Bradykinin-induced Internalization of the Human B2 Receptor Requires Phosphorylation of Three Serine and Two Threonine Residues at Its Carboxyl Tail*

Anne PizardDagger §, Andree Blaukatparallel , Werner Müller-Esterl, François Alhenc-GelasDagger , and Rabary M. RajerisonDagger

From Dagger  INSERM Unité 367, 17 rue du Fer à Moulin 75005 Paris, France and the  Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg University at Mainz, Duesbergweg 6, D-55099 Mainz, Federal Republic of Germany

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ABSTRACT
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The binding of bradykinin (BK) to B2 receptor triggers the internalization of the agonist-receptor complex. To investigate the mechanisms and the receptor structures involved in this fundamental process of receptor regulation, the human B2 receptor was mutated within its cytoplasmic tail by complementary strategies of truncation, deletion, and amino acid substitution. Ligand binding, signal transduction, internalization as well as phosphorylation were studied for the mutated receptors expressed in COS, CHO, and HEK 293 cells. Truncation of 44 out of 55 amino acid residues of the receptor's cytoplasmic tail corresponding to positions 321-364 did not alter the kinetics of BK binding and the receptor coupling to phospholipase C and phospholipase A2. By contrast, truncations after positions 320 and 334, deletions within the segment covering positions 335-351, as well as alanine substitution of serine and threonine residues within segment 335-351 diminished the internalization capacity of the mutant receptors. Mutants with a markedly reduced internalization potential failed to produce BK-induced receptor phosphorylation suggesting that phosphorylation may be involved in receptor internalization. The mutagenesis approaches converged at the conclusion that three serines in positions 339, 346, and 348 and two threonines in positions 342 and 345, contained in a sequence segment that is highly conserved between species, have a critical role in the ligand-dependent internalization and phosphorylation of kinin receptors and can intervene in these processes in an alternative manner. However, mutants lacking these residues were still sensitive to dominant-negative forms of beta -arrestin and dynamin, suggesting the existence of additional receptor structure(s) involved in the receptor sequestration through clathrin-coated vesicles.

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ABSTRACT
INTRODUCTION
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The capacity of most G protein-coupled receptors (GPCRs)1 to respond to agonists becomes altered after previous exposure to their ligands, a phenomenon referred to as desensitization. Desensitization can be a consequence of receptor endocytosis (1) and may also result from receptor phosphorylation that prevents further activation of G proteins (2, 3). Negative cooperativity in bradykinin (BK) binding due to receptor-receptor interaction in the cell membrane can also contribute to the desensitization of some receptors including the BK B2 receptor (4). For few GPCRs, internalization appears to be a prerequisite for resensitization prior to receptor recycling to the plasma membrane (5). Internalization is believed to involve clathrin-coated vesicles and/or caveolin-rich vesicles, and to result from an interaction of components of the endocytic machinery with specific motifs located in the cytoplasmic domains of the receptor (6). For many receptors, the carboxyl tail has been found to play a critical role in receptor internalization (2, 3, 7) but other critical motifs have been identified in the second or third intracellular loops (8, 9) as well as in the seventh transmembrane domain (10).

The molecular mechanisms that trigger internalization of the agonist-occupied receptor have been studied for only a few receptors. Phosphorylation has been implicated in the desensitization process uncoupling the receptor from G proteins, and in the initiation of internalization sequestering the receptor from the cell surface (2, 3). Indeed, mutations of potential phosphorylation sites of the GLP-1, muscarinic m3, and the cholecystokinin receptors have been shown to reduce their internalization (11-13). However, receptor internalization and desensitization are not always causally linked, e.g. mutations of certain GPCRs suppressed desensitization but not internalization (14, 15). Conversely mutations in some GPCRs altered internalization but not desensitization (10, 15). Clearly, the two processes can proceed independently although they may involve the same type of post-translational modifications such as phosphorylation.

Prototypical GPCRs that differ by their ligand-induced densensitization, phosphorylation, and internalization are the receptors for the vasoactive kinin peptides. Two subtypes of mammalian kinin receptors, B1 and B2, have been recognized so far (16-18). The B2 receptor is responsible for most of the physiological actions of BK including vasodilation (19) through activation of G proteins (20) that stimulate the activity of phospholipase C (PLC) and phospholipase A2 (PLA2) and increase the cytoplasmic calcium concentration, [Ca2+]i (21). More recently, other signaling pathways such as the mitogen-activated protein (MAP) kinase pathway were found to be triggered via the B2 receptor (22, 23). BK-induced B2 receptor internalization has been reported in several cell systems including cultured human fibroblasts HF-15 (24, 25) and CHO-K1 cells transfected with the human B2 receptor cDNA (4), and we have recently reported that in HF-15 cells BK induces the phosphorylation of serine and threonine residues located in the COOH terminus of the B2 receptor (21). The time course of BK-stimulated phosphorylation paralleled the kinetics of desensitization/resensitization and of internalization/recycling of the receptor, suggesting that these phenomena may be interrelated (21).

The present work was aimed at studying the role of the COOH-terminal tail of the human renal B2 bradykinin receptor (referred as wild-type, B2wt) in BK binding, coupling to signaling pathways, internalization, and phosphorylation. For this purpose, we generated a series of human B2 receptor mutants and transfected them into COS-7 cells, CHO-K1, and HEK 293 cells. Three complementary strategies were followed for mutagenesis, by generating mutants with truncated COOH-terminal tail, mutants with deletion of internal regions of the COOH-terminal tail, and mutants with serine and/or threonine residue(s) replaced by alanine. This allowed us to demonstrate that a cluster of three serine and two threonine residues located in the center portion of the COOH-terminal tail region that becomes phosphorylated in response to BK is involved in the internalization of the B2 receptor. Co-transfection of the B2wt and its mutants with beta -arrestin and dynamin mutants documented a role of clathrin-coated pit pathway in receptor internalization.

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Construction of the Mutant Receptor cDNAs-- Mutant cDNAs were constructed by using the previously cloned B2wt cDNA placed under the control of cytomegalovirus promoter into the eucaryotic expression vector pcDNA3 (Invitrogen, Leek, Netherlands) (4) as a template in site-directed mutagenesis using the TransformerTM Site-directed Mutagenesis Kit (2nd version, CLONTECH, Palo Alto, CA). All mutations were sequenced using an AmplicycleTM sequencing kit (Perkin Elmer, Langen, Germany). Three truncated B2 mutants, tR351, tI334, and tY320 were created by single nucleotide substitutions (indicated by bold face in the sequences given below) at the appropriate sites so as to create stop codons (underlined) terminating translation at positions 351, 334, and 320, respectively, of the protein sequence (numbering according to Hess et al. (17)). The nucleotide sequences of the oligonucleotide primers used were: 5'-CGTGGAACGCTAGATTCACAAACTG-3' for the mutant tR351, 5'-CAGAACCCATTTAGATGGAGAACTCC-3' for tI334, and 5'-GGAGGTGTACTAGGGAGTGTG-3' for tY320. Hence deletion mutants tR351, tI334, and tY320 lacked the terminal 13, 30, and 44 amino acid residues, respectively, of the human B2 receptor. Deletions of regions within the COOH-terminal tail of the B2 receptor were obtained by mutagenesis with the following oligonucleotide primers: 5'-CAGGTCAGAACCCATTCAGATTCACAAACTGC-3' for the mutant del[335-351], 5'-CAGGTCAGAACCCATTCTGCGGACCTCCATC-3' for del[335-342] and 5'-CTCCATGGGCACACAGATTCACAAACTGC-3' for del[343-351]. The resulting mutants del[335-351], del[335-342], and del[343-351] lacked 17, 8, and 9 amino acid residues, respectively; their relative positions within the protein sequence are given in brackets. Point mutations were created by the same procedure to replace Ser and/or Thr residues in the COOH-terminal tail by Ala using the following oligonucleotide primers: 5'-GGAGATCCAGGCCGAGAGGAGG-3' for the mutant T237A, 5'-CGAAAGAAGGCTTGGGAGGTG-3' for S316A, 5'-GGCTGCAGGGCAGAACCC-3' for S331A, 5'-GATGGAGAACGCCATGGGCAC-3' for S339A, 5'-CTCCATGGGCGCACTGCGGAC-3' for T342A, 5'-GCACACTGCGGGCCGCCATCTCCGTG-3' for T345A/S346A, 5'-GACCTCCATCGCCGTGGAAC-3' for S348A, 5'-GGAGAACGCCATGGGCGCACTGCGG-3' for S339A/T342A and 5'-GAACGCCATGGGCGCACTGCGGGCCGCCATCGCCGTGG-3' for B2Delta ST in which Ser339, Thr342, Thr345, Ser346, and Ser348 were simultaneously substituted by Ala. The mutant S339A/T345A/S346A was obtained by combining the primers used for the S339A and T345A/S346A mutants.

Cell Culture and Receptor Expression-- COS-7 and HEK 293 cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's medium (Biological Industries, Israel) and CHO-K1 cells in Ham's F-12 medium (Seromed, Berlin, Germany) both supplemented with 10% fetal calf serum, 2 mM glutamine, and 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 0.25 µg/ml amphotericin B. For immunoprecipitation and phosphorylation studies, transient receptor expression was achieved by transfecting COS-7 cells grown in 6-well plates using the LipofectAMINETM method (Life Technologies). Otherwise, transfections were performed with cells grown in T75 flasks (COS-7 cells) by using the DEAE-dextran method (26) and the cells were then subcultured into 24-well plates where binding, internalization, PLC, and PLA2 activation experiments were performed or with cells directly grown in 24-well plates (HEK 293 and CHO-K1 cells) by using Superfect Transfection Reagent (Qiagen, Courtaboeuf, France). CHO-K1 cell clones that were selected with geneticin (0.75 µg/ml) for stable expression and with [3H]BK binding and coupling assays for receptor expression, were expanded to obtain the CHO-K1 cell lines used in some experiments. All cell types were maintained at 37 °C in a humidified water-jacketed incubator with 5% CO2. Functional studies of the transfected cells were done at confluence. Control cells were created by transfecting the pcDNA3 vector without insert.

Radioligand Binding and Internalization of [3H]BK-- Cells were incubated as described previously (4) in Hank's balanced salt solution (HBSS, 0.33 mM Na2HPO4, 0.44 mM KH2PO4, 127 mM NaCl, 5 mM KCl, 4 mM NaHCO3, 20 mM HEPES, 1 mM MgCl2, 0.8 mM MgSO4, 1.5 mM CaCl2, 5 mM glucose, 10 mM sodium acetate, pH 7.4) containing protease inhibitors (10-5 M captopril, 0.08 unit/ml aprotinin, 0.1 mg/ml bacitracin), 0.1% fatty acid-free bovine serum albumin (BSA), and [3H]BK (110 Ci/mmol, NEN, Leblanc Mesnil, France). For equilibrium studies and determination of receptor density or apparent affinity for [3H]BK, incubation was carried out at 4 °C for 6 h in the presence of 1.25 × 10-11 to 2.5 × 10-8 M [3H]BK (COS cells) or 10-11 to 10-7 M [3H]BK (CHO cells). The cells were extensively washed with HBSS and bound [3H]BK was determined in a liquid scintillation counter. Internalization of [3H]BK was studied by incubating the cells with 2 nM [3H]BK at 37 °C for 2 to 70 min (4). Cells were washed with HBSS before cell surface-bound radioligand was separated from internalized radioactivity by an acidic washing step (27) with 0.2 M acetic acid, 0.5 M NaCl, pH 2.5. To study the role of protein kinase C (PKC) activity, cells were preincubated with 10-7 M phorbol 12-myristate 13-acetate (PMA) or 10-7 M staurosporine (Sigma, St-Quentin-Fallavier, France) for 30 min before and during the incubation with [3H]BK. To monitor the effect of phosphatase inhibitor, cells were incubated with 10-7 M okadaic acid (Sigma) 5 min prior to [3H]BK challenge, and internalization was followed for 10 min in the continuous presence of the phosphatase inhibitor. In some internalization experiments, cells were treated with hypertonic saline solution containing 0.4 M sucrose to study the contribution of clathrin-coated vesicle pathway (28). In addition, internalization was examined in cells co-transfected with the receptor and with rat beta -arrestin cDNA or its inactive fragment (319-418) mutant in pEGFP obtained from Bunnett and co-workers (29) and pcDNA3 plasmids encoding for bovine beta -arrestin fragment (319-418) mutant and inactive human dynamin K44A both obtained from Benovic and co-workers (30). Co-transfection was done using 15 µg of each plasmid. For determination of specific binding, each assay included measurements of nonspecific binding in the presence of 1000-fold excess of unlabeled BK; nonspecific binding was substracted from total binding determined in the absence of unlabeled BK. In each culture plate, the protein content was determined according to the manufacturer's instructions in three wells using BSA as the standard protein (Bio-Rad, München, Germany).

Inositol Phosphate Production-- Cells were loaded for 18 h at 37 °C with 3 µCi/ml myo-[2-3H]inositol (10-20 Ci/mmol, Amersham International) added to the culture medium. The cells were washed twice with HBSS, preincubated for 10 min at 37 °C with 10 mM LiCl in HBSS containing 0.1% BSA and the protease inhibitors used for binding assay, and stimulated for 15 min with BK at varying concentrations (10-10 to 10-7 M). To study the role of PKC, cells were treated with PMA (10-7 M) or staurosporine (10-7 M) for 30 min before and during the incubation with BK. Reactions were terminated by addition of 3% (w/v) ice-cold perchloric acid and total [3H]inositol phosphate radioactivity was isolated using AG1-X8 anion exchange column chromatography (formate form, 100-200 mesh, Bio-Rad) after the radioactivity contained in phospholipids had been extracted with chloroform (4). Results are expressed as the ratio between the radioactivity measured in the inositol phosphates and the total radioactivity incorporated into the overall compounds labeled with myo-[3H]inositol.

Measurement of Phospholipase A2 activity-- Cells were labeled to equilibrium with 1 µCi/ml [3H]arachidonic acid (150-230 Ci/mmol; Amersham International) for 18 h. After washing steps to eliminate unbound radiolabel (4), phospholipase A2 activation experiments were performed at 37 °C for 10 min in HBSS containing 0.1% BSA, protease inhibitors, and the test compounds or vehicles. The medium radioactivity containing the released [3H]arachidonic acid plus derived 3H-labeled metabolites was counted and expressed in percent of the total radioactivity, i.e. the medium plus cell associated radioactivity.

35S Labeling and Immunoprecipitation-- Cells were washed twice with sulfur-free HEPES-buffered Dulbecco's modified Eagle's medium, incubated for 30 min at 37 °C in the same medium, and labeled with 0.1 mCi/ml 35S-labeled amino acids (Prox-mixTM, Amersham International) for 8 h (21). After three washes with 50 mM Tris, pH 7.5, 150 mM NaCl (Tris-buffered saline), cells were scraped into ice-cold lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% (w/v) Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 0.1 mM Pefabloc SCTM, and 10 µg/ml each of 1,10-phenantroline, aprotinin, leupeptin, and pepstatin A, and incubated for 45 min at 4 °C under gentle rocking. The resulting lysates were centrifuged for 10 min at 13,000 rpm and the supernatants were precleared with 50 µl of Staphylococcus aureus cell suspension (PansorbinTM, Calbiochem).

To immunoprecipitate the receptor, 2.5 µl of antiserum AS346 (21) diluted in 0.1 ml of 5% BSA in lysis buffer was added. After 15 min at room temperature, 50 µl of PansorbinTM was added, and the suspension incubated for 10 min at room temperature. The precipitate was recovered by a centrifugation for 2 min at 6,000 rpm and washed three times with lysis buffer and once with water. SDS sample buffer (25 µl) was added to the immunoprecipitate followed by a 15-min incubation at 45 °C. Proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE) in the presence of 5 M urea. After fixation with 20% (w/v) trichloroacetic acid for 20 min, the gels were washed several times with water and subjected to fluorography using 15% (w/v) sodium salicylate as the fluorophor. For control, the antiserum was preadsorbed for 90 min at room temperature with the peptide antigen coupled to Affi-Gel 10 (Bio-Rad).

Receptor Phosphorylation-- Cells were washed twice with phosphate-free HEPES-buffered Dulbecco's modified Eagle's medium, incubated for 1 h at 37 °C, and labeled with 0.25 mCi/ml [32P]orthophosphate (ICN) for 8 h in the same medium. After a 5-min exposure to 1 µM BK or vehicle at 37 °C, cells were scraped into 1 ml of ice-cold lysis buffer containing protease inhibitors (see above) and phosphatase inhibitors (50 mM sodium fluoride, 25 mM sodium pyrophosphate, 1 mM sodium orthovanadate). To study the role of PKC and the effect of phosphatase inhibitor, cells were exposed to 0.1 µM PMA and 0.1 µM okadaic acid, respectively, in the absence or presence of 1 µM BK before the cell lysis. Solubilization, immunoprecipitation, and gel electrophoresis were carried out as above. Proteins labeled by [32P]orthophosphate were revealed by autoradiography.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Our strategy to analyze the role of the COOH-terminal domain of the B2 receptor in ligand binding, signal transduction, sequestration, and phosphorylation involved the production of mutants with progressive truncations of the COOH-terminal tail of the receptor, deletions of defined portions of the tail region, or exchange of single or multiple residues of serine and threonine against alanine at defined positions of the COOH-terminal domain of the receptor.

Endogenous Receptor of Cells Used for Transfection-- Initially we tested for the presence of endogenous B2 receptor in the COS cells using mock-transfected cells and comparing them with transfected cells expressing the wild-type human B2 receptor (COSB2wt). The receptor density measured by [3H]BK binding and the BK-sensitive PLC activity determined by IPs production in mock-transfected COS cells represented <5% of the values measured in COSB2wt, whereas the ligand-induced [Ca2+]i transients of mock-transfected cells were similar to those obtained with COSB2wt (data not shown). No PLA2 activation was observed in mock-transfected or COSB2wt cells indicating the absence of this transduction pathway in COS-7 cells. Therefore we choose [3H]BK binding and IPs production to exemplify the signaling properties of the recombinant human B2 receptors expressed in COS-7 cells. No endogenous B2 receptor was detected with [3H]BK binding, PLC, or PLA2 assays in the mock-transfected CHO-K1 and HEK 293 cells.

Generation of Mutant B2 Receptors-- To study the functional role of the COOH-terminal tail region corresponding to intracellular domain ID-4 of the B2wt we produced three truncation variants with progressive deletions of the COOH-terminal portion of the receptor. These truncation mutants are designated by "t" followed by the name (single letter code) and position of their carboxyl-terminal amino acid, i.e. tR351, tI334, and tY320; they lack the COOH-terminal 14, 30, or 44 residues of the native receptor (Fig. 1). Three deletion mutants are designated by "del" followed by the positions delimiting the deleted region, i.e. del[335-351], del[335-342], and del[343-351] lacking 17, 8, and 9 residues, respectively, of the COOH-terminal tail region (Fig. 1). We also created 7 receptor mutants in which alanine (Ala) was substituted for serine (Ser) or threonine (Thr) residue(s), singly or combined (Table I). In the mutant B2Delta ST we exchanged 5 Ser/Thr residues located in the center portion of the COOH-terminal domain, i.e. at positions 339, 342, 345, 346, and 348 (Fig. 1). We have also constructed 3 mutants where Ser/Thr residues have been replaced by Ala in other regions of the receptor, i.e. T237A, S316A, and S331A (not shown). The rationale for the design of the various mutants is given below.


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Fig. 1.   Schematic representation of the COOH-terminal tail of B2wt and mutant receptors. Top, mutated amino acid residues are indicated by their position within the B2wt sequence; the numbering is according to Hess et al. (39). Ser and Thr residues which were replaced by Ala are marked by asterisks. Center, cytoplasmic tail truncations were performed at 3 sites to generate tR351, tI334, and tY320. Bottom, deletion of segments holding 17, 8, and 9 residues generated mutants del[335-351], del[335-342], and del[343-351], respectively; the deleted segments are identified by their positions (given in brackets) and marked by bold lines.

                              
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Table I
[3H]BK binding and BK-induced PLC activation characteristics of B2wt and mutant receptors in COS cells
In each binding experiment, up to eight mutants were studied together with the B2wt as control. Cells were used 72 h after transfection, and incubated at 4 °C for 6 h with seven increasing concentrations of [3H]BK (from 1.25 × 10-11 to 2.5 × 10-8 M), before determination of specific binding (see "Experimental Procedures"). Data were first plotted using Scatchard coordinates. Given the curvilinear character of the plots obtained, binding values at the three highest [3H]BK concentrations were used to estimate the maximal binding capacity (Bmax). All binding values were then plotted using Hill coordinates to estimate the [3H]BK concentration (KDapp) corresponding to half-saturation of binding sites. In each PLC activation experiment out of the 14 performed, up to six mutants were studied together with the B2wt as control. Cells at 72 h after transfection were incubated at 37 °C for 15 min with 10-11 to 10-7 M BK and 10 mM LiCl, before determination of IPs production (see "Experimental Procedures"). For each mutant receptor, results are the mean ± S.E. of at least three independent experiments, each performed in triplicate.

Expression and Characterization of Mutant B2 Receptors-- The mutant receptors were characterized for their ligand binding and signal transduction properties. Ligand binding was assessed by incubating transfected cells with a wide range of [3H]BK concentrations at 1.25 × 10-11 M to 2.5 × 10-8 M for COS-7 cells (Table I) and at 10-11 M to 10-7 M for CHO-K1 cells (Table II). The incubation was carried out for 6 h under equilibrium conditions at 4 °C to suppress internalization. In COS cells (Table I) the kinetics of [3H]BK binding displayed negative cooperativity as previously observed for the human B2wt in CHO-K1 cells (4). The receptor density determined by Scatchard analysis (31) was similar for the B2wt and mutant receptors except for tY320 and B2Delta ST which were 10- and 5-fold less expressed, respectively. The kinetics of [3H]BK binding estimated from Hill analysis of the binding data (32) were similar for the various receptors since half-maximum binding (KD,app) occurred within a narrow range (2.3 to 6.3 nM) of [3H]BK concentrations. Regarding the signal transduction properties, the basal IPs production was similar for B2wt and the mutant receptors demonstrating that none of the mutations resulted in a constitutive activation of PLC. All mutant receptors were able to trigger PLC activation in response to 10-11 to 10-7 M BK and responded to BK with maximum stimulation (4.1-8.5-fold) and EC50 values (0.2-0.8 nM) in the same order of magnitude as those observed with B2wt with the exception of tY320 which had a higher EC50 value (2.2 ± 0.9 nM). Then, we choose to stably express in CHO-K1 cells the mutant tY320 bearing the most pronounced structural change and also the largest deleted mutant, del[335-351] lacking the 5 Ser and Thr residues. The B2wt CHO cell line was obtained as described previously (4). In these cells (Table II), tY320 also exhibited a higher EC50 value for PLC activation, a greater EC50 value for PLA2 activation and a lower affinity for [3H]BK. By contrast, the mutant del[335-351] exhibited a slightly higher affinity for [3H]BK and exhibited a 30- and 50-fold lower EC50 value for PLC and PLA2 activation, respectively, compared with B2wt (Table II). These data indicate that the COOH-terminal tail is not necessary for signal transduction but that its conformation influences the efficiency of BK binding and coupling to PLC and PLA2.

                              
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Table II
[3H]BK binding and BK-induced PLC and PLA2 activation characteristics of B2wt and mutant receptors in CHO-K1 cells
For each type of experiment mutants were studied together with the B2wt as control. CHO-K1 stable transfected cells were incubated overnight at 4 °C with 30 increasing concentrations of [3H]BK (from 10-11 to 10-7 M), before determination of specific binding (see "Experimental Procedures"). Data were obtained as explained in the legend of Table I. In coupling experiments, cells were incubated at 37 °C with 10-11 to 10-7 M BK for 10 min before measurement of PLA2 activation and 15 min in presence of 10 mM LiCl before determination of IPs production (see "Experimental Procedures"). For each mutant receptor, results are the mean ± S.E. of at least three independent experiments.

Internalization of [3H]BK by Mutant B2 Receptors Expressed in COS-7 Cells-- In the COSB2wt which were transfected with 15 µg of cDNA and challenged with 2 nM [3H]BK (Fig. 2), the internalization occurred with a t1/2 of 12 min at 37 °C and represented 60.3 ± 2.5% (n = 22) of the bound [3H]BK at 70 min. The same internalization rate was observed in COS cells transfected with 0.75, 1.5, 3, 5, or 10 µg of cDNA and displaying a wide range of receptor density (from 0.9 to 5.0 pmol/mg of protein) (data not shown). This is also the case for CHO cells which expressed the B2wt stably at different density (0.3 to 2.5 pmol/mg protein) (not shown) with the remark that the internalization was more efficient (72.6 ± 1.5% (n = 7) of the bound radioactivity) in CHO than in COS cells. Regarding the effects of the receptor mutations, most of the data presented below were from COS-7 cells transfected with 15 µg of cDNA. In these cells, it was apparent that the serial truncations of the COOH-terminal tail region gave distinct phenotypes: tR351 which lacks the extreme 14 COOH-terminal residues had an unchanged internalization capacity whereas tI334 displayed a markedly reduced capacity to internalize [3H]BK, i.e. 35% of that of the B2wt (100%). Further truncations of the receptor exemplified by mutant tY320 did not result in a greater reduction of the internalization capacity (Fig. 2). These findings suggest that the COOH-terminal portion distal to Arg351 is dispensable for receptor internalization while the segment spanning positions 335 to 351 comprising 17 residues appears to be of critical importance to receptor sequestration. To further test this hypothesis we constructed a deletion mutant, del[335-351] which selectively lacks segment Gln335 to Arg351 and retains the portion distal to Arg351 (Fig. 1). The internalization capacity of del[335-351] was similar to that of tI334 demonstrating that the 13 most COOH-terminal residues of the B2 receptor (positions 352 to 364) cannot rescue this phenotype. In addition, the same internalization rate was observed over a wide range of density of this mutant receptor del[335-351] that was established by transfecting cells with 0.75 to 15 µg of cDNA (not shown). To pinpoint the region(s) important for internalization we generated mutants where the proximal 8 residues of the critical Gln335 right-arrow Arg351 segment are removed, del[335-342], or where the distal 9 residues are deleted, del[343-351]. The effect of the largest deletion mutant (del[335-351]) was almost quantitatively reproduced by deletion mutant del[335-342] (24.7 ± 5.2 and 30.7 ± 5.2%, respectively, of B2wt). However, del[343-351] also resulted in a reduced internalization capacity (56.5 ± 4.5% of B2wt), suggesting that the adjacent segments spanning positions 335 to 342 and 343 to 351 do not function in a simple additive manner for receptor internalization.


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Fig. 2.   [3H]BK internalization in COS-7 cells transfected with B2wt and mutant receptors. Cells (used 72 h after transfection) were incubated with 2 nM [3H]BK at 37 °C for the times indicated. Unbound radioactivity was removed at 4 °C before the cell surface-associated and the internalized radioactivities were separated and quantitated as described under "Experimental Procedures." Specific binding was calculated as the difference of total binding and nonspecific binding in the presence of unlabeled BK (10 µM). Internalized radioactivity represents the acid-resistant fraction (%) of the total specific binding. Results are given as the percentage of the control, i.e. B2wt internalization at 70 min representing 60.3 ± 2.5% of the [3H]BK bound (100% corresponding to 0.18 ± 0.01 pmol/mg of protein). For point mutations, the data for five representative mutants (out of 10) are presented. Values are means ± S.E. from at least three independent experiments each performed in duplicate. TS345-356A, T345A/S346A.

Because the segment of the COOH-terminal domain of the B2wt that appears to interfere with receptor internalization contains 5 residues of serine/threonine that may be critical for receptor sequestration we constructed mutant receptors where a single residue was changed to alanine, i.e. S339A, T342A, and S348A, or two adjacent residues were substituted at the same time, i.e. T345A/S346A. The internalization capacity was reduced in the order Thr342 > Thr345, Ser346 > Ser339 > Ser348 although the level (41.1 ± 4.5% to 88.3 ± 3.1% of B2wt) was not as dramatic as for del[335-351] and tI334 (24.7 and 35.1% of B2wt, respectively) (Fig. 2). By contrast, the simultaneous replacement of distant Ser/Thr residues, i.e. S339A/T342A and S339A/T345A/S346A, produced a markedly reduced internalization (27.6 ± 2.6 and 32.4 ± 1.8% of B2wt, respectively) similar to that observed for del[335-351] and tI334 (Fig. 2). This increment was not further increased by the substitution of all 5 Ser/Thr residues of the Gln335 right-arrow Arg351 segment, i.e. S339A/T342A/T345A/S346A/S348A (Fig. 2, note that this mutant B2Delta ST represents a full-length receptor protein). Together these findings suggest that Ser and Thr residues within the 17-residue segment spanning positions 335 to 351 contribute differentially to receptor endocytosis and can very likely intervene in this process in an alternative manner. Mutations of Ser/Thr residues external to this segment, e.g. T237A, S316A, and S331A neither altered the binding capacity nor the internalization rate of the resultant receptor mutants (data not shown).

Internalization of [3H]BK by Mutant B2 Receptors Expressed in CHO-K1 and HEK 293 Cells-- Additionally to their characterization in the COS-7 cells, the wild type and some mutant receptors were tested for their ability to internalize [3H]BK when expressed in CHO-K1 cells (stably and transiently) and in HEK 293 (transiently). The results summarized in Fig. 3 demonstrated that in those cells the truncation mutant tY320 and the deletion mutant del[335-351] as well as the point mutation B2Delta ST receptor all exhibited a markedly decreased ability to internalize [3H]BK compared with the B2wt, like in COS-7 cells. However, the fraction of the [3H]BK binding which was internalized differed slightly from one cell type to another. For the control B2wt, this fraction was of 72.6 ± 1.5% in CHO-K1, 57. ± 3.1% in HEK 293, and 60.3 ± 2.5% in COS-7. These observations can be linked to the differential amount of GRKs and arrestins expressed in these cell types reported by Menard et al. (33). It should be pointed out that the deletion mutant del[335-351] exhibited in every cell type the same reduced internalization rate than B2Delta ST supporting our hypothesis that phosphorylation of Ser and Thr within the segment 335-351 is crucial for an optimal internalization of the BK B2 receptor.


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Fig. 3.   [3H]BK internalization in HEK 293 and CHO cells with transient or stable expression of the B2wt or mutant receptors. Cells cultured during 48 h after transfection for transient expression or stable CHO cell lines at confluence were incubated with 2 nM [3H]BK at 37 °C for 70 min. Data were obtained as described in the legend of Fig. 2. Results (mean ± S.E.) are given as the percent of the control, i.e. B2wt internalization which represents 57.4 ± 3.1% of the corresponding total specific binding in HEK 293 cells and 72.6 ± 1.5% and 89.6 ± 1.9% in transiently and stably expressing CHO cells, respectively; ***, p < 0.005, test ANOVA.

Immunoprecipitation of Mutant Receptors-- The above observations made it of interest to study the receptor phosphorylation, especially to demonstrate whether the Ser and Thr residues discussed above are implicated. To this end, transfected cells with the various truncated, deleted and point mutated receptor mutants were labeled with [35S]methionine or [32P]orthophosphate, and incubated with or without 1 µM BK for 5 min prior to cell lysis. For immunoprecipitation of the receptor we applied antiserum AS346 which has been raised against a peptide derived from the COOH terminus of the B2wt (positions 329 to 364) (21). In 35S labeling experiments (Fig. 4d), immunoprecipitation of B2wt resulted in a diffuse band of 60-100 kDa that was superimposable with the immunoreactive band found in 32P labeling experiments (Fig. 4, a-c). All mutant receptors including B2Delta ST were readily immunoprecipitated by the antiserum except for deletion mutants del[343-351] and del[335-351] which were precipitated at a 3-4-fold lower efficiency, and truncation mutants tR351, tI334, and tY320 which failed to react. These findings localize major immunogenic epitope(s) recognized by antiserum AS346 to the extreme COOH-terminal receptor portion distal of residue Arg351.


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Fig. 4.   BK-induced phosphorylation of B2wt and mutant receptors. COS cells used 40 h after transfection were radiolabeled with 35S-labeled amino acids (panel d) or with [32P]orthophosphate and incubated for 5 min with (+) or without (-) 1 µM BK. The cells were lysed, and the receptors immunoprecipitated with antiserum AS346. The radiolabeled proteins were analyzed by reducing 10% SDS-PAGE and autoradiography. Relative molecular masses of standard proteins (not shown) are indicated on the left. Data are representative of three independent experiments with similar results. TS345-346A, T345A/S346A; S339T342A, S339A/T342A; S339TS345-346A, S339A/T345A/S346A.

Phosphorylation of Mutant Receptors-- Under basal conditions, i.e. in the absence of BK, a significant phosphorylation was observed for B2wt and mutant receptors except for del[335-351], del[343-351], and S348A suggesting that Ser at position 348 may represent a major target site for ligand-independent phosphorylation. BK dramatically increased the phosphorylation level of B2wt as well as of T237A, S316A, and S331A where Ser and Thr residues external to the Gln335 right-arrow Arg351 segment had been mutated (data not shown). In marked contrast, BK failed to increase the phosphorylation level of del[335-351], B2Delta ST, and S339A/T345A/S346A (Fig. 4); these mutants are characterized by a markedly reduced internalization capacity (Figs. 2 and 3). In all mutants exhibiting an "intermediate" internalization phenotype such as del[335-342], del[343-351], S339A, T342A, S348A, S339A/T342A, and T345A/S346A BK was still able to increase the phosphorylation (Fig. 4). Together these results demonstrate that the Ser and Thr residues phosphorylated upon ligand stimulation are located within the Gln335 right-arrow Arg351 segment. The data also point to a close relationship between internalization and phosphorylation implying that BK promotes receptor internalization by homologous phosphorylation. To further test this hypothesis, we investigate with the B2wt whether PKC is involved in the internalization and phosphorylation processes. It was apparent that [3H]BK binding was not sensitive to application, before and during the ligand incubation, of 0.1 µM PMA or staurosporine, an activator and inhibitor of PKC, respectively (not shown). Under the same conditions, there was also no change in [3H]BK internalization and BK stimulation of PLC. Concurrently, an immunoprecipitation of the B2wt showed that, unlike BK, PMA did not increase the receptor phosphorylation (not shown). Thus, PKC regulation of B2wt was not detected in COS-7 cells like in CHO-K1 cells (4). As discussed elsewhere in detail (4), this is in contrast with the reported role of PKC in B2 receptor modulation in other cell types (17, 34-36) and can be explained by a low PKC content of COS-7 and CHO-K1 cells facing receptor overexpression.

Interestingly, the treatment of COSB2wt with 0.1 µM okadaic acid (a potent phosphatase 2A/1 inhibitor) for 5 min prior to and during the 10-min incubation period with 2 nM [3H]BK (Fig. 5) resulted in a considerable increase in the internalization rate: 64.6 ± 6.7% of the total [3H]BK bound was internalized under okadaic acid treatment compared with 34.4 ± 2.2% under control conditions (p < 0.05). Okadaic acid also resulted in a marked increase in the phosphorylation level of B2wt in the presence of BK (Fig. 5). By contrast, the same treatment applied to the cells expressing the mutant B2Delta ST had no effect on the [3H]BK internalization rate and the phosphorylation level (Fig. 5). These findings further support the hypothesis of a close relationship between internalization rates and phosphorylation levels of the B2 receptor.


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Fig. 5.   Effects of phosphatase inhibitor treatment on B2wt and B2Delta ST receptor internalization and phosphorylation. COS cells used 72 h after transfection were (a) incubated with 0.1 µM okadaic acid for 5 min prior and during a 10-min incubation period with 2 nM [3H]BK at 37 °C. Results are expressed in percent of internalized radioactivity in untreated B2wt cells which represents 34.4 ± 2.2% of total bound radioactivity. b, 32P radiolabeled transfected cells were exposed to 0.1 µM okadaic acid in the presence or absence of 1 µM BK for 5 min before analysis of antiserum AS346-immunoprecipitated proteins as described in the legend to Fig. 4; *, p < 0.05, test ANOVA.

Relationship between B2 Receptor Phosphorylation and Internalization-- It has been reported that sucrose inhibits receptor endocytosis through clathrin-coated vesicles and that phosphorylated Ser/Thr residues in the COOH-terminal tail region of the beta 2-adrenergic receptor couple to the endocytic machinery via the adaptor protein arrestin (5, 6, 33). We here wondered whether the critical residues in the Gln335 right-arrow Arg351 segment are essential for receptor endocytosis through clathrin-coated vesicles. For this purpose, we exposed cells expressing the B2wt or the mutant receptor del[335-351] to a standard solution or to hypertonic solution containing 0.4 M sucrose for 30 min prior to and for 10 min after the addition of [3H]BK, and measured the internalization of the radioligand. Both the B2wt as well as the mutated receptor showed reduction of [3H]BK internalization by roughly 70% (not shown). The same results were obtained whether receptors were expressed in COS-7 cells or CHO-K1 cells. This suggests that the clathrin-mediated, sucrose-sensitive endocytosis of the bradykinin B2 receptor might not require the Ser/Thr phosphorylation residues in the Gln335 right-arrow Arg351 segment. To further document this hypothesis, we tested the ability of wild type and dominant-negative mutant beta -arrestin and dynamin to influence the sequestration of B2wt and the residual sequestration of B2Delta ST receptors in COS-7 cells. Fig. 6 shows that unlike for the beta -adrenergic receptor (33), the co-transfection with beta -arrestin did not increase the internalization of [3H]BK by either the B2wt or B2Delta ST receptors, but co-expression of beta -arrestin (319-418) or dynamin K44A mutants exhibited inhibited [3H]BK endocytosis for both receptors. One possibility to explain the inability of the overexpression of wild type beta -arrestin to increase significantly the internalization rate of both receptors, is that the endogenous proteins may be sufficient to mediate maximal internalization of these receptors. In any case these observations indicate that although clathrin-mediated endocytosis may be important for B2 receptor internalization this mechanism does not exclusively involve the phosphorylation of Ser/Thr residues in the Gln335 right-arrow Arg351 segment.


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Fig. 6.   Effects of beta -arrestins or/and dynamin co-transfection on B2wt and B2Delta ST receptor internalization. COS cells were transfected with 15 µg of expression vector for B2wt and B2Delta ST, together with 15 µg of empty vector or wt rat beta -arrestin, rat, or bovine beta -arrestin (319-418) mutants, or human dynamin-K44A mutant, as indicated. Then, 72 h after the transfection, the cells were incubated with 2 nM [3H]BK at 37 °C for 10 min. Results are given as the percent of the control, i.e. B2wt or B2Delta ST-mediated internalization which represents 26.8 ± 4.1 and 4.1 ± 0.8% of the total bound radioactivity at 10 min, respectively. Data are the mean ± S.E. of eight determinations. They were analyzed using unpaired Student's t test (*, p < 0.05, when compared with the corresponding B2wt and B2Delta ST control cells).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Post-stimulatory desensitization and cellular redistribution of GPCRs are important mechanisms that regulate the availability and signaling capacity of hormonal effector systems. For instance, ligand-induced internalization of the beta -adrenergic receptor allows the assembly of a scaffold of signaling factors of the mitogen-activated protein kinase system on intracellular vesicles and thus connects this receptor to the mitogenic pathways of the cell (37). Accumulating evidence suggests that specific sequence motifs of the intracellular loops and/or reversible modifications such as acylation and phosphorylation of the cytoplasmic domains of the receptors play critical roles in these processes (38). However, the precise molecular mechanisms driving desensitization and internalization of GPCRs have often remained unknown.

The present work demonstrates that a short sequence segment covering positions 335 to 351 of the bradykinin B2 receptor COOH-terminal tail plays a major role in the agonist-induced internalization and phosphorylation of the receptor, but is not necessary for PLC and PLA2 activation. Five amino acid residues, namely Ser in positions 339, 346, and 348 and Thr in positions 342 and 345, were shown to be involved in these functions and their respective contribution was analyzed. Complementary approaches for receptor modifications, i.e. truncation, deletion, and mutation of single or multiple residues were used to get insights into the structure-function relationships within the COOH-terminal tail. Most of the mutations resulted in alteration in the receptor capacity to undergo internalization and these alterations were consistent in different cell types. A reduction was always observed, indicating that the COOH-terminal tail possesses positive rather than negative motifs for internalization; at maximum we observed almost 80% reduction in the receptor sequestration in COS-7 cells. This contrasts with the findings that the human beta 2-adrenergic receptor truncated at position 365 was internalized to a greater extent than the wild type receptor in these cells (14). Our data show that the COOH-terminal tail contributes significantly to the internalization of the B2wt, although other structures such as the intracellular loops may also be involved, albeit with a reduced efficiency (39).

The maximum reduction in receptor internalization was obtained with three distinct types of mutations and the effect of these mutations was consistent in the three different cell types tested COS-7, CHO-K1, and HEK 293. Substitution of the 5 Ser/Thr residues in the Gln335 right-arrow Arg351 segment (B2Delta ST) produced a similar impairment in internalization as the deletion of the entire segment (del[335-351]) or the truncation in position 334 (tI334). Furthermore, substitution of only two (Ser339 and Thr342) or three (Ser339, Thr345, and Ser346) of these residues resulted in the same effect. This signifies that substitution of the critical Ser/Thr residues rather than a global structural change was responsible for the observed effects. Deletions which remove the proximal residues Ser339 and Thr342 (del[335-342]) or the distal residues Thr345, Ser346, and Ser348 (del[343-351]) each gave a phenotype intermediate to that of B2wt and del[335-351]. Intermediate phenotypes were also observed for individual (S339A, T342A, and S348A) or combined replacements (T345A/S346A). Of note, the sum of the effects of partial deletions 335-342 and 343-351 exceeded the effect of the full deletion 335-351. Likewise, the combined effects of individual substitutions exceeded the effect of a simultaneous mutation of all 5 residues in B2Delta ST. This indicates that the contribution of the non-mutated residues is smaller in the mutated receptors than in the intact receptor and suggests that the Ser/Thr residues of the Gln335 right-arrow Arg351 segment do not operate in a simple additive manner but operate in coordination to optimize receptor sequestration.

The present study shows that B2 receptor internalization is independent of the affinity for BK or of the efficiency of PLC and PLA2 coupling as reported for the truncated human platelet activating factor receptor (40). The COOH-terminal tail segment downstream of Tyr320 as well as the proximal residues Thr237, Ser316, and Ser331 do not function in the coupling of the receptors to G proteins stimulating the PLC pathway. These observations are in agreement with those of Faussner et al. (7) who reported that truncations either at Gly327 or Lys315 diminished the capacity to internalize BK but left the capacity to activate PLC unchanged. These and our results are, however, inconsistent with a recent report (39) claiming compromised ligand internalization associated with reduced PLC activity for a rat B2 receptor mutant devoid of the terminal 34 residues (corresponding to tR331 for the human receptor). Differences in species (rat versus man) and expression systems (Rat-1 versus COS-7, CHO-K1, and HEK 293) may help explain some of the observed discrepancies.

