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J. Biol. Chem., Vol. 276, Issue 44, 40431-40440, November 2, 2001

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Determination of Bradykinin B₂ Receptor in Vivo Phosphorylation Sites and Their Role in Receptor Function^*

Andree Blaukat^§, Anne Pizard^¶, Andreas Breit^**, Christer Wernstedt, François Alhenc-Gelas^¶, Werner Müller-Esterl^**, and Ivan Dikic

From the  Ludwig Institute for Cancer Research, Box 595, S-75124 Uppsala, Sweden, ^¶ INSERM U367, 17, rue du Fer à Moulin, F-75005 Paris, France, and the ^** Institute for Biochemistry II, University of Frankfurt Hospital, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany

Received for publication, July 24, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. Using metabolic ³²P labeling and phosphopeptide sequencing we provide a complete phosphorylation map of the human bradykinin B₂ receptor in its native cellular environment. We identified three serine residues, Ser³³⁹, Ser³⁴⁶, and Ser³⁴⁸, at the C-terminal tail as principal phosphorylation sites. Constitutive phosphorylation occurs at Ser³⁴⁸, while ligand-induced phosphorylation is found at Ser³³⁹ and Ser³⁴⁶/Ser³⁴⁸ that could be executed by several G protein-coupled receptor kinases. In addition, we found a protein kinase C-dependent phosphorylation of Ser³⁴⁶ that was mutually exclusive with the basal phosphorylation at Ser³⁴⁸ and therefore may be implicated in differential regulation of B₂ receptor activation. Functional analysis of receptor mutants revealed that a low phosphorylation stoichiometry is sufficient to initiate receptor sequestration while a clustered phosphorylation around Ser³⁴⁶ is necessary for desensitization of the B₂ receptor-induced phospholipase C activation. This was further supported by the specifically reduced Ser³⁴⁶/Ser³⁴⁸ phosphorylation observed upon stimulation with a nondesensitizing B₂ receptor agonist. The differential usage of clustered phosphoacceptor sites points to distinct roles of multiple kinases in controlling G protein-coupled receptor function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

G protein-coupled receptors (GPCRs)¹ constitute the largest family of proteins converting external stimuli into intracellular activity. They share a common deduced structure comprising seven -helical transmembrane domains connected by extra- and intracellular loops. Through their intracellular domains they interact with heterotrimeric G proteins, which in turn modulate the activity of various effectors, such as adenylate cyclases, phospholipases, and ion channels. These effectors generate the intracellular second messengers, which ultimately evoke cellular responses (1). Signal transduction of GPCRs is carefully controlled: continuous or repeated agonist stimulation leads to an attenuation of the response, a phenomenon called desensitization. Although desensitization of receptor/G protein/effector systems generally involves perturbations of all three components, the impairment of the ability of receptors to activate G proteins appears to be the most important and seems to involve an agonist-induced receptor phosphorylation (2-4).

Much of the knowledge about the molecular mechanisms governing desensitization has come from studies of rhodopsin and ₂-adrenergic receptors (5-7). Rhodopsin, which is available in much greater quantities than any other GPCR, has been successfully subjected to mapping of in vitro (i.e. purified and reconstituted components) and in vivo (i.e. in cultured cells) phosphorylation sites in its C-terminal tail (8, 9). More recently, the identification of phosphorylation sites in the reconstituted ₂-adrenergic receptor by G protein-coupled receptor kinase (GRK) 2 and GRK5 was reported (10). The functional relevance of these in vitro phosphorylation sites has subsequently been challenged by mutagenesis studies that failed to correlate the presence of the mapped residues with cellular receptor-mediated functions (11). This discrepancy could be explained by the rather poor substrate specificity of GRKs in reconstituted systems resulting in the phosphorylation of sites not used in vivo (2). For instance, rhodopsin kinase (GRK1) phosphorylates the ₂-adrenergic receptor in vitro, and rhodopsin is an excellent substrate for GRK2 in reconstituted systems (4, 12). However, due to their differential tissue distribution both combinations are very unlikely to play a relevant role under physiological conditions. Thus, phosphorylation sites of GPCRs identified in vitro do not necessarily correlate with sites that tune receptor functions in a native cellular environment.

In many studies site-directed mutagenesis and total phosphorylation of GPCRs has been performed to screen for key amino acids involved in signal transduction. Using this approach, several mutant receptors have been generated that were useful to corroborate a role of GPCR phosphorylation in adaptation processes (5, 13, 14). For example, four consecutive serine residues in the third intracellular loop were identified as the major phosphorylation sites of the _2A-adrenergic receptor in Chinese hamster ovary cells, and their phosphorylation correlated in an additive manner with the desensitization of the receptor-mediated reduction of cellular cAMP levels (13). A general limitation of the mutagenesis approach is that truncations, deletions, or exchanges of amino acids often affect receptor structure, trafficking, localization, and stability or intervene with GRK recognition. Therefore, mutagenesis studies may allow valuable deductions about in vivo phosphorylation sites but the unequivocal identification of these sites requires receptor purification and biochemical analysis. Because of the hydrophobic nature and inherently low expression of GPCRs this particularly challenging task has not yet been accomplished for a single non-rhodopsin GPCR.

In an effort to identify the in vivo phosphorylation sites of a prototypical GPCR, we chose to study the human bradykinin B₂ receptor (B₂R). Previous work has demonstrated that the B₂R desensitizes upon prolonged or repeated agonist stimulation (15-19), and that the agonist-induced B₂R phosphorylation and dephosphorylation correlate with its de- and resensitization (15). Furthermore, a cluster of serine and threonine residues located in the C-terminal tail of the B₂R has been suggested to hold potential phosphorylation sites (15, 17, 18). Using two-dimensional phosphopeptide mapping and Edman sequencing we report the complete in vivo phosphorylation pattern of the human B₂R and demonstrate that the differential usage of clustered phosphoacceptor sites contributes to the complex regulation of receptor sequestration and desensitization.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- [³H]Bradykinin (1.48-4.07 TBq/mmol), [³²P]orthophosphate (360 MBq/ml), and myo-[³H]inositol (2.96-4.44 TBq/mmol) were from Amersham; bradykinin was from Bachem; aprotinin (Trasylol^TM) was from Bayer; AG-X8 anion exchanger resin was from Bio-Rad; GF109203X and PMA were from Calbiochem; bacitracin and Pefabloc^TM were from FLUKA; FR190997 was a kind gift from Fujisawa Pharmaceutical Co., LipofectAMINE^TM and protein markers were from Life Technologies; cellulose thin layer chromatography (TLC) plates were from Merck; sequencing grade trypsin was from Promega; leupeptin was from Roche Molecular Bioscience; nitrocellulose membranes were from Schleicher & Schuell; phosphoamino acid standards were from Sigma; and protein A-agarose was from Zymed Laboratories Inc. All tissue culture reagents were from Sigma and Life Technologies.

Mutagenesis-- Mutants of the human B₂R were generated using the Transformer site-directed mutagenesis kit from CLONTECH. The following constructs have been previously described: S339A, S348A, and ST (17). Point mutations were created by the same procedure to replace serine residues (by single nucleotide substitution) by alanine using the following oligonucleotide primers: 5'-GCACACTGCGGACCGCCATCTCCGTG-3' for S346A, 5'-GAACGCCATGGGCACACTGCGGACCGCCATCTCCGTGG-3' for S339A/S346A, 5'-GCGGACCGCCATCGCCGTGG-3' for S346A/S348A, and 5'-GAACGCCATGGGCACACTGCGGACCGCCATCGCCGTGG-3' for S. All mutations were confirmed by sequencing using the Amplicycle^TM kit (PerkinElmer Life Sciences).

Cell Culture and Transfection-- Human embryonic kidney cells HEK293T were grown to about 50-70% confluence in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Transfections were done in serum-free medium with the indicated cDNAs using 0.2-0.4 µg/well of a 24-well plate, 0.4-1 µg/well of a 12-well plate, 1-2 µg/well of a 6-well plate or 10 µg/10-cm dish by the LipofectAMINE^TM method according to the suppliers manual. Cells were used for experiments 48-60 h after transfection. HF-15 human foreskin fibroblasts were grown to confluence in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum.

Ligand Binding and Receptor Internalization-- Ligand binding and internalization were measured according to established procedures using [³H]bradykinin (20). HEK293T cells grown on 12-well plates were used 40 h after transfection with B₂R constructs. Cells were washed twice with phosphate-buffered saline supplemented with 0.2% bovine serum albumin, 2 mM bacitracin, and incubated for 90 min at 4 °C in the same solution containing 5 nM [³H]bradykinin in the absence ("total binding") or presence ("nonspecific binding") of 5 µM unlabeled bradykinin. Internalization of the receptor was initiated by incubating cells for different time periods at 37 °C. Cells were washed twice to remove free ligand, and the cell-bound [³H]bradykinin was extracted with 0.2 M acetic acid, pH 2.8, 0.5 M NaCl, 0.2% bovine serum albumin, and radioactivity of the extract was measured ("surface-associated bradykinin"). The acid-stripped cells were dissolved in 1 M NaOH and the radioactivity of the lysate was determined ("intracellular bradykinin").

Phospholipase C Assays-- Phospholipase C activity was measured by analyzing inositol phosphate accumulation (21). Cells grown on 24-well plates were labeled with 1 µCi/ml myo-[³H]inositol for 24 h in inositol-free Ham's F-12 including 0.1% (w/v) bovine serum albumin, treated for 5 min with 10 mM LiCl and then challenged with 0.01-1 µM bradykinin for 10 min in the presence of 10 mM LiCl. Reactions were stopped by addition of 1 ml of ice-cold 10 mM formic acid. Water-soluble inositols were extracted for 2-12 h at 4 °C and separated by anion exchange chromatography using AG-X8 as a resin. Inositol phosphates were eluted with 2 M ammonium formate and quantified by liquid scintillation counting. Results were normalized for total labeling of lipid pools, which was calculated from the radioactivity in water-soluble extracts and cells. To follow desensitization, cells were pretreated with 100 nM bradykinin for 5 min. Following removal of excess of ligand by washing cells three times with medium, 10 mM LiCl was added 10 or 15 min after the initial stimulation and inositol phosphate levels were measured as described above.

³²P Labeling and Two-dimensional Mapping of Phosphorylation Sites-- ³²P labeling of cells (1-2 mCi/ml for 6-8 h), solubilization, and immunoprecipitation of B₂R using an antiserum against the C-terminal receptor domain were done as detailed previously (15). For HF-15 cells with endogenous B₂R, a 10-cm dish was used, whereas for transiently transfected HEK293T and COS-7 cells a well of a 6-well plate was sufficient for the following procedure. After 10% SDS-PAGE proteins were transferred onto nitrocellulose membranes using a semi-dry unit from Bio-Rad. Radiolabeled B₂R was detected by PhosphorImager (BAS2000, Fuji) analysis and tryptic digests were performed as described by Boyle et al. (22) with minor modifications. Briefly, membrane pieces containing the ³²P-labeled B₂R were cut out and blocked with 0.5% polyvinylpyrrolidon 40 in 0.6% acetic acid for 30 min at 37 °C. Following extensive washes with water, membrane bound B₂R was cleaved in situ with 1 µg of modified sequencing grade trypsin in 200 µl of 50 mM (NH₄)HCO₃ for 12 h at 37 °C. Released tryptic peptides were vacuum-dried and oxidized with 50 µl of performic acid for 1 h on ice. Reactions were stopped by dilution with 500 µl of 20% (v/v) ammonia solution. Thereafter samples were frozen, vacuum-dried, and a second digest was performed with 1 µg of trypsin in 50 µl of 50 mM (NH₄)HCO₃ for 12 h at 37 °C. Following vacuum drying, samples were dissolved in 5 µl of electrophoresis buffer (formic acid:acetic acid:water, 46:156:1790 (v/v/v)) and phosphopeptides were separated by electrophoresis on cellulose thin layer plates in a first dimension (2000 V, 40 min, electrophoresis buffer) and ascending chromatography in a second dimension (15 h, isobutyric acid, 1-butanol, pyridine, acetic acid, water, 1250:38:96:58:558 (v/v/v/v/v)). Phosphopeptides were detected by PhosphorImager analysis and eluted from the cellulose matrix with 2 × 250 µl of 20% (v/v) acetonitrile in a sonicated water bath for 15 min. Part of the extract (25-100 cpm) was hydrolyzed with 6 M HCl for 1 h at 110 °C and subjected to a phosphoamino acid analysis (15, 22). The second fraction (50-500 cpm) was sequenced by Edman degradation using a solid phase sequencer (ABI 477). Twenty sequencing cycles were collected, dried, and analyzed for their content of ³²P radioactivity using a PhosphorImager. Data obtained from Edman degradation (position of phosphoamino acids in tryptic peptides) and phosphoamino acid analysis (phosphorylated amino acid), the known sequence of tryptic peptides (Table I) and the anticipated transmembrane topography of the B₂R (23) were used to predict phosphorylation sites. The prediction was verified by in vitro mutagenesis of corresponding phosphoacceptor sites followed by two-dimensional phosphopeptide mapping.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mapping of Serine 339, 346, and 348 as Major in Vivo Phosphorylation Sites of B₂ Receptor-- To molecularly characterize the importance of receptor phosphorylation in signal transduction of the B₂R, we applied an analytical two-dimensional phosphopeptide mapping strategy to identify in vivo phosphorylation sites of the receptor (22). HF-15 human fibroblasts expressing 400-750 fmol of endogenous B₂R/mg of protein were labeled with [³²P]orthophosphate, stimulated with bradykinin, lysed, and B₂R was immunoprecipitated with a specific anti-peptide antibody (15). Following SDS-PAGE, proteins were transferred onto nitrocellulose membranes and the radiolabeled B₂R was detected as a 55-68-kDa band by PhosphorImager analysis (Fig. 1A). Notably the broadness of the band and an occasionally observed doublet are likely due to heterogeneous glycosylation (24). In average a 4.0 ± 0.5-fold increase of the basal B₂R phosphorylation was observed in bradykinin-stimulated cells. In situ digestion with trypsin-generated phosphopeptides that were separated on thin layer plates by high voltage electrophoresis and ascending chromatography. PhosphorImager analysis revealed a major phosphopeptide in untreated cells that contained more than 80% of the total radioactivity (Fig. 1A, peptide 1). The same phosphopeptide was present in bradykinin-stimulated cells as well as three additional ³²P-labeled peptides (Fig. 1A, peptides 2-4). Total hydrolysis and phosphoamino acid analysis performed with a fraction of the material isolated from the spots revealed that peptides 1, 2, and 3 contained exclusively phosphoserine whereas peptide 4 carried the ³²P label both on serine and threonine (Fig. 1B). The major fraction of the peptides was used for solid phase Edman sequencing. Because peptide quantities were too low for chemical conversion and identification of released amino acids, cleavage products of 20 sequencing cycles were collected and analyzed for their ³²P content using a PhosphorImager. The majority of radioactivity from peptide 1 eluted in cycle 4 (Fig. 1C). Together with the finding that phosphate in peptide 1 is exclusively attached to serine (cf. Fig. 1B), we conclude that the fragment covering amino acid positions 345-351 of the human B₂R sequence (Ref. 23, Table I) is the only one present in the tryptic digest that can be serine phosphorylated at position 4, namely at Ser³⁴⁸ (pS348). Peptide 2 generated a peak of radioactivity in sequencing cycle 9 thus identifying Ser³³⁹ as the phosphorylation site (Fig. 1C, Table I). In the case of peptide 3, peaks of radioactivity were detected in cycles 2 and 4, whereas peptide 4 released ³²P in cycles 9 and 12 (Fig. 1C). Alignment with the sequence of tryptic peptides (Table I) and consideration of the data from the phosphoamino acid analysis (Fig. 1B) allowed the identification of Ser³⁴⁶ and Ser³⁴⁸ (peptide 3) and Ser³³⁹ and Thr³⁴² (peptide-4) as B₂R phosphorylation sites.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Mapping of serine 339, 346, and 348 as the major phosphorylation sites of B2 receptor. A, HF-15 cells grown on 10 cm-plates were labeled with [32P]orthophosphate and left untreated or stimulated with 1 µM bradykinin (Bk) for 10 min. Following immunoprecipitation and 10% SDS-PAGE, isolated proteins were transferred onto nitrocellulose membranes and analyzed using a PhosphorImager (BAS2000, Fuji). The 32P-labeled B2R was in situ digested with trypsin and resulting peptides were separated on thin layer chromatography (TLC) plates by high voltage electrophoresis and ascending chromatography. A cross illustrates where samples were applied; + and  indicate the polarity during electrophoresis. Thereafter, phosphopeptides were localized by PhosphorImager analysis. B, peptides were eluted and a fraction was hydrolyzed, subjected to phosphoamino acid analysis followed by two-dimensional electrophoretic separation on TLC plates and PhosphorImager analysis. Phosphorylated amino acids were identified by commercial standards (locations indicated by dashed circles). C, major fractions of phosphopeptides were subjected to 20 cycles of Edman degradation and cleaved amino acids were collected and analyzed using a PhosphorImager to locate the position of the phosphorylation site(s) as exemplified for peptide 1. The content of 32P radioactivity of each sequencing cycle was quantified and expressed in arbitrary units (AU). D, phosphopeptides on TLC plates were quantified using a PhosphorImager and the relative contributions of individual phosphopeptides to total basal (light) as well as bradykinin-mediated (dark) phosphorylation were determined. Mean ± S.D. from eight independent experiments are shown.

For a more quantitative comparison, two-dimensional maps from several experiments were analyzed using a PhosphorImager. The contribution of the individual ³²P-labeled peptides to total phosphorylation was calculated for untreated and bradykinin-stimulated cells and corrected for the increase in total phosphorylation. The contribution of peptide 4 containing pS339/T342 to total receptor phosphorylation was always very low (<5%), and therefore this peptide was excluded from quantitative studies. As shown in Fig. 1D, phosphorylation of Ser³⁴⁸ in peptide 1 did not significantly change during agonist challenge on this relative scale. In contrast, phosphorylation of Ser³³⁹ and the double-labeled peptide 3 containing pS346/S348 increased about 10- and >40-fold, respectively, in the presence of bradykinin (Fig. 1, A and D).

Phosphorylation of B₂ Receptor Mutants Lacking Identified Phosphorylation Sites-- To validate the assignment of B₂R phosphorylation sites we used receptor mutants in which Ser³³⁹, Ser³⁴⁶, and Ser³⁴⁸ individually or in combinations were replaced by alanine residues. Comparable expression levels of all mutants were confirmed by binding assays and immunoprecipitation of ³⁵S-labeled proteins (not shown). Immunoprecipitation of the ³²P-labeled B₂R mutants revealed that the ligand-induced phosphorylation of S339A and S346A was slightly reduced whereas the basal phosphorylation in the absence of a ligand was essentially unchanged over the wild-type receptor (Fig. 2A). In contrast, we observed a complete lack of basal phosphorylation in S348A and S346A/S348A mutants, together with a significantly decreased bradykinin-induced phosphorylation. Basal phosphorylation of the double mutant S339A/S346A was slightly increased, and only a minor increment was observed upon bradykinin stimulation. Finally, the S (S339A/S346A/S348A) and ST (S339A/S346A/S348A-T342A/T345A) mutants failed to produce any significant phosphorylation above background (Fig. 2A).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Phosphorylation of B2 receptor mutants lacking the mapped phosphorylation sites. HEK293T cells were transfected with the wild-type human B2R or the following mutants: S339A, S346A, S348A, S339A/S348A, S346A/S348A, S339A/S346A/S348A (S) or S339A/S346A/S348A-T342A/T345A (ST). A, cells were labeled with [32P]orthophosphate, stimulated with 1 µM bradykinin (Bk) for 5 min or not (), lysed, and B2R was analyzed by immunoprecipitation and autoradiography. B, tryptic phosphopeptides were separated and visualized using a PhosphorImager. A representative two-dimensional map including phosphoamino acid analysis and quantification of Edman sequencing cycles of the major spot (arrow) from the S348A mutant is shown. C, the relative contributions of individual phosphopeptides to total receptor phosphorylation were determined and expressed in arbitrary units (AU). Bars in the stack diagram indicate phosphopeptides pS339 (white), pS346 (light gray), pS348 (gray), and pS346/S348 (black). Means calculated from at least three independent experiments are shown.

Mutant B₂R were further subjected to two-dimensional phosphopeptide mapping and the resultant phosphopeptides were characterized by phosphoamino acid analysis and Edman degradation. Surprisingly, a major phosphorylated peptide was seen in the S348A mutant in a similar location as peptide 1 in wild-type B₂R (Fig. 2B, arrow). Sequence and phosphoamino acid analysis revealed that the S348A mutant had a compensatory phosphorylation at Ser³⁴⁶ in position 2 of the corresponding peptide (Fig. 2B). In the single mutants S339A and S346A as well as in the double mutants S339A/S346A and S346A/S348A, phosphopeptides corresponding to peptides 2 and 3 of wild-type B₂R (cf. Fig. 1A) were absent from the two-dimensional maps (Fig. 2C) thus confirming our identification of vivo B₂R phosphorylation sites. Under these conditions, no phosphopeptide(s) of significant quantity was detected in the two-dimensional maps of the S and ST variants. Taken together, we have identified (i) graded phosphorylation of four closely spaced residues, Ser³⁴⁸ > Ser³⁴⁶ > Ser³³⁹ Thr³⁴², (ii) basal phosphorylation at a single site, Ser³⁴⁸, (iii) bradykinin-induced phosphorylation at two major sites, Ser³⁴⁶ and Ser³³⁹, and (iv) combined phosphorylation at Ser³⁴⁶ and Ser³⁴⁸.

Identification of Kinases That Can Phosphorylate the B₂ Receptor-- Co-expression of receptors with GRKs is commonly used to obtain information about the nature of kinases involved in GPCR phosphorylation (25-29). To identify potential kinase(s) executing B₂R phosphorylation and to locate their corresponding substrate sites we analyzed the phosphopeptide patterns of B₂R co-transfected with human GRK2-6 in HEK293T cells. Immunoprecipitates from ³²P-labeled cells indicated that total basal and bradykinin-mediated B₂R phosphorylation did not significantly change upon co-expression with GRK2, GRK3, GRK5, or GRK6 (Fig. 3A). In contrast, expression of GRK4 drastically increased the basal level of ³²P incorporation into B₂R. However, phosphopeptide maps revealed quantitative changes in the distribution of phosphopeptides for the various GRKs. For example, ³²P labeling of peptide 3 containing pS346/pS348 was enhanced 1.5-3-fold as compared with mock-transfected cells in the order GRK6 < GRK5 < GRK2 < GRK4 < GRK3. Most prominently, GRK4 elevated the basal phosphorylation of Ser³³⁹ and Ser³⁴⁶/Ser³⁴⁸ 15- and 24-fold, respectively. These results suggest that several endogenous GRKs may phosphorylate the B₂R and that the various GRKs, even without apparent effect on total GPCR phosphorylation levels, may induce distinct phosphorylation patterns with possible functional consequences for receptor desensitization and sequestration.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Identification of kinases that can phosphorylate the B2 receptor. A, HEK293T cells grown on 6-well plates were co-transfected with the human B2R and human GRK2, GRK3, GRK4, GRK5, and GRK6. Cells were labeled with [32P]orthophosphate, left untreated () or stimulated with 1 µM bradykinin (Bk) for 5 min, lysed, and B2R was isolated by immunoprecipitation. Following 10% SDS-PAGE and transfer onto nitrocellulose, B2R was cleaved with trypsin and the resulting phosphopeptides were separated. Spots were quantified using a PhosphorImager and relative contributions of individual phosphopeptides (white, pS339; gray, pS348; and black, pS346/S348) to total receptor phosphorylation were calculated and expressed in arbitrary units (AU). Mean from three independent experiments are shown. B, HF-15 cells or HEK293T cells transfected with the S346A mutant were labeled with [32P]orthophosphate and left untreated () or stimulated with 1 µM PMA (+PMA) for 10 min. Following immunoprecipitation, SDS-PAGE, transfer onto nitrocellulose membranes, and tryptic digest resulting peptides were separated and localized by PhosphorImager analysis. C, Edman sequencing and phosphoamino acid analysis of peptides 1 and 5. D, phosphopeptides were quantified and relative contributions of the individual phosphopeptides to total receptor phosphorylation were calculated and expressed in arbitrary units (AU). Mean ± S.D. from three independent experiments are shown.

We have previously observed that activators of protein kinase C may induce a ligand-independent phosphorylation of the B₂R (15). Under identical conditions PMA pretreatment significantly reduced the bradykinin-induced PLC stimulation (not shown), indicating that an agonist-independent ("heterologous") receptor phosphorylation may negatively affect signal transduction of the B₂R.

To identify residue(s) in the B₂R sequence targeted by PKC we analyzed two-dimensional phosphopeptide maps from ³²P-labeled HF-15 cells treated with PMA. PhosphorImager analysis revealed a new spot (peptide 5) that partially overlapped with the major phosphopeptide 1 containing pS348 (Fig. 3B). Peptides 1 and 5 were isolated avoiding cross-contamination, and sequence and phosphoamino acid analysis confirmed the identity of pS348 in peptide 1 (Fig. 3C). Peptide 5 showed ³²P-labeled serine in position 2 suggesting residue Ser³⁴⁶ in peptide 345-351 as potential PKC phosphorylation site (Table I). When HEK293T cells expressing the S346A mutant B₂R were treated with PMA, total phosphorylation did not change and spot 5 failed to appear whereas spot 1 was present (Fig. 3B). Together these findings point to Ser³⁴⁶ as the major PKC target site in the B₂R. This notion was confirmed by the repression of PMA-induced Ser³⁴⁶ phosphorylation in the presence of PKC inhibitors (not shown). Quantitative evaluation of two-dimensional maps revealed a 3-fold increase in Ser³⁴⁶ phosphorylation upon PMA treatment, and a 40% decrease in ³²P incorporation in the major B₂R phosphorylation site Ser³⁴⁸ (Fig. 3D). PKC stimulation did not produce any double phosphorylated peptide 4 bearing pS346/S348 (Fig. 3, B and D), suggesting that the basal phosphorylation at Ser³⁴⁸ and the PKC-mediated phosphorylation at Ser³⁴⁶ are mutually exclusive. A thorough review of two-dimensional phosphopeptide maps revealed the presence of low amounts of peptide 5 with a single phosphorylation at Ser³⁴⁶, partially overlapping with highly abundant peptide 1 comprising pS348 (cf. Figs. 1A and 3B). This indicates that a PKC-mediated phosphorylation of Ser³⁴⁶ also occurs under physiological conditions in the absence of exogenous PKC activators.

Time Course and Dose Dependence of Bradykinin-stimulated B₂ Receptor Phosphorylation-- Having identified the principal phosphoacceptor sites in intact cells we tested whether the phosphopeptide pattern of the B₂R changes during the time course of stimulation. We followed the kinetics of phosphorylation of the endogenous B₂R in HF-15 fibroblasts over a period of 60 min and found that bradykinin-induced B₂R phosphorylation is a fast process reaching a maximum after 5 min and decreasing almost to basal levels after 60 min (Fig. 4A, bottom panel). The appearance of pS339 and pS346/S348 strictly followed this time course, whereas pS346 or pS348 remained constant over the entire period of the experiment (Fig. 4A, top panel).

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Dose dependence and time course of agonist stimulated B2 receptor phosphorylation. HF-15 cells grown on 6-well plates were labeled with [32P]orthophosphate and stimulated for different time periods (0, 2.5, 5, 15, 30, and 60 min) with 1 µM bradykinin (A) or challenged with increasing concentrations (0, 0.001, 0.01, 0.1, 1, and 10 µM) of bradykinin for 5 min (B). Following immunoprecipitation, tryptic digest and two-dimensional separation, phosphopeptides were quantified using a PhosphorImager. The relative contributions of individual phosphopeptides (pS339, ; pS346 ; pS348, shaded diamond; and pS346/S348, ) to total receptor phosphorylation of typical experiments expressed in arbitrary units (AU) are shown.

Analyzing the dose-dependence of B₂R phosphorylation, a half-maximum effect was observed with ~10 nM bradykinin, and 100 nM of the ligand was sufficient to trigger full receptor phosphorylation (Fig. 4B, bottom panel). Whereas pS348 was constant and independent of the applied bradykinin concentration, single phosphorylation at Ser³⁴⁶ increased up to 2.5-fold during stimulation with low ligand concentrations (1 nM Bk), remained constant at intermediate bradykinin concentrations (1-100 nM), and decreased at the highest agonist concentrations (100 nM). Phosphorylation of Ser³³⁹ and Ser³⁴⁶/Ser³⁴⁸ increased markedly in correlation with the dose of the ligand (Fig. 4B, top panel). While pS339 reached saturation levels at 0.1-1 µM bradykinin, pS346/S348 strongly increased up to 10 µM bradykinin, i.e. the maximum ligand concentration used in our experiments. Thus, a PKC-dependent B₂R phosphorylation at Ser³⁴⁶ preferentially prevailed at low agonist concentrations, whereas pS339 and pS346/S348 were induced by moderate agonist concentrations, and phosphorylation at Ser³⁴⁶/Ser³⁴⁸ was dominant at high bradykinin concentrations.

Role of Specific Phosphorylation Sites in Receptor Sequestration and Desensitization-- Next we analyzed the role of the various B₂R phosphorylation sites in modulating receptor functions. First we studied B₂R internalization in HEK293T cells transfected with different B₂R constructs using [³H]bradykinin as a probe. The internalization process of wild-type B₂R was fast with a half-maximal effect after 5-10 min and a maximum of almost 70% internalized receptors after 60 min (Fig. 5A). Single or double mutations of serine residues in B₂R caused only minor effects on the internalization capacity of the corresponding constructs. The time course and the extent of S sequestration were initially similar to wild-type B₂R, but diverged beyond 20 min of incubation such that only 40% of receptors were internalized after 60 min. In contrast, internalization of the ST mutant was clearly reduced at all time points tested, and the majority of mutant receptors (>80%) remained surface exposed during the whole experiment. These results suggest that the initiation of B₂R sequestration requires only a low stoichiometry of phosphorylation without any obvious prevalence for specific residue(s) and that additional, probably phosphorylation-independent processes may be involved in the relocation of the receptor (17).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Role of specific phosphorylation sites in the regulation of receptor sequestration and desensitization of the bradykinin-induced phospholipase C activation. A, HEK293T cells were transfected with the wild-type human B2R () or the following mutants: S339A (), S346A (), S348A (), S339A/S346A (), S346A/S348A (shaded diamond), S (×), and ST (shaded cross). Internalization was studied after binding of 5 nM [3H]bradykinin at 4 °C by shifting the temperature to 37 °C for the indicated time periods. Extracellular and internalized ligand was separated and quantified by liquid scintillation counting. Means from a typical experiment performed in triplicates are shown. B, desensitization of the B2R-induced PLC activation was studied by following intracellular inositol phosphate accumulation in HEK293T cells transfected with the indicated B2R mutants and pretreated with 100 nM bradykinin for 5 min. After removal of excess of ligand and a lag phase of 10 (gray bars) or 15 min (black bars) 10 mM LiCl was added and inositol phosphate (IPn) accumulation was measured for 10 min. Mean ± S.D. from a typical experiment performed in triplicates are shown. C, 32P-labeled HEK293T cells were stimulated with increasing doses of the non-peptidic B2R agonist FR190997, B2R was immunoprecipitated, resolved by 10% SDS-PAGE, and visualized using a PhosphorImager. D, typical two-dimensional maps of tryptic phosphopeptides of B2R from cells treated either with 10 nM bradykinin (left panel) or 10 nM FR190997 (right panel) are shown.

We also determined desensitization of the bradykinin-mediated PLC activation of wild-type B₂R and various phosphorylation-deficient mutants. To circumvent the problem that bradykinin, which has a high affinity to the B₂R cannot be properly washed out after receptor stimulation, we adapted an alternative protocol that monitors signal duration as a measure of receptor desensitization (21). In transfected HEK293T cells inositol phosphate levels triggered by wild-type B₂R were reduced by ~20% after 10 or 15 min of delayed accumulation as compared with control (Fig. 5B). Minor variations were seen with S339A and S348A mutants although difference to wild-type B₂R did not reach statistical significance. Under the same conditions the S346A mutant showed a slight increase of inositol phosphates, while an augmented second messenger accumulation was particularly evident and significant for the S339A/S346A, S346A/S348A, S, and ST mutants. We conclude that Ser³⁴⁶ is a critical residue for desensitization of the B₂R, and that a clustered phosphorylation of Ser³⁴⁶ and at least one additional serine residues seems to be necessary for full desensitization of B₂R-mediated PLC activation.

To further confirm the role of receptor phosphorylation in desensitization we analyzed the pattern of B₂R phosphorylation upon stimulation of cells with FR190997. This synthetic non-peptidic agonist that has been reported to mediate a sustained activation of B₂R indicative of reduced desensitization (30, 31). Stimulation of B₂R-expressing HEK293T cells with increasing concentrations of FR190997 led to a dose-dependent rise in receptor phosphorylation (Fig. 5C) comparable to that observed with bradykinin (cf. Fig. 4B). However, analysis of the phosphopeptide pattern revealed a significantly reduced ³²P incorporation in peptide 3 containing pS346/S348 as well as in peptide 2 representing pS339 (Fig. 5D). These findings are in accord with the observation of a sustained B₂R signaling upon FR190997 stimulation (30, 31), and they lend further support to our hypothesis that phosphorylation of Ser³⁴⁶ in tandem with Ser³⁴⁸ is an important event during desensitization of B₂R-mediated signal transduction.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The implication of receptor phosphorylation in regulation of GPCR functions has been studied for more than a decade (2-4, 32). Most of the knowledge has come from in vitro studies using purified components for reconstitution and/or from mutagenesis approaches targeting anticipated phosphoacceptor sites (2). To date the biochemical identification of in vivo phosphorylation sites of a GPCR and characterization of their biological role(s) has not been successful for any GPCR but rhodopsin (2, 8, 33, 36). In this report we present the precise mapping of phosphorylation sites of the human bradykinin B₂ receptor in its native cellular environment. This approach has allowed us to discriminate between (i) constitutive phosphorylation of human B₂R on Ser³⁴⁸, (ii) homologous phosphorylation at Ser³³⁹ and/or Ser³⁴⁶ in tandem with Ser³⁴⁸, (iii) and heterologous phosphorylation of Ser³⁴⁶ (Fig. 6). We were also able to follow discrete, but important changes in the phosphopeptide pattern of the B₂R upon GRK co-expression, during the kinetics of agonist stimulation and over a broad range of ligand concentrations. At last, the phosphorylation of specific residues was correlated with the initiation of receptor internalization and the regulation of its desensitization. This is thus the first report about two-dimensional mapping of in vivo phosphorylation sites of a non-rhodopsin GPCR with a detailed analysis of the importance of specific phosphoacceptor sites in controlling GPCR functions.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   In vivo phosphorylation sites present in the B2R tail domain. A, schematic representation of the amino acid sequence (single letter code) of the B2R C terminus starting from the potential palmitoylation sites (indicated by serpentine lines). Identified phosphoacceptor sites are numbered and highlighted, and candidate kinases are indicated. The thickness of the arrows point to the relative quantity of phosphate incorporation. B, phosphorylation reactions occurring upon stimulation of B2R with bradykinin and PMA and S348A mutant with bradykinin.

A highly sensitive strategy combining two-dimensional phosphopeptide mapping together with Edman degradation was employed to directly identify basal and agonist-induced phosphorylation sites of the human B₂R. A recent mass spectrometry study described the constitutive phosphorylation of six out of seven possible serine/threonine residues in the C terminus of rat B₂R that had been purified from transfected CHO cells (35). However, this report did not provide any quantitative information about the relative frequency of the identified phosphopeptides and did not distinguish between basal, heterologous, and agonist-induced phosphorylation. The present study was performed in an analytical scale, which enabled us to follow B₂R phosphorylation with temporal resolution in different physiological situations. We could clearly discriminate a basal phosphorylation at Ser³⁴⁸ and an agonist-mediated phosphorylation of Ser³³⁹ and/or Ser³⁴⁶ in tandem with Ser³⁴⁸ in native and recombinant cells. These markedly distinct phosphorylation patterns were not expected, since the two-state model of receptor activation would suggest rather quantitative differences that should reflect the equilibrium between inactive and active, subsequently phosphorylated receptors. The finding that various GRKs affect the relative abundance of phosphorylation of specific phosphoacceptor sites in the B₂R underlines the power of our approach in identifying subtle positional changes that would escape total phosphorylation studies but may affect receptor fine-tuning. An unanticipated result of our experiments is the functionally compensatory phosphorylation of Ser³⁴⁶ that was found when the major phosphorylation site Ser³⁴⁸ had been mutated to alanine. This alternative phosphorylation points to a rather relaxed substrate specificity of the B₂R kinase(s) that could scan from the receptor C terminus toward the membrane-inserted region for appropriate phosphoacceptor sites. Such a "sliding kinase" mechanism is also supported by the observed quantitative differences in the contribution of identified serine residues to total B₂R phosphorylation, i.e. Ser³⁴⁸ > Ser³⁴⁶ > Ser³³⁹ (Fig. 6). Based on mass spectrometry studies, a similar GRK-driven sequential modification has been suggested for the C-terminal portion of rhodopsin (36). However, in other GPCRs such as the _2A-adrenergic receptor the phosphorylation of multiple serines in the third intracellular loop seems to occur independently of any directional order (13).

Data from GRK co-expression experiments and the finding that ³²P labeling of Ser³⁴⁸ remained largely invariable during agonist challenge suggests that either a non-GRK activity constantly provides new pS348 for subsequent GRK-mediated Ser³⁴⁶ phosphorylation or that synchronous de novo phosphorylation of Ser³⁴⁶/Ser³⁴⁸ occurs (Fig. 6B). The former hypothesis would support a priming function of pS348 as it has been proposed for other GPCRs such as rhodopsin, the µ-opioid and the A₃ adenosine receptor (36-38). Such a priming phosphorylation of B₂R at Ser³⁴⁸ would also convert Thr³⁴⁵ and Ser³⁴⁶ to consensus sites for further GRK-mediated phosphorylation (29).

In contrast to the bradykinin-induced dual phosphorylation of Ser³⁴⁶ and Ser³⁴⁸, PKC selectively triggered phosphorylation of Ser³⁴⁶ that appeared to block rather than promote subsequent Ser³⁴⁸ phosphorylation. The PKC-induced phosphorylation of Ser³⁴⁶ in the B₂R could be involved in agonist-independent, heterologous desensitization as it has been suggested for other GPCRs (2, 28, 33, 34, 37, 39). This hypothesis is supported by our finding that carbachol stimulation of co-expressed G_q-coupled m₁ and m₃ but not of G_i-coupled m₂ muscarinic receptors resulted in a moderate but significant increase of B₂R single Ser³⁴⁶ phosphorylation (data not shown). However, results from our analysis of the dose dependence of B₂R phosphorylation also suggest that PKC-mediated phosphorylation contributes to homologous B₂R desensitization upon stimulation with low doses of bradykinin. Based on inhibitor studies, such a scenario was earlier proposed for protein kinase A in regulating ₂-adrenergic receptor signaling (40).

Receptor internalization has been implicated in desensitization of GPCRs, although it is often too slow (t_1/2  5-20 min) for a significant contribution to acute desensitization that usually occurs within the first few minutes of agonist challenge (2, 4). The sequestration of ₂-adrenergic receptors and the acidification of the corresponding intracellular compartments have been suggested to constitute the initial steps of resensitization, because both processes were found to be essential for GPCR dephosphorylation (41, 42). Indeed B₂R internalization seems to be necessary for its full dephosphorylation and subsequent resensitization (15, 43) but in addition, receptor phosphorylation was shown to initiate internalization of the B₂R (17). The fact that receptor mutants with deletions of two principal phosphorylation sites (S339A/S346A or S346A/S348A) do not display significant changes in their sequestration kinetics demonstrates that a low stoichiometry of phosphorylation is sufficient to trigger B₂R internalization. A relaxed phosphorylation requirement with respect to the location of phosphoacceptor sites and stoichiometry has been proposed for internalization of m₂ muscarinic and N-formylpeptide receptors (14, 44). Even a B₂R mutant with all three major phosphorylation sites replaced (S) allowed internalization of a sizable receptor fraction (40%). The finding that this fraction was further reduced in the ST variant, where five potential phosphorylation sites (3 serines and 2 threonines) have been replaced, could be explained by the minute levels of Thr³⁴² and Thr³⁴⁵ phosphorylation in the S mutant that became obvious after pretreatment of cells with serine/threonine phosphatase inhibitors (data not shown).

Unlike the low stoichiometry phosphorylation requirement for receptor internalization, we found that tandem phosphorylation of Ser³⁴⁶ with Ser³³⁹ or Ser³⁴⁸ is necessary and sufficient to desensitize the B₂R-mediated PLC activation. These data correlate well with the findings of Leeb-Lundberg and co-workers (18) who described an increased spontaneous activity of a B₂R mutant replacing, among other residues, Ser³⁴⁶ and Ser³⁴⁸. However, single phosphorylation of the major acceptor site Ser³⁴⁸ that was also found in the absence of ligand does not affect receptor signaling by itself, but may prime the B₂R for desensitization. Other examples for the critical role of clustered phosphoserine and phosphothreonine residues in desensitization have been reported for the m₂ muscarinic and N-formylpeptide receptors (14, 44). Furthermore, the observation that FR190997, an agonist capable of sustained signaling, is a weak inducer of Ser³⁴⁶/Ser³⁴⁸ phosphorylation points out the importance of this tandem phosphorylation for B₂R desensitization and provides an intuitive explanation for the delayed B₂R desensitization upon FR190997 stimulation (30, 31).

The results from this comprehensive two-dimensional mapping study of in vivo phosphorylation sites demonstrate the power of this analytical method to reveal subtle temporal and positional changes in the phosphorylation pattern that translate into substantial alterations in the functional capacity of a prototypic GPCR. Future studies will unravel whether the molecular insights into differential phosphorylation requirements for internalization and desensitization of B₂R hold for GPCRs in general.

	ACKNOWLEDGEMENTS

We thank Lars Rönnstrand for advice on the two-dimensional phosphopeptide mapping technique and Carl-Henrik Heldin, Stefan Offermans, and Lars Rönnstrand for critical reading of the manuscript.

	FOOTNOTES

^* 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 postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. Present address: Institute for Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, D-61290 Heidelberg, Germany.

Supported by the Fondation pour la Recherche Medicale and the Association de secours des amis des Sciences. Present address: Dept. of Genetics and Howard Hughes Medical Institute, Harvard Medical School, Warren Alpert Building, 200 Longwood Ave., Boston, MA 02115.

Supported by the Boehringer Ingelheim Fonds. To whom correspondence should be addressed. Tel.: 46-18-160403; Fax: 4618-160-420; E-mail: Ivan.Dikic@licr.uu.se.

Published, JBC Papers in Press, August 21, 2001, DOI 10.1074/jbc.M107024200

	ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; B₂R, bradykinin B₂ receptor; PMA, phorbol 12-myristate 13-acetate; PKC, protein kinase C; PLC, phospholipase C; PAGE, polyacrylamide gel electrophoresis; pS, phosphorylated serine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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