect any effect of the anti-I-II antibodies on binding of the classical B 2 agonists, bradykinin or kallidin to the B 2 receptor. Chemical cross-linking studies (27) and site-directed mutagenesis (28,29) suggest that the agonist-and antagonist-binding sites to the B 2 receptor are not identical and may be only partially overlapping. When we tested the effect of a 1000-fold molar excess of the B 2 antagonist HOE140 on the binding of anti-I-II antibodies to the B 2 receptor, we found that in contrast to B 2 agonists, HOE140 almost completely suppressed antibody binding.…”

Section: Discussionmentioning

confidence: 99%

Investigation of the Extracellular Accessibility of the Connecting Loop between Membrane Domains I and II of the Bradykinin B2 Receptor

Quitterer¹,

Zaki

AbdAlla

1999

Journal of Biological Chemistry

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In analogy to the structure of rhodopsin, the seven hydrophobic segments of G-protein-coupled receptors are supposed to form seven membrane-spanning ␣-helices. To analyze the topology of the bradykinin B 2 receptor, we raised site-directed antibodies to peptides corresponding to the loop regions and the amino and carboxyl terminus of this receptor. We found that a segment with predicted intracellular orientation according to the rhodopsin model, the connecting loop between membrane domains I and II of the bradykinin B 2 receptor, was accessible to site-directed antibodies on intact fibroblasts, A431 cells, or COS cells expressing human B 2 receptors. Extracellular orientation of this loop was further confirmed by the substituted cysteine accessibility method which showed that exchange of cysteine 94 for serine on this loop by point mutagenesis suppressed the effect of thiol modification by a membrane impermeant maleimide. In addition, this segment seemed to be involved in B 2 receptor activation, since (i) thiol modification of cysteine 94 partially suppressed B 2 receptor activation, and (ii) site-directed antibodies to the connecting loop between membrane domains I and II were agonists. The agonistic activity of the antibodies was suppressed by the B 2 antagonist HOE140 confirming the B 2 specificity of the antibody-generated signal. The extracellular orientation of the connecting loop between membrane domains I and II suggests a topology of the B 2 receptor different from rhodopsin, consisting of five (instead of seven) transmembrane domains and two hydrophobic segments with both ends facing the extracellular side.The seven hydrophobic segments of G-protein-coupled receptors are supposed to form seven transmembrane-spanning ␣-helices in analogy to the three-and two-dimensional crystal structures of bacteriorhodopsin (1) and rhodopsin (2). For the ␤-adrenergic receptor and the vasopressin V 2 receptor the rhodopsin-like topology has been confirmed by site-directed antibodies (3) or by a gene fusion approach (4). Charged amino acids determine the orientation of integral membrane proteins. Both prokaryotic and eukaryotic membrane spanning stretches generally have a net positive charge on the cytoplasmic side and few arginine or lysine residues in extracellular domains (5-7). The connecting loop between membrane domains I and II of the rat and human B 2 receptor lacks a net positive charge (8,9), although the rhodopsin model predicts the intracellular orientation of this loop. Thus, for the bradykinin B 2 receptor the seven transmembrane domain topology is in conflict with the "positive inside" rule (7). We therefore determined the orientation of this loop on the intact receptor. We chose an approach which did not alter the receptor's primary sequence since insertion of additional reporter sequences may disturb the correct orientation and/or membrane insertion of the protein (10, 11). To this end antibodies to a peptide corresponding to the connecting loop region between membrane domains I and II were raised. Sit...

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Mutation of Aspartate Residues in the Third Extracellular Loop of the Rat B2 Bradykinin Receptor Decreases Affinity for Bradykinin

Cited by 37 publications

References 0 publications

Mutations in the B2 Bradykinin Receptor Reveal a Different Pattern of Contacts for Peptidic Agonists and Peptidic Antagonists

Mutations in the B2 Bradykinin Receptor Reveal a Different Pattern of Contacts for Peptidic Agonists and Peptidic Antagonists

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

Investigation of the Extracellular Accessibility of the Connecting Loop between Membrane Domains I and II of the Bradykinin B2 Receptor

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