HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 279, Issue 30, 31268-31276, July 23, 2004

This Article Abstract Full Text (PDF) All Versions of this Article: 279/30/31268    most recent M401796200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kalatskaya, I. Articles by Faussner, A. Search for Related Content PubMed PubMed Citation Articles by Kalatskaya, I. Articles by Faussner, A. Social Bookmarking                      What's this?

Mutation of Tyrosine in the Conserved NPXXY Sequence Leads to Constