J Biol Chem, Vol. 274, Issue 37, 26079-26084, September 10, 1999

Involvement of the Amino Terminus of the B₂ Receptor in Agonist-induced Receptor Dimerization^*

Said AbdAlla, Essam Zaki, Heinz Lother^§, and Ursula Quitterer^¶

From the  Genetics Engineering and Biotechnology Research Institute, Alexandria, Egypt, the ^§ Heinrich Pette Institut für Experimentelle Virologie und Immunologie, Martinistrasse 52, 20251 Hamburg, Germany, and the ^¶ Institut für Pharmakologie und Toxikologie der Universität, Versbacher Strasse 9, 97078 Würzburg, Germany

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mechanisms and the functional importance of G-protein-coupled receptor dimerization are poorly understood. We therefore analyzed dimerization of the bradykinin B₂ receptor. The binding of the agonist bradykinin to the B₂ receptor endogenously expressed on PC-12 cells led to the formation of receptor dimers, whereas the B₂ antagonist HOE140 did not induce dimerization, suggesting that B₂ receptor dimerization was linked to receptor activation. Addition of a peptide corresponding to the amino terminus of the receptor reduced the amount of detected B₂ receptor dimers, whereas peptides derived from the extracellular loops had no effect. To further analyze the role of the amino terminus of the receptor in receptor dimerization, we created two different rat B₂ receptor variants with truncated amino termini, B₂⁵³ and B₂⁶⁵, starting at amino acids 53 and 65. In contrast to the wild-type B₂ receptor and to B₂⁵³, bradykinin did not induce dimerization of the B₂⁶⁵ receptor. Both receptor variants were similar to the wild-type B₂ receptor with respect to agonist binding and signal generation. However, B₂⁶⁵ was not phosphorylated, did not desensitize, and was not downregulated upon bradykinin stimulation. Likewise, antibodies directed to the amino terminus of the receptor partially reduced internalization of [³H]bradykinin on PC-12 cells. These findings suggest that the amino terminus of the B₂ receptor is necessary for triggering agonist-induced B₂ receptor dimerization, and receptor dimers are involved in receptor-mediated signal attenuation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mechanism and the function of agonist-induced receptor dimerization of G-protein-coupled receptors is still a matter of debate. For a variety of G-protein-coupled receptors, receptor dimers have been detected recently, e.g. for the ₂-adrenergic receptor (1), the dopamine D₂ receptor (2), the metabotropic glutamate receptor 5 (3), or the calcium sensing receptor (4). G-protein-coupled receptors seem to dimerize via two different mechanisms. Whereas dimerization of the dopamine D₂ and the ₂-adrenergic receptor occurs via transmembrane regions, the metabotropic glutamate receptor and the calcium sensing receptor dimerize via their large amino terminus and, to a lesser extent, via their transmembrane regions (1-4). G-protein-coupled receptors are classified into three main families according to the structure and length of their amino terminus and the localization of the agonist binding site (5). The metabotropic glutamate receptor and the calcium sensing receptor are members of family 3 receptors. In family 3 receptors, the amino terminus is not only involved in dimerization but also essential for agonist binding. In contrast, adrenaline and dopamine, which are agonists on family 1 receptors, bind within the seven transmembrane helices (5). The transmembrane regions are also involved in dimerization of these receptors (1, 2). Thus, the mechanism of receptor dimerization seems to be related to agonist binding. To further investigate this hypothesis, we analyzed dimerization of the bradykinin B₂ receptor. The B₂ receptor belongs also to family 1. However, receptors for catecholamines or dopamine belong to the 1a subfamily, whereas receptors for peptides like bradykinin are classified into the subfamily 1b (5). In contrast to family 1a receptors, 1b-type receptors are characterized by an agonist binding site within the amino terminus of the receptor and the extracellular loops (5). In accordance with its classification as a family 1b receptor, a binding site(s) for the agonist bradykinin was identified within the extracellular loop regions, e.g. the connecting loops between membrane domains IV and V and domains VI and VII (6-9). Because the agonist binding site of 1b receptors differs from that of 1a family receptors, we asked whether the sites necessary for receptor dimerization were also different between members of these two subfamilies. Here, we report that binding of bradykinin to the B₂ receptor endogenously expressed on PC12 cells or transiently expressed in HEK293 cells led to the formation of receptor dimers/oligomers, which were irreversibly captured by covalent cross-linking. The amino terminus of the receptor seemed to be involved in receptor dimerization because a B₂ receptor variant lacking the entire amino terminus, B₂⁶⁵ was impaired in agonist-induced receptor dimerization.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [2,3-prolyl-3,4-³H]Bradykinin (specific activity, 98 Ci/mmol), myo-[2-³H]inositol (specific activity, 17 Ci/mmol), Na¹²⁵I (specific activity, 17.4 Ci/mg), and the chemiluminescence detection kit were from Amersham Pharmacia Biotech; bradykinin, desArg⁹-Lys⁰-bradykinin, and HOE140 (D-Arg⁰-Hyp³-Thi⁵-D-Tic⁷-Oic⁸-bradykinin) were from Bachem; fura-2/AM was from Calbiochem; and m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),¹ 1,5-difluoro-2,4-dinitrobenzene (DFDNB), and iodogen (1,3,4,6-tetrachloro-3-6-diphenyl-glycoluril) were from Pierce.