Our work provides strong evidence that phosphorylation is the triggering signal that enters the B2wt in the internalization process. Several lines of evidence support this notion: (i) BK stimulation induces internalization and homologous receptor phosphorylation; (ii) the kinetics of receptor internalization and recycling parallel those of receptor phosphorylation and dephosphorylation in HF-15 cells (24); (iii) inhibition of phosphatase activity which delays B2wt receptor dephosphorylation increases [3H]BK internalization but neither alters the internalization of [3H]BK by B2Delta ST nor the phosphorylation state of this mutant receptor; (iv) PMA and staurosporine treatment which fail to change [3H]BK internalization do not affect receptor phosphorylation; (v) mutations tY320, del[335-351], B2Delta ST, and S339A/T345A/S346A that suppress homologous receptor phosphorylation impair receptor internalization in all cell types tested; (vi) mutation of three phosphorylation sites, i.e. Ser339, Thr345, and Ser346, suffices to abolish homologous receptor phosphorylation and to markedly reduce internalization capacity of the human B2 receptor. Remarkably Ala substitution of 3 out of 14 potential phosphorylation sites (Ser, Thr, and Tyr) present in the 4 intracellular domains are sufficient to abolish ligand-induced phosphorylation and to markedly reduce the internalization capacity of the human B2 receptor studied in COS-7 cells. This observation points to the fact that the remaining non-mutated Ser/Thr residues cannot rescue the altered phosphorylation and internalization phenotype. On the contrary, the contributions of the non-mutated residues to internalization appear to be curtailed in these mutants.

The question whether BK-sensitive phosphorylation sites can serve as recognition motifs for receptor interaction with clathrin-coated vesicles was not elucidated in the present work. Indeed, the mutant del[335-351] with reduced internalization capacity and lacking these sites still exhibited a sensitivity to the clathrin-coated vesicle disrupting sucrose (data not shown) and to the effect of co-expression of dominant-negative forms of beta -arrestin and dynamin. The experiments presented here are the first to address the involvement of arrestins and dynamin in the internalization of the bradykinin B2 receptor. The magnitude of the inhibition of the B2Delta ST internalization induced by co-expression of dominant-negative forms of beta -arrestin or dynamin in COS-7 cells was of the same extent as that observed for B2wt in the same cell line. This suggests that the B2Delta ST receptor has a affinity for arrestins comparable to those of the B2wt receptor and its residual internalization still involves clathrin-coated vesicles. The internalization that was not sensitive to beta -arrestin and dynamin mutants could reflect sequestration of the receptor in caveolae, like that described for the B2 receptor in DDT1 MF-2 smooth muscle cells by de Weerd et al. (25), and in A431 cells by Haasemann et al. (41). Interestingly, the desensitization that we have previously characterized in the CHO-K1 cells expressing the B2wt (4) did not occur in COS-7 expressing either the B2wt or mutant receptors, probably because of a differential expression of the human B2 receptor desensitization machinery between the two cell types. The lack of desensitization in COS-7 cells, together with the decreased internalization observed in some mutants, is, however, of interest because it indicates that receptor internalization is not a prerequisite for the desensitization, as reported by others (10).

In conclusion, this work has allowed us to establish that optimal internalization and phosphorylation of the B2wt require the integrity of Ser339, Ser346, Ser348, Thr342, and Thr345 residues, located in a short segment of 17 residues (Gln335 right-arrow Arg351) in the center portion of the COOH-terminal tail. However, the mutation of these residues is not sufficient to completely suppress the sequestration through clathrin-coated vesicles, suggesting that the internalization of the [3H]BK-B2 receptor complex proceeds from different mechanisms involving distinct receptor structures. The critical Ser/Thr residues are flanked by 2 acidic residues, Glu in positions 337 and 350 delimiting a core sequence of 14 residues, Glu337 right-arrow Glu350. Previous in vitro studies with the beta -adrenergic receptor have pointed to the potential importance of acidic residues juxtaposed to Ser/Thr residues (42). Comparison of the human Glu337 right-arrow Glu350 segment with the sequences of other vertebrate kinin receptors (17, 18, 43) demonstrates that this cassette is well conserved among the kinin receptors: 9 of the 14 residues (65%) are invariant including all serine and threonine residues (Ser339, Ser346, Ser348, Thr342, and Thr345), a centrally located positive residue of arginine (Arg344), and the proximal acidic residue (Glu337) whereas the distal acidic residue is conserved (Glu350 or Asp350). By contrast the overall sequence identity of intracellular domain ID4 is poor (15/64 corresponding to 23%). Our notion that the Glu337 right-arrow Glu350 cassette plays a central role for receptor phosphorylation and internalization is further strengthened by the finding that the replacement of COOH-terminal tail region of the wild-type human B1 receptor which fails to undergo ligand-induced phosphorylation and internalization, by the homologous region of the B2 receptor holding the Glu337 right-arrow Glu350 cassette confers the capacity to the B1/B2 receptor chimera for ligand-induced internalization (7) and phosphorylation.2

One limitation of the present approach is highlighted by the finding of alternative phosphorylation of closely spaced Ser/Thr residues within the putative phosphorylation cassette. Hence we cannot draw firm conclusions as to relative importance, functional hierarchy, and/or sequential modification of the critical Ser/Thr residues. Studies aimed at the precise mapping of the phosphorylation sites in vivo and at the elucidation of the sequence of phosphorylation events in the human B2 bradykinin receptor are underway.

    ACKNOWLEDGEMENTS

We thank Dr. Benovic for the generous gift of pcDNA3 plasmids encoding for bovine beta -arrestin fragment (319-418) mutant and inactive human dynamin K44A and Dr. Bunnett for wt rat beta -arrestin and its inactive fragment (319-418) mutant in pEGFP.

    FOOTNOTES

* This work was supported by INSERM and grants from the Bristol-Meyers Squibb Institute for Medical Research (Princeton, NJ) and the Deutsche Forschungsgemeinschaft.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.

§ Supported by a grant from La Fondation Pour la Recherche Medicale. To whom correspondence should be addressed. Tel.: 33-1-45-87-61-00; Fax: 33-1-45-35-66-29; E-mail: pizard{at}ifm.inserm.fr.

parallel Present address: Ludwig Institute for Cancer Research, P.O. Box 595, S-75124 Uppsala, Sweden.

2 A. Faussner and A. Blaukat, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: GPCRs, G protein-coupled receptors; HBSS, Hank's buffered saline solution; BK, bradykinin; B2wt, human B2 BK wild-type receptor; COSB2wt, COS-7 cells overexpressing the B2wt; PLC, phospholipase C; PLA2, phospholipase A2; BSA, bovine serum albumin; IPs, inositol phosphates; [Ca2+]i, intracellular Ca2+; CHO, Chinese hamster ovary; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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