Identification of a Key Region of Kinin B 1 Receptor for High Affinity Binding of Peptide Antagonists*

To investigate the molecular basis for the specificity of ligand recognition in human kinin B 1 (B 1 R) and B 2 (B 2 R) receptors, we constructed a series of chimeric re- ceptors by progressively replacing, from the N to the C terminus, the human B 2 R domains by their B 1 counter- parts. The chimeric construct possessing the C-terminal tail and the transmembrane domain VII (TM VII) of the B 2 R (construct 6) displayed 7- and 20- fold decreased affinities for the B 1 agonist [ 3 H]desArg 10 -kallidin (de sArg 10 -KD) and the B 1 antagonist [ 3 H]desArg 10 -[Leu 9 ]-KD respectively, as compared with the wild-type B 1 R. Moreover, the substitution of the B 1 TM VII by its B 2 homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567. High affinity for desArg 10 -KD binding was fully regained when the B 2 residue Thr 287 was replaced in construct 6 by the corresponding B 1 Leu 294 residue. When the B 2 residue Tyr 295 was exchanged with the corresponding B 1 Phe 302 , high affinity binding for both agonist and antagonist was recovered. Moreover, the L294T and F302Y mutant B 1 R exhibited 69- and 6.5-fold increases, respectively, in their affinities for the B 2 receptor antagonist, Hoe140. Therefore we proposed that Leu 294 and Phe 302 residues, which may not be directly involved in the binding of B 1 R ligands and, hence, their Thr 287 and Tyr The Netherlands). Chimeric receptor constructs were made using a PCR-ligation-PCR protocol. T7 and SP6 were chosen as flanking primers, and internal primers were designed according to the desired fusion point in the chimeric construct and contained a Ksp 632I site. Appropriate B 1 and B 2 fragments were amplified for 10 cycles in a 50- m l PCR reaction containing 10 ng of template DNA, 200 m M dNTPs, 100 pmol of each primer, 1.5 m M MgCl 2 , 5 units of Red Goldstar polymerase, and the reaction buffer supplied by the manufacturer (Eurogentec, Angers, France). Another 10 cycles of amplification was performed in the presence of 5-methyldeoxycytosine ( m5 dCTP) to protect already-existing internal Ksp 632I sites from sub-sequent cleavage by the endonuclease. The isolated PCR products were

To investigate the molecular basis for the specificity of ligand recognition in human kinin B 1 (B 1 R) and B 2 (B 2 R) receptors, we constructed a series of chimeric receptors by progressively replacing, from the N to the C terminus, the human B 2 R domains by their B 1 counterparts. The chimeric construct possessing the C-terminal tail and the transmembrane domain VII (TM VII) of the B 2 R (construct 6) displayed 7-and 20-fold decreased affinities for the B 1 agonist [ 3 H]desArg 10 -kallidin (de-sArg 10 9 ]-KD respectively, as compared with the wild-type B 1 R. Moreover, the substitution of the B 1 TM VII by its B 2 homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567. High affinity for desArg 10 -KD binding was fully regained when the B 2 residue Thr 287 was replaced in construct 6 by the corresponding B 1 Leu 294 residue. When the B 2 residue Tyr 295 was exchanged with the corresponding B 1 Phe 302 , high affinity binding for both agonist and antagonist was recovered. Moreover, the L294T and F302Y mutant B 1 R exhibited 69-and 6.5-fold increases, respectively, in their affinities for the B 2 receptor antagonist, Hoe140. Therefore we proposed that Leu 294 and Phe 302 residues, which may not be directly involved in the binding of B 1 R ligands and, hence, their Thr 287 and Tyr 295 B 2 counterparts, are localized in a receptor region, which plays a pivotal role in the binding selectivity of the peptide or pseudopeptide kinin ligands.

-KD) and the B 1 antagonist [ 3 H]desArg 10 -[Leu
Bradykinin (BK) 1 and kallidin (Lys-bradykinin (KD)) are biologically active peptides derived from large precursors (kininogens) by the action of serine proteases named kallikreins (1). Kinins are released in response to tissue injury and activate sensory pain fibers, contract smooth muscle, cause endotheliumdependent vasodilatation, and induce plasma extravasation (1,2). Most of the acute effects of kinins seem to be mediated by the activation of constitutive B 2 receptors (2, 3), whereas some chronic responses to kinins are mediated by B 1 receptors (4). B 1 and B 2 kinin receptors have been cloned (5,6) and are members of the G-protein-coupled seven-transmembrane receptor superfamily. B 1 and B 2 receptors display 36% sequence identity; they can be discriminated by BK, which binds primarily to the B 2 receptor, and the C-terminal truncated carboxypeptidase fragments desArg 9 -BK and desArg 10 -KD, which bind selectively to the B 1 receptor.
Little is known about the receptor domains or amino acids involved in determining subtype-specific ligand recognition. Some residues important for agonist binding to the B 2 receptor have been located in transmembrane domains (TMs) III, VI, and VII as well as extracellular domains I, II, and III (7)(8)(9)(10)(11)(12)(13). Recent progress has been made in delineating the determinants of ligand binding to the kinin B 1 receptor (12,13). TM VI was shown to be directly involved in conferring B 1 and B 2 receptors with their ability to distinguish between the subtypeselective peptide agonists. TM VI might contain residues important for the binding of the B 1 -selective agonist desArg 10 -KD, as its high affinity binding was suppressed in the chimeric construct possessing the homologous B 2 domain (12). Fathy et al. (13) also report the complete loss of high affinity of the B 1 receptor for desArg 10 -KD upon Lys 118 mutation to Ser in TM III, suggesting that Lys 118 provides a counter-ion for the C terminus of B 1 -selective desArg peptides. Concerning the human B 2 receptor, Phe 259 and Thr 263 located near the extracellular surface of the TM VI are crucial for the recognition of the B 2 -selective agonist, BK. To further investigate the determinants of ligand recognition, we applied a two-step strategy: (i) construction of a series of chimeric receptors by progressively replacing, from the N to the C terminus, the human B 2 TMs by their B 1 homologous domains, (ii) site-directed mutagenesis to target essential amino acids inside the discriminating TMs. The pharmacological profiles of the various constructs were determined in transfected cells by radioligand binding and phosphoinositide hydrolysis assay.  9 ]-kallidin, and desArg 10 -Hoe140 were from Neosystem (Strasbourg, France). All molecular biology and cell culture reagents were purchased from Life Technologies, Inc. All other chemicals were supplied by Sigma.

Materials-[
Construction of Chimeric and Mutant Receptors cDNA-The original B 1 and B 2 receptor cDNA were subcloned into the mammalian expression vector pcDNA3.0 (Invitrogen, Leek, The Netherlands). Chimeric receptor constructs were made using a PCR-ligation-PCR protocol. T7 and SP6 were chosen as flanking primers, and internal primers were designed according to the desired fusion point in the chimeric construct and contained a Ksp632I site. Appropriate B 1 and B 2 fragments were amplified for 10 cycles in a 50-l PCR reaction containing 10 ng of template DNA, 200 M dNTPs, 100 pmol of each primer, 1.5 mM MgCl 2 , 5 units of Red Goldstar polymerase, and the reaction buffer supplied by the manufacturer (Eurogentec, Angers, France). Another 10 cycles of amplification was performed in the presence of 5-methyldeoxycytosine ( m5 dCTP) to protect already-existing internal Ksp632I sites from subsequent cleavage by the endonuclease. The isolated PCR products were * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Epitope Tagging-A 9-amino acid sequence (hemagglutinin (HA) tag) derived from the influenza virus HA protein was inserted at the extracellular N terminus of the chimeric constructs and the wild-type B 1 receptor. Two reverse complementary oligonucleotides were designed to include the HA-tag sequence followed by the first eight codons encoding the N-terminal part of the B 1 receptor and to generate a HindIII restriction site at the 5Ј end and a XbaI site at the 3Ј end. Both nucleotides were annealed and inserted in the wild-type pcDNA3 vector. The cDNA encoding for the B 1 receptor and for all chimeric constructs were then subcloned into the pcDNA3-HA vector.
10 g of vector containing the cDNA fragment of interest were then transfected into the cells using the calcium phosphate precipitation method (14) or the electroporation transfection method as described elsewhere (15). Native pcDNA3 vector was used as a negative control. COS-7 cells were allowed to recover in a standard growth medium for 48 h before membrane preparations or functional studies.
Receptor Binding Assays-Membrane preparations and binding assays were performed as described previously (16). Briefly, for saturation experiments, radioligands were incubated for 1 h at room temperature in a final volume of 500 l. The assay was terminated by filtration on Whatman GF/B filters, and the radioactivity was determined by liquid scintillation counting. Nonspecific binding was determined in the presence of 10 M corresponding unlabeled ligand. Competition binding experiments were carried out in the presence of [ 3 H]BK or [ 3 H]des-Arg 10 -KD with varying concentrations of kinin analogs. All assays were carried out in duplicate. Protein concentration was measured by the method of Bradford (17).
Assay of Phosphoinositide Hydrolysis-Subconfluent transiently transfected COS-7 cells maintained in 12-well plates were labeled with 0.5 Ci/well of myo-[ 3 H]inositol in serum-free Eagle's minimum essential medium). After 24 h, the cells were washed with phosphate-buffered saline and incubated 15 min at 37°C in phosphoinositide (PI) buffer of the following composition: 5 mM HEPES, 136 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO 4 , 2.5 CaCl 2 , 1.2 mM KH 2 PO 4 , 11 mM glucose, pH 7.4, supplemented with 10 mM LiCl (500 l/well). Stimulation of PI hydrolysis was induced by desArg 10 -KD for 20 min. After removal of PI buffer, the reaction was stopped by the addition of a 5% (v/v) HClO 4 solution containing phytic acid, 5 g/ml (500 l/well). After 10 min on ice, wells were rinsed with 500 l of 2.5% HClO 4 . The acidic extracts were pooled and neutralized with 155 l of 2 M K 2 CO 3 and 25 l of 1 M HEPES. Samples were further applied on anion exchange columns (Dowex AG1-X8), and the different inositol phosphates were separated as described elsewhere (18).
Antibody Preparation-A specific polyclonal antibody raised against the N-terminal part of the B 1 receptor was generated. Briefly, a 120base pair cDNA encoding the N-terminal part of the B 1 receptor was amplified by PCR and subcloned into a glutathione S-transferase fusion protein expression vector, pGEX-4 (Amersham Pharmacia Biotech). Glutathione S-transferase-fused protein and antibodies were prepared as described by Morohashi et al. (19).
Western Blot Analysis-Membrane preparations were fractionated using 10% Tris-glycine precast gel (Novex, Frankfurt, Germany) and were assayed by Western blotting (20). HA-tagged chimeric constructs were detected using a monoclonal anti-HA antibody (Roche Molecular Biochemicals). The secondary antibody horseradish peroxydase-conjugated rabbit anti-rat IgG (Southern Biotechnology Associates, Montrouge, France) was used in 1:50,000 dilution. Non-tagged mutant receptors were detected using the polyclonal antibody raised against the N-terminal part of the B 1 receptor as a primary antibody. The specific secondary antibody (goat anti-rabbit IgG, Caltag, Tebu, Le Perray en Yvelines, France) was conjugated to horseradish peroxydase. The chemiluminescence immunoassay was performed with ECL kit (Amersham Pharmacia Biotech).
Data Analysis-Binding experiments and concentration-response curves for PI hydrolysis were analyzed using GraphPADInPlot (Graph-PAD Software, San Diego, CA). Statistical analysis were performed using Statview (Abacus Concept, Palo Alto, CA). A one-way analysis of variance followed by a Student's t test was used to establish significant differences. A P value less than 0.05 was considered as statistically significant.  Fig. 2A) and failed to induce inositol phosphate formation upon desArg 10 -KD treatment (Fig. 2B). We assessed that the observed losses in ligand recognition were not the result of impaired receptor expression; for this purpose, Western blot analysis of membrane proteins of COS-7 cells transfected with the human B 1 receptor and all chimeric constructs, using an antibody raised against the N-terminal part of the human B 1 receptor, showed bands with similar intensity with an apparent molecular mass of approximately 40 kDa (data not shown). This molecular weight is close to the calculated one of the human B 1 receptor (6). To confirm our results, all chimeric receptors were tagged at their N termini with a nine-amino acid HA epitope after checking that the tagged HA-B 1 receptor displayed the same affinity for [ 3 H]desArg 10 -KD and [ 3 H]desArg 10 -[Leu 9 ]-KD as the wild-type receptor (data not shown). Receptor immunodetection through Western blotting analysis of plasma membrane extracts from transfected COS-7 cells revealed that all tagged chimeric constructs were adequately expressed and confirmed that there were similar levels of receptor antigen expression (data not shown).

Construction and Characterization of Chimeric B 1 /B 2 Receptors in COS
A B 1 -type binding and functional behavior (Fig. 2, A and B) was restored for chimeric receptors possessing the six first B 1 TMs (Constructs 6 and 7). Saturation binding experiments were carried out on membrane preparation from COS-7 cells transfected with Constructs 6 and 7. B max values were within the same range as those obtained for the wild-type receptor, indicating a normal chimeric receptor expression (data not shown).
The  (Table I). As expected, these binding affinities values were not changed when the C-terminal tail of the B 1 R was substituted with the corresponding region of the B 2 R (Construct 7). The Construct 6, which bears both the C-terminal tail and the transmembrane domain VII (TM VII) of the B 2 R bound [ 3 H]desArg 10 -KD and [ 3 H]desArg 10 -[Leu 9 ]-KD with a 7-and 20-fold lower affinity, respectively, compared with the wild type B 1 R (Table II). The EC 50 values relative to desArg 10 -KDinduced inositol phosphate accumulation were 37-and 14-fold higher for Constructs 6 and 7, respectively, than for the wildtype B 1 R (Table IV). In addition, the maximal response to
Therefore, the ability of Constructs 6 and 7 to bind B 1 -and B 2 -specific kinin analogues (structures depicted in Table I) was determined through competition binding assays using [ 3 H]des-Arg 10 -KD as a radioligand.
The K i values were not changed when the C-terminal tail of the B 1 R was substituted with the corresponding region of the B 2 R (Table II). The affinities of Construct 6 for the B 1 R agonist, desArg 10 -KD, and of the B 1 R antagonists desArg 9 -[Leu 8 ]-BK and desArg 10 -[Leu 9 ]-KD were 6-, 50-, and 20-fold lower, respectively, than those for B 1 R and Construct 7. Interestingly, chimeric B 1 receptors possessing the B 2 TM VII significantly increased the affinity of the pseudopeptide antagonists, Hoe140 and desArg 10 -Hoe140, by 13-and 2-fold, respectively, without affecting the binding of BK and desArg 9 -BK (Table II).
Role of Specific TM VII Amino Acids in the Selectivity of Kinin Ligand Recognition-To identify specific residues involved in discrimination of kinin peptides, four nonconserved amino acid residues (Thr 287 , Ser 291 , Met 293 , and Tyr 295 ) located in the upper portion of the TMVII of Construct 6 were mutated to their homologous B 1 amino acids (Leu 294 , Asn 298 , Phe 300 , and Phe 302 ) as shown in Fig. 4 (Table III). B max values were within the same range as for the wild-type B 1 receptor (data not shown). When Thr 287 was replaced by the corresponding Leu 294 residue (Construct 6 [T287L]), high affinity for des-Arg 10 -KD binding was fully restored (K D ϭ 0.61 Ϯ 0.16 nM) compared with the wild-type B 1 receptor (Table III) (Table III). The four mutants retained functional activity as shown by the capacity of desArg 10 -KD to induce phosphoinositide hydrolysis (table IV). Consistent with binding data, the desArg 10 -KD potency was partially recovered in cells expressing Construct 6 [T287L] and Construct 6 [Y295F], whereas EC 50 values relative to desArg 10 -KD activation of other Constructs remained 6to 250 -fold higher than those obtained with the wild type B 1 R. The maximal response to desArg 10 -KD was significantly re-duced for all mutant receptors (Table IV).
To confirm the involvement of Leu 294 and Phe 302 in the high affinity binding of desArg 10 -KD and desArg 10 -[Leu 9 ]-KD, mutations of Leu 294 to Thr and Phe 302 to Tyr were introduced in the TM VII of the wild-type B 1 receptor (Fig. 5). The binding properties of the mutated receptors were compared with those of the wild-type B 1 and B 2 receptors. The B 1 [L294T]R displayed a pharmacological profile similar to the wild-type B 1 receptor (Table III and (Table III) and a 33-fold decrease in the desArg 10 -KD potency to induce inositol phosphate (   Table IV. with a low affinity to the B 1 receptor and to the various mutants. Interestingly, the mutation of Leu 294 to Thr had a profound effect on the binding affinity of pseudopeptide B 2 and B 1 /B 2 receptor antagonists. This single amino acid exchange increased the affinity of Hoe140, desArg 10 -Hoe140, and NPC 567, respectively, by 70-, 12-, and 32-fold (Table V). Substitution of Phe 302 by Tyr also resulted in an increase of binding affinity for the aforementioned antagonists (Fig. 6). In contrast, the non-peptide B 2 antagonist, FR 173657, selectively bound to the B 2 receptor and had no affinity for the wild-type B 1 receptor as well as for the various chimeric or mutated receptors (Table V).

DISCUSSION
Human kinin B 1 and B 2 receptors, which display 36% identity in their amino acid sequence, are recognized by specific endogenous peptide ligands, which differ solely by the presence or absence of an arginine at the C-terminal position. Little is known regarding the domains or individual residues involved in determining subtype-specific ligand binding. However, it has been recently proposed that residues important for the binding of the B 1 receptor agonist desArg 10 -KD may be located in TM VI, since the construction produced by substitution of B 2 TM VI into the B 1 receptor did not support high affinity binding of desArg 10 -KD (12). Fathy et al. (13) also report the complete loss of high affinity for desArg 10 -KD binding following the substitution of the Lys 118 by a residue Ser in the TM III of the human B 1 receptor, a result leading to the suggestion that Lys 118 provides a counter-ion for the C terminus of B 1 -selective desArg peptides.
To investigate epitopes of the human kinin B 1 receptor involved in the selective ligand recognition, we constructed a series of chimeric receptors by progressively replacing transmembrane, N-and C-terminal domains of the B 2 receptor by their corresponding B 1 counterparts. We first demonstrated that N-terminal parts of the B 1 R and B 2 R, although markedly different in amino acid composition, are not essential in determining ligand selectivity, since the pharmacological properties were unchanged upon B 2 /B 1 N terminus exchange. This is in contrast with some other G-protein-coupled receptors such as chemokine receptors in which the N-terminal region markedly influences ligand binding (21,22).

Epitope Binding Pocket for Kinin Antagonists
The major finding of this study concerns the involvement of TM VII in the specificity of ligand recognition by the human B 1 receptor. Not only was the affinity of desArg 10 -KD and des-Arg 10 -[Leu 9 ]-KD decreased in the chimeric receptor possessing the B 2 TM VII (Construct 6), but a high affinity binding of desArg 10  A role for TM VII in the efficacy of G-protein coupling has been described for other receptors (23)(24)(25). However, the reduced efficacy of desArg 10 -KD to stimulate inositol phosphate production in COS-7 cells expressing Constructs 6 and 7 could at least partly be ascribed to the presence of the cytoplasmic carboxyl tail of the B 2 receptor, which is involved in internalization and signal transduction (26 -28). In agreement with binding data, the functional potency of desArg 10 -KD was partially recovered when Thr 287 and Tyr 295 were replaced by the corresponding Leu and Phe in the TMVII of the Construct 6. Moreover, the converse mutation of Phe 302 to Tyr in the wildtype B 1 receptor resulted in a loss of desArg 10 -KD-activating properties. Taken together, our results support a role of TM VII and, more specifically, of Leu 294 and Phe 302 in inducing a B 1 receptor conformation appropriate for ligand recognition and signal transduction. We also evidenced that TMVII is a key region for the recognition of pseudopeptide kinin antagonists. Indeed, the replacement of the B 1 receptor TM VII by the corresponding B 2 receptor significantly increased the binding of both pseudopeptide antagonists Hoe140 and desArg 10 -Hoe140. In addition, the replacement of Leu 294 or Phe 302 in the wild-type B 1 receptor by the corresponding Thr or Tyr of the B 2 receptor, respectively, was found to be sufficient to confer high affinity binding of pseudopeptide antagonist: NPC567, representative of the first generation of peptide B 2 receptor antagonists (29) exhibiting a very low selectivity between the receptor subtypes; Hoe140, belonging to the second generation of B 2 receptor antagonists and displaying increased affinity and selectivity for the B 2 receptor (30); desArg 10 -Hoe140, possessing a drastically decreased affinity for the B 2 receptor and increased affinity for the B 1 receptor (31). Even if their pharmacological profile is somewhat different, these three ligands share the common D-Arg 0 -[Hyp 3 ] motif and a replacement of L-Pro 7 by an aromatic structure. It might constitute the basis for some common features, suggested by our results, in the interaction of NPC567, Hoe140, and desArg 10 -Hoe140 with the B 1 receptor. As the binding of the synthetic non-peptide B 2 receptor antagonist, FR 173657, was not affected by the described point mutations in the B 1 receptor, the recognition of this new class of ligands should involve a different specific mechanism.
The delineation of amino acids located in TM VII and conferring high affinity binding for kinin pseudopeptide receptor antagonists is consistent with previous data showing that mutations in this domain can drastically change the selectivity of several G-protein-coupled receptors: binding of SMS 201-995, a somatostatin peptide analogue, to the human somatostatin receptors SSTR1 and SSTR2 (32); species adenosine A 1 receptor differences in the binding of various ligands (33); subtypespecific ligand binding to a ␣2and ␤2adrenergic receptors (34); subtype specificity and major pharmacological differences between human and rodent 5-hydroxytryptamine receptors as-  cribed to a single amino acid of TM VII (35); changes in 5HT1 Badrenergic specificity (36). Interestingly, the two mutations conferring high affinity binding for kinin pseudopeptide antagonists did not improve BK binding, suggesting a different binding site for kinin agonists and antagonists. Our results are consistent with previous data indicating that the binding pocket for Hoe140 might be deeper in the transmembrane domain and closer to TM VII than the BK binding site (11). In this study, a number of alanine amino acid replacements in the rat B 2 receptor were found to affect Hoe140 binding but not BK binding, including substitution of Tyr 297 , which corresponds to Tyr 295 of the human B 2 receptor. Another approach using antibodies raised against peptides derived from the putative extracellular domains of the B 2 receptor also suggested that the binding sites for peptidic agonists and antagonists onto the B 2 receptor did not overlap (9).
By combining chimeric constructions and a mutagenesis approach, we have demonstrated that the TMVII of the human B 1 receptor is involved in the efficacy of G-protein coupling and also possesses important determinants for the binding of B 1selective ligands such as desArg 10 -KD and desArg 10 -[Leu 9 ]-KD. In the B 2 receptor, extensive analysis of the transmembrane and the extracellular domains by alanine-scanning mutagenesis or epitope-specific antibodies binding experiments has identified a few residues important for agonist binding (7,9,11). In the B 1 receptor, recent studies proposed that TMIII and TMVI bear important residues for the binding of the B 1 agonist desArg 10 -KD (12,13). However, no information has yet been published on determinants important for pseudopeptide antagonists binding such Hoe140 in the kinin receptors. We propose that the residues Leu 294 and Phe 302 in the upper part of the B 1 TMVII and their homologous residues Thr 287 and Tyr 295 in the B 2 receptor contribute to the induction of conformations that selectively recognize the various peptidic or pseudopeptidic kinin antagonists.