Cell Culture and Cell Transfection-- HEK293 and PC-12 cells (ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum and kept in a humidified 7.5% CO₂/92.5% air atmosphere at 37 °C. Absence of mycoplasma infection was routinely controlled. HEK293 cells were transfected by Ca²⁺ phosphate precipitation with 20 µg of DNA/10⁶ cells (10). For B₂ receptor desensitization and down-regulation, the B₂ receptor was expressed at low levels (0.05-0.1 pmol/mg of protein) to avoid saturation of the downstream effector components of the cells. The amount of B₂-cDNA used for these experiments was 0.1-0.2 µg of DNA/10⁶ cells, and the total amount of transfected DNA was held constant by plasmid pcDNA3. The transfection efficiency was usually 70-90%, as determined by staining of the cells by X-Gal (5-bromo-4-chloro-3-indoyl-d-galactopyranoside) after transfection with a plasmid encoding -galactosidase.

Binding Studies of [³H]Bradykinin-- Binding affinities of B₂ receptors expressed in HEK293 cells for [2,3-prolyl-3,4-³H]bradykinin (specific activity, 98 Ci/mmol) were determined on cell membranes. Membranes were prepared as described previously in the presence of protease inhibitors (6). Incubations were carried out in binding buffer (20 mM K⁺-Hepes, pH 7.4, containing 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 2 µM enalaprilate, 2 µM leupeptin, 1 mM bacitracin, 1 µg/ml N-[N-(L-3-transcarboxyoxiran-2-carbonyl)-L-leucyl] agmatin at 4 °C for 2 h with increasing concentrations of [³H]bradykinin (0.01-10 nM) as described previously (6). At higher concentrations of [³H]bradykinin (>10 nM) a low affinity binding site was detected with B₂⁶⁵.

Construction of Expression Vectors-- The cDNAs coding for the rat B₂ receptor variants starting at amino acid 53 (B₂⁵³) and 65 (B₂⁶⁵) were constructed by polymerase chain reaction and subcloned into pcDNA3. An initiating ATG was introduced to start translation at the indicated amino acid. The identity of the constructs was confirmed by DNA sequencing.

Desensitization of the Bradykinin-induced Rise in [Ca²⁺]_i-- Bradykinin-induced changes in the intracellular free Ca²⁺ concentration, [Ca²⁺]_i, were determined in fura-2-loaded HEK293 cells expressing the different B₂ receptor variants. Cells (1 × 10⁶ cells/ml) suspended in incubation buffer (138 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.6 mM CaCl₂, 1 g/liter glucose, 20 mM Na⁺-HEPES, pH 7.3) were loaded with fura-2/AM as described previously (11). Desensitization of the B₂ receptor was initiated by stimulation of fura-2-loaded HEK293 cells with 1 µM bradykinin for 5 min at 25 °C. Subsequently, cells were washed three times with prewarmed incubation buffer and used for [Ca²⁺]_i determination. Changes in [Ca²⁺]_i are given as the ratio between 340 and 380 nm.

Down-regulation of the Bradykinin-stimulated Increase in Inositol Phosphate Levels-- For determination of total inositol phosphate levels, HEK293 cells expressing the different B₂ receptor variants were seeded on six-well plates and labeled with myo-[2-³H]inositol (2 µCi/ml; specific activity, 17 Ci/mmol) for 16 h in inositol-free RPMI medium containing 0.2% dialyzed fetal calf serum. Prior to the experiment, cells were washed with incubation buffer (138 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.6 mM CaCl₂, 1 g/liter glucose, 20 mM Na⁺-HEPES, pH 7.3), and stored for 5 min in incubation buffer with 10 mM LiCl. Then, the cells were placed at 37 °C, and the experiment was started by addition of buffer or bradykinin as indicated. For B₂ receptor down-regulation, cells were incubated with 10 µM bradykinin for 6 h and washed three times with incubation buffer before the experiment was started as described above by the addition of bradykinin (0.1 nM to 1 µM). After 20 min, total inositol phosphates were extracted as described previously (12).

B₂ Receptor Phosporylation-- B₂ receptor phosphorylation was performed as described previously (13) with minor modifications. Briefly, B₂ receptor-enriched membranes from transiently transfected HEK293 cells were prepared at 4 °C by sucrose density gradient centrifugation (14) in the presence of protease inhibitors (6). Membranes containing 3-5 pmol of B₂ receptor/100 µg of protein were washed by 5 M urea and stored at 80 °C. Recombinant G-protein-coupled receptor kinase 2 (GRK2) was purified from baculovirus-infected Sf9 cells as described (15). For B₂ receptor phosphorylation, reaction mixtures (50 ul) contained 20 nM B₂ receptor, 50 nM GRK2, 200 nM complexes purified from bovine brain (16), 5 mM MgCl₂, 50 µM [-³²P]ATP (NEN Life Science Products), and 20 mM Tris, pH 7.6. Incubations were carried out at 30 °C for 25 min in the presence or absence of 1 µM bradykinin. Reaction was stopped with an equal volume of 2× SDS sample loading buffer, and samples were electrophoresed on SDS/10% polyacrylamide gels containing 6 M urea. Proteins were visualized by autoradiography.

Detection of Agonist-induced B₂ Receptor Dimerization on PC-12 Cells and on HEK293 Cells Expressing Different B₂ Receptor Variants-- Bradykinin-induced formation of receptor dimers on PC12 cells and on HEK293 cells expressing wild-type B₂ receptor, B₂⁵³, and B₂⁶⁵ was detected similarly. Membranes of PC-12 or transfected HEK293 cells were prepared at 4 °C in the presence of protease inhibitors as described previously (6). The membranes were incubated with 10 nM bradykinin or 10 nM HOE140 for 30 min at 25 °C in incubation buffer (20 mM HEPES, 138 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, pH 7.4). For cross-linking, buffers were routinely degassed and handled under N₂ atmosphere. Cross-linking was initiated by the addition of 0.2 mM MBS and incubation for 15 min at room temperature. A new batch of MBS was used for each experiment to avoid inactivation of the cross-linker. Cross-linking was terminated by addition of 100 mM Tris, pH 8.0, and 10 mM dithiothreitol. Membranes were collected by centrifugation and solubilized in SDS sample loading buffer for 15 min at 25 °C instead of boiling to avoid unspecific receptor aggregation. Solubilized proteins were separated by SDS-PAGE, including 6 M urea. Cross-linked bradykinin-B₂ receptor complexes were detected by anti-bradykinin antibodies (7). Similarly, HOE140-B₂ receptor complexes were detected by anti-HOE140 antibodies (6). To control B₂ receptor specificity, a 500-fold molar excess of HOE140 was used as a control for bradykinin cross-linking and a 500-fold molar excess of bradykinin as a control for HOE140 cross-linking. Bound antibodies were visualized with peroxidase-labeled secondary antibodies with enhanced chemiluminescence detection.

Effect of Peptides Corresponding to the Extracellular Domains of the Rat B₂ Receptor on B₂ Receptor Dimer Formation-- PC-12 membranes containing 0.5-1 pmol of B₂ receptor/mg of protein were suspended in phosphate-buffered saline including protease inhibitors (6) and incubated without or with 10 µM of a peptide corresponding to the sequence of the connecting loop between membrane domains II and III (ED2), IV and V (ED3_C), and VI and VII (ED4_N) (6) and to the amino terminus of the B₂ receptor (ED1) for 1 h at 4 °C. The position of the peptides in the rat B₂ receptor and their synthesis has been described previously (6). To induce receptor dimerization, 10 nM [¹²⁵I]Tyr⁸bradykinin was added to the membranes for 30 min at 25 °C. Preparation of [¹²⁵I]Tyr⁸bradykinin was performed as described previously (14), and cross-linking by 0.2 mM DFDNB was performed for 15 min at room temperature. The cross-linking reaction was terminated by 100 mM Tris, pH 8.0, and unbound ligand was removed by washing with the same buffer. The membranes were solubilized in SDS-sample buffer, and proteins were separated by 10% SDS-PAGE including 6 M urea. [¹²⁵I]Tyr⁸bradykinin-labeled B₂ receptors were visualized by autoradiography.

Effect of Site-directed Antibodies on the Internalization of [³H]Bradykinin-- PC-12 cells on six-well plates were incubated for 2 h at 4 °C in the absence (control) or presence of 100 nM affinity-purified antibodies to the amino terminus of the B₂ receptor (-ED1) or to the connecting loop between membrane domains II and III ( -ED2), IV and V (-ED3_C), and VI and VII ( -ED4_N) in incubation buffer (138 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.6 mM CaCl₂, 1 g/liter glucose, 20 mM Na⁺-HEPES, pH 7.3). The generation of the antibodies has been performed essentially as described previously (6). Then, 5 nM [³H]bradykinin was added and incubated for an additional 1 h at 4 °C. To induce internalization, cells were shifted to 37 °C. At the time points indicated, cells were washed three times with 0.2 M glycine, 0.5 M NaCl, pH 3.0, followed by three washing steps with incubation buffer. Then, cells were dissolved by 1 M NaOH, and internalized [³H]bradykinin was determined in a -counter.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Detection of Agonist-induced B₂ Receptor Dimerization on PC-12 Cells-- To determine a potential functional role of receptor dimerization, we attempted to detect B₂ receptor dimers in a system with endogenously expressed B₂ receptors, and we chose PC-12 cells (11). To detect bradykinin-stimulated receptor dimerization, we stimulated cell membranes by bradykinin at 25 °C for 30 min and irreversibly captured receptor dimers by addition of the cross-linker MBS (0.2 mM). Cross-linked bradykinin-B₂ receptor complexes were detected by anti-bradykinin antibodies (7). And indeed, bradykinin binding to the B₂ receptor lead to the formation of receptor dimers and higher oligomerization states as shown in Fig. 1A, lane 1. The B₂ antagonist HOE140 did not significantly stimulate the formation of receptor aggregates when HOE140 was cross-linked to the B₂ receptor under similar conditions as described above and detected by anti-HOE140 antibodies (Fig. 1A, lane 3). To control B₂ receptor specificity, a 500-fold molar excess of HOE140 was used as a control for bradykinin cross-linking (Fig. 1A, lane 2) and a 500-fold molar excess of bradykinin as a control for HOE140 cross-linking (Fig. 1A, lane 4). The presence of a 500-fold molar excess of HOE140 or bradykinin abolished the specific labeling of the B₂ receptor. Thus, B₂ receptors had been labeled by bradykinin and by HOE140, but only bradykinin had induced the formation of receptor dimers/oligomers. This finding suggests that B₂ receptor dimerization/oligomerization was dependent on the binding of agonist to the B₂ receptor.

View larger version (83K): [in this window] [in a new window]   Fig. 1.   A, bradykinin-induced B2 receptor dimerization/oligomerization on PC-12 cells. Membranes of PC-12 cells (0.5-1 pmol of B2 receptor/mg of protein) were incubated with 10 nM bradykinin (lanes 1 and 2) and with 10 nM HOE140 (lanes 3 and 4) in the absence (lanes 1 and 3) or presence of a 500-fold molar excess of HOE140 (lane 2) or bradykinin (lane 4) for 30 min at 25 °C. Ligands were cross-linked with the B2 receptor by 0.2 mM MBS. Proteins were separated by SDS-PAGE; cross-linked bradykinin-B2 receptor complexes were detected by anti-bradykinin antibodies (lanes 1 and 2), and cross-linked HOE140-B2 receptor complexes were detected by anti-HOE140 antibodies (lanes 3 and 4) in Western blot. B, detection of B2 receptor monomers/dimers by [125I]Tyr8bradykinin (10 nM) cross-linked by 0.2 mM DFDNB to PC-12 membranes preincubated without (lanes 4 and 5) or with 10 µM of a peptide corresponding to the connecting loop between membrane domains II and III (ED2) (lane 1), IV and V (ED3C) (lane 2), and VI and VII (ED4N) (lane 3). As a control, the cross-linking was performed in the presence of 5 µM unlabeled bradykinin (lane 5). C, B2 receptor monomers/dimers detected on PC-12 cell membranes by [125I]Tyr8bradykinin (10 nM) cross-linked with B2 receptors by 0.2 mM DFDNB and preincubated in the absence (lanes 1 and 3) or presence (lane 2) of 10 µM of a peptide derived from the amino terminus of the B2 receptor (ED1). As a control, the cross-linking was performed in the presence of 5 µM HOE140 (lane 3). Labeled receptors were detected by autoradiography.

Effect of Peptides Corresponding to the Extracellular Domains of the B₂ Receptor on B₂ Receptor Dimer Formation-- We asked whether the extracellular domains of the B₂ receptor might be involved in B₂ receptor dimerization, and we analyzed the effect of peptides derived from the extracellular domains of the B₂ receptor. The position of the peptides in the rat B₂ receptor has been described previously (6). Membranes of PC-12 cells were preincubated without (Fig. 1B, lanes 4 and 5) or with 10 µM of a peptide corresponding to the connecting loop between membrane domains II and III, ED2 (Fig. 1B, lane 1); to the connecting loop between membrane domains IV and V, ED3_C (Fig. 1B, lane 2); to the connecting loop between membrane domains VI and VII, ED4_N (Fig. 1B, lane 3); and to the amino terminus of the B₂ receptor, ED1 (Fig. 1C, lanes 1 and 2) (cf. Ref. 6 for sequence of the peptides). B₂ receptor monomers and dimers were identified by cross-linking of [¹²⁵I]Tyr⁸bradykinin to B₂ receptors on PC-12 cell membranes by 0.2 mM DFDNB. The presence of 10 µM peptide corresponding to the extracellular loops of the B₂ receptor did not reduce the ratio between labeled B₂ receptor monomers versus dimers compared with the control without peptide (Fig. 1B, lanes 1-3 versus lane 4). Interestingly, no oligomeric states larger than dimers were found when we used the homobifunctional cross-linker DFDNB (Fig. 1B) compared with the heterobifunctional cross-linker MBS (cf. Fig. 1A). This finding may be due to different length and different specificity of the applied cross-linkers. The specificity of the cross-linking was confirmed by the finding that a 500-fold molar excess of unlabeled bradykinin suppressed the labeling of the B₂ receptor by [¹²⁵I]Tyr⁸bradykinin (Fig. 1B, lane 5). Together, these findings suggest that peptides corresponding to the extracellular loops of the B₂ receptor did not affect the capturing of receptor dimers by DFDNB.

In contrast, a peptide derived from the amino terminus of the B₂ receptor (ED1) significantly reduced the ratio between labeled B₂ receptor monomers versus dimers (Fig. 1C, lane 2) compared with the control without peptide (Fig. 1B, lane 4, and Fig. 1C, lane 1) or compared with the controls with peptides directed to the extracellular loops (Fig. 1B, lanes 1-3). The presence of a 500-fold molar excess of the B₂ antagonist HOE140 abolished the labeling by [¹²⁵I]Tyr⁸bradykinin, demonstrating B₂ receptor specificity of the signal(s) (Fig. 1C, lane 3). Together, these findings may suggest that the amino terminus of the B₂ receptor was involved in triggering B₂ receptor dimerization induced by bradykinin.

Expression of Amino-terminally Truncated B₂ Receptor Variants in HEK293 Cells-- To further analyze the role of the amino terminus of the B₂ receptor in receptor dimerization, we created two rat B₂ receptor variants with truncated amino termini, B₂⁵³, starting with amino acid 53 after the conserved cysteine 52 (17), and B₂⁶⁵, lacking the entire amino terminus. The indicated positions of the amino acids are derived from the rat B₂ receptor sequence with extended amino terminus (17). The wild-type B₂ receptor and the two B₂ receptor variants were expressed in HEK293 cells. Expression of the B₂ receptor protein was detected in Western blot by anti-HOE140 antibodies after cross-linking of HOE140 to the B₂ receptor. The antibodies detected a major protein of 55 ± 5 kDa in wild-type receptor-expressing cells (Fig. 2, A, lane 1, and B, lane 1), and proteins of 45 ± 6 and 43 ± 4 kDa were detected in cells expressing B₂⁵³ (Fig. 2B, lane 2) and B₂⁶⁵ (Fig. 2B, lane 3), respectively. No significant amounts of receptor dimers were detected (Fig. 2, A, lane 1, and B, lanes 1-3). The labeling of the B₂ receptors by HOE140 was suppressed when the cross-linking was performed in the presence of a 500-fold molar excess of bradykinin, as demonstrated for the wild-type B₂ receptor (Fig. 2A, lane 2). These findings demonstrate that binding of HOE140 to the different B₂ receptors expressed in HEK293 cells did not induce the formation of receptor dimers, although HOE140 was efficiently cross-linked to these receptors, revealing similar expression levels.

View larger version (39K): [in this window] [in a new window]   Fig. 2.   Bradykinin-induced B2 receptor dimerization/oligomerization on HEK293 cells. A and B, membranes of HEK293 cells (0.5-1 pmol of B2 receptor/100 µg of protein) expressing wild-type B2 receptor (A, lanes 1 and 2; B, lane 1), B253 (B, lane 2), and B265 (B, lane 3) were incubated with 10 nM HOE140 in the absence (A, lane 1; B, lanes 1-3) or presence (A, lane 2) of a 500-fold molar excess of bradykinin for 30 min at 25 °C. Ligands were cross-linked with the B2 receptor by 0.2 mM MBS. Proteins were separated by SDS-PAGE and cross-linked HOE140-B2 receptor complexes were detected by anti-HOE140 antibodies in Western blot. C, detection of bradykinin-induced B2 receptor dimerization/oligomerization. Membranes of HEK293 cells transfected with wild-type B2 receptor (lane 1), with B253 (lane 2), and with B265 (lane 3) were stimulated by 10 nM bradykinin for 30 min at 25 °C. Bradykinin-induced B2 receptor dimers/oligomers were stabilized by 0.2 mM MBS, and proteins were separated by SDS-PAGE and analyzed in Western blot for cross-linked bradykinin-B2 receptor complexes by anti-bradykinin antibodies as above.

Agonist-induced Receptor Dimerization of B₂ Receptors Expressed in HEK293 Cells-- Next we asked whether bradykinin stimulated receptor dimerization of the wild-type B₂ receptor and the truncated B₂ receptor variants expressed on HEK293 cells. Similarly as on PC-12 cells, B₂ receptor dimers were irreversibly captured by MBS and detected by anti-bradykinin antibodies in Western blot. Bradykinin induced the formation of receptor dimers and higher oligomerization states on membranes of HEK293 cells expressing wild-type B₂ receptors (Fig. 2C, lane 1) and on membranes of cells expressing B₂⁵³ (Fig. 2C, lane 2). Although no oligomeric states larger than dimers were found with B₂⁵³, the ratio between B₂ receptor monomers versus dimers was similar for the wild-type B₂ receptor and for B₂⁵³.

In contrast, there were no significant amounts of receptor dimers detectable following stimulation of the B₂⁶⁵ variant by bradykinin (Fig. 2C, lane 3). Bradykinin was efficiently cross-linked to the monomeric form of B₂⁶⁵ (Fig. 2C, lane 3), confirming that the truncation of further 12 amino acids in B₂⁶⁵ compared with B₂⁵³ did not reduce cross-linking efficiency (Fig. 2C, lane 3 versus lane 2). A 500-fold molar excess of HOE140 suppressed the specific labeling of the wild-type B₂ receptor, of B₂⁵³ and of B₂⁶⁵ by bradykinin, confirming B₂ receptor specificity (not shown). Together, these findings suggest that 12 amino acids upstream of membrane domain I were sufficient to support agonist-induced dimerization/oligomerization of the B₂ receptor.

Functional Characterization of the B₂ Receptor Variants-- Next, we analyzed the interrelationship between B₂ receptor dimerization and receptor function, and we determined the K_D values for the binding of [³H]bradykinin. The binding affinity of bradykinin for the wild-type B₂ receptor, for B₂⁵³, and for B₂⁶⁵ was similar. The K_D values of the wild-type B₂, of B₂⁵³, and of B₂⁶⁵ were 0.5 ± 0.4 × 10⁹, 0.3 ± 0.4 × 10⁹, and 0.5 ± 0.4 × 10⁹ M, respectively. This finding suggests that the entire amino terminus of the B₂ receptor was not essential for binding of the agonist bradykinin.

Next, we determined the bradykinin-induced rise in inositol phosphate levels of HEK293 cells expressing the different B₂ receptor variants. The concentration of bradykinin necessary to produce the half-maximum increase in inositol phosphate levels (EC₅₀ value) was 34 ± 7 × 10⁹, 22 ± 8 × 10⁹, and 38 ± 6 × 10⁹ M for the wild-type B₂ receptor, for B₂⁵³, and for B₂⁶⁵, respectively (cf. Fig. 3C). Thus, the amino terminus of the B₂ receptor is not essential for B₂ receptor activation.

View larger version (47K): [in this window] [in a new window]   Fig. 3.   A, bradykinin-induced phosphorylation of the B2 receptor. Membranes of HEK293 cells (20 nM B2 receptor) expressing wild-type B2 (lanes 1-3), B253 (lanes 4-6), and B265 (lanes 7 and 8) were phosphorylated by GRK2 (50 nM) in the absence (lanes 1, 4, and 7) or presence of 1 µM bradykinin (lanes 2, 5, and 8) or desArg10kallidin (lanes 3 and 6) at 30 °C for 25 min. Proteins were separated by SDS-PAGE (7.5%), and 32P-labeled proteins were visualized by autoradiography. The autoradiography is representative of three independent experiments with similar results. B, desensitization of the bradykinin-induced increase in [Ca2+]i. HEK293 cells (1 × 106 cells/ml) expressing wild-type B2 (panels 1 and 4), B253 (panels 2 and 5), and B265 (panels 3 and 6) were loaded with fura-2 and suspended in incubation buffer, and fluorescence was recorded. At the time point indicated by an arrow, 50 nM bradykinin was added. To desensitize the B2 receptor (panels 4-6), cells were treated with 1 µM bradykinin for 5 min prior to the experiment. After removal of bradykinin, changes in [Ca2+]i were determined as above. The data presented are representative of three independent experiments each with similar results. C, down-regulation of the bradykinin-induced increase in inositol phosphate levels. HEK293 cells expressing wild-type B2, B253, and B265 were labeled with myo-[2-3H]inositol. Six hours prior to the experiment, cells were stimulated with 10 µM bradykinin (open circles) or received buffer as a control (filled circles). The concentration-response relationship for the bradykinin-induced rise in inositol phosphate levels was determined. The bradykinin-stimulated increase in inositol phosphate levels is given as percentage of control, i.e. cells that received buffer instead of bradykinin 6 h before the experiment and that were stimulated by 1 µM bradykinin. The control values were 5210 ± 430, 6220 ± 590, and 5010 ± 410 cpm for cells expressing wild-type B2, B253, and B265, respectively. The basal value of unstimulated control cells was 460 ± 80 cpm. Data (± S.E.) are the means of three independent experiments.

Interrelationship between Agonist-induced Receptor Dimerization and Desensitization-- B₂ receptor phosphorylation is the initial step in B₂ receptor desensitization (12, 13). Therefore, phosphorylation of the B₂ receptor variants by GRK2 was analyzed. GRK2 phosphorylated the B₂⁵³ variant upon agonist stimulation (Fig. 3A, lane 5) similarly to the wild-type B₂ receptor (Fig. 3A, lane 2). No significant increase in B₂ receptor phosphorylation was detected after stimulation by a B₁-specific agonist, desArg¹⁰kallidin (Fig. 3A, lanes 3 and 6) compared with the unstimulated control (Fig. 3A, lanes 1 and 4). In contrast, GRK2 did not significantly phosphorylate the B₂⁶⁵ variant upon bradykinin stimulation (Fig. 3A, lane 8) compared with the unstimulated control (Fig. 3A, lane 7). This experiment strongly suggests that agonist-induced phosphorylation of the B₂⁶⁵ receptor by GRK2 was impaired.

Receptor phosphorylation is assumed to precede receptor desensitization (18, 19). Therefore, we next asked whether desensitization of the B2⁶⁵ variant was also impaired. The desensitization of the bradykinin signal was analyzed for the bradykinin-stimulated rise in [Ca²⁺]_i on fura-2-loaded HEK293 cells expressing the different B₂ receptor variants. The bradykinin-induced rise in [Ca²⁺]_i was similar in wild-type B₂ receptor-expressing cells, in cells expressing B₂⁵³, and in cells expressing B₂⁶⁵ (Fig. 3B, panels 1-3). To initiate receptor desensitization, the cells were preincubated for 5 min with 1 µM bradykinin. After removal of bradykinin, the cells were given a second pulse of bradykinin. A second stimulation by bradykinin did not significantly increase [Ca²⁺]_i in wild-type B₂ receptor-expressing cells (Fig. 3B, panel 4) or in cells expressing the B₂⁵³ receptor (Fig. 3B, panel 5), indicating that the receptors were efficiently desensitized after prestimulation by 1 µM bradykinin. In contrast, the B₂⁶⁵ receptor was not desensitized after the initial bradykinin stimulation, as revealed by a rise in [Ca²⁺]_i following a second pulse of bradykinin (Fig. 3B, panel 6).

Down-regulation of the Different B₂ Receptor Variants-- The B₂⁶⁵ receptor poorly dimerized, was not significantly phosphorylated, and did not desensitize upon bradykinin stimulation. Therefore, we asked whether the B₂⁶⁵ receptor could be down regulated. To this end, the B₂ receptor-expressing cells were prestimulated by 10 µM bradykinin for 6 h. After washing, the bradykinin-stimulated increase in inositol phosphate levels was determined compared with the unstimulated control. After long-term bradykinin stimulation, the B₂ receptor-mediated signal was down-regulated on wild-type B₂ receptor-expressing cells and on B₂⁵³ expressing cells. The signal was reduced by 28 ± 3 and 25 ± 5%, respectively (Fig. 3C, panels 1 and 2).

In contrast, the B₂ receptor-mediated signal was not down regulated on B₂⁶⁵-expressing cells (Fig. 3C, panel 3). Thus, a B₂ receptor variant lacking the entire amino terminus had lost its capability to undergo agonist-induced dimerization, phosphorylation, desensitization, and down-regulation.

Effect of Site-directed Antibodies on the Internalization of [³H]Bradykinin on PC-12 Cells-- Finally, we asked whether the amino terminus of the B₂ receptor was also involved in the signal attenuation process of a B₂ receptor expressed in a native system. To this end, we initially tested the effect of the peptide ED1 derived from the amino terminus of the B₂ receptor, which has been shown to reduce the ratio between detected B₂ receptor monomers versus dimers (cf. Fig. 1C). However, this peptide (10 µM) had no significant effect on B₂ receptor phosphorylation and internalization under the conditions applied (not shown). Therefore, we next analyzed the effect of site-directed antibodies to the extracellular domains of the B₂ receptor. The generation, immunoselection, and characterization of these antibodies was performed as described previously (6). The site-directed antibodies used for this study did not affect the binding of [³H]bradykinin or the activation of the B₂ receptor (6). The antibodies to the amino terminus of the B₂ receptor (-ED1) caused a significant reduction of internalized [³H]bradykinin (Fig. 4, A and B) by 24 ± 4%, whereas site-directed antibodies to the extracellular loops (-ED2, -ED3_C, and -ED4_N) had no effect on internalization of [³H]bradykinin (Fig. 4A). Results obtained with the site-directed antibodies in phosphorylation studies were not consistent, probably due to the moderate effect of the antibodies (not shown). Nevertheless, our data indicate that the amino terminus of the B₂ receptor is also involved in B₂ receptor-mediated signal attenuation processes on PC-12 cells.

View larger version (41K): [in this window] [in a new window]   Fig. 4.   A, effect of site-directed antibodies on the internalization of [3H]bradykinin. PC-12 cells were incubated for 2 h at 4 °C in the absence (control) or presence of 100 nM affinity-purified antibodies to the amino terminus of the B2 receptor (-ED1), to the connecting loop between membrane domains II and III (-ED2), IV-V (-ED3C), and VI-VII (-ED4N). Then, 5 nM [3H]bradykinin was added and incubated for 1 h at 4 °C. To induce internalization, cells were shifted to 37 °C. After 20 min, internalized [3H]bradykinin was determined. B, PC-12 cells were incubated for 2 h at 4 °C in the absence (control) or presence of 100 nM affinity-purified antibodies to the amino terminus (-ED1). Then, 5 nM [3H]bradykinin was added and incubated for 1 h at 4 °C. To induce internalization, cells were shifted to 37 °C, and internalized [3H]bradykinin was determined at the time points indicated. Data are the means of three independent experiments (±S.E.).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Although the detailed mechanism of receptor dimerization is far from being understood, different regions of G-protein-coupled receptors have been identified to be involved in receptor dimerization. Dimerization of the dopamine D₂ and the ₂-adrenergic receptor occur via transmembrane regions (1, 2). In contrast, the metabotropic glutamate receptor and the calcium sensing receptor dimerize via their large extracellular amino terminus and to a lesser extent via their transmembrane regions (3, 4). Although the B₂ receptor, as a family 1b receptor, lacks a large extracellular domain that is characteristic of family 3 receptors, agonist-induced B₂ receptor dimerization also needs the amino terminus of the receptor because (i) a peptide corresponding to the amino terminus of the B₂ receptor partially suppressed bradykinin-induced receptor dimerization on PC-12 cells, and (ii) a B₂ receptor variant lacking the entire amino terminus, B₂⁶⁵, was impaired in agonist-induced receptor dimerization. The amino terminus, which was necessary for agonist-stimulated B₂ receptor dimerization, was not essential for agonist binding. Such a finding is in contrast to previous studies with the ₂-adrenergic, the dopamine, the calcium sensing, or the metabotropic glutamate receptor where the region(s) involved in receptor dimerization were overlapping with the agonist binding sites (1-4). This potential mechanistic difference of B₂ receptor dimerization may be related to the predicted topology of the B₂ receptor, which is different from rhodopsin with five transmembrane domains instead of seven and two membrane reentrant segments, membrane domains I and II (20). Thus, membrane domain I, which follows the amino terminus, is not membrane-spanning.

In addition to the mechanism, the functional importance of receptor dimerization is not clear. Receptor dimerization of the ₂-adrenergic receptor seems to be related to receptor activation because the amount or receptor dimers increased upon agonist stimulation, and a peptide derived from transmembrane domain VI partially suppressed receptor dimerization and receptor activation (1). Several other lines of evidence suggest, however, that receptor dimerization does not seem to be essential for receptor activation. The stoichiometry of the interaction between the receptor, G-protein, and effector molecule is assumed to be 1:1:1 (21), and a single receptor with a single G_s protein fused to its carboxyl terminus is functionally active (22). Therefore, the potential role of receptor dimerization may lie apart from receptor activation. And indeed, a B₂ receptor variant (B₂⁶⁵) with impaired agonist-induced receptor dimerization was "normal" with respect to signal generation. In contrast, this B₂ receptor variant was defective in agonist-induced receptor phosphorylation, desensitization, and down-regulation. Likewise, affinity-purified antibodies to the region that is necessary for receptor dimerization, the amino terminus of the B₂ receptor, partially reduced internalization of [³H]bradykinin on PC-12 cells. Thus, agonist-induced dimerization of the B₂ receptor seems to follow receptor-induced G-protein activation and to precede receptor phosphorylation, desensitization, and down-regulation.

Twelve amino acids upstream of membrane domain I may be sufficient to support agonist-induced B₂ receptor dimerization because B₂⁵³ was still capable of dimerizing after bradykinin stimulation, whereas B₂⁶⁵, lacking the entire amino terminus, did not significantly dimerize after bradykinin stimulation. Bradykinin-induced B₂ receptor dimerization did not depend on receptor overexpression because bradykinin-induced B₂ receptor dimerization was also detected on PC-12 cells with endogenously expressed B₂ receptors. Thus, agonist-induced B₂ receptor dimerization via the amino terminus of the receptor is a characteristic of B₂ receptor signaling.

In addition to agonist-induced dimerization, G-protein-coupled receptors tend to dimerize under native conditions via hydrophobic interactions (7). Receptor clustering via hydrophobic interactions within the (trans)membrane domains may be supported by high receptor density. Future work will have to determine whether, in addition to agonist-induced receptor dimerization, which has been shown to regulate signal attenuation processes, receptor clustering under basal conditions represents an additional mechanism of regulating B₂ receptor activity.

	ACKNOWLEDGEMENTS

We thank Drs. H. Moawad (Alexandria, Egypt) and A. El Massiery (Cairo, Egypt) for help in generating site-directed antibodies and M. J. Lohse (Würzburg, Germany), for support in the field of G-protein-coupled receptors.

	FOOTNOTES

^* This work was supported in part by the Deutsche Forschungsgemeinschaft Grant Lo 371/2-2.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 0049-931-201-3982; Fax: 0049-931-201-3539.

	ABBREVIATIONS

The abbreviations used are: MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; DFDNB, 1,5-difluoro-2,4-dinitrobenzene; GRK, G-protein-coupled receptor kinase; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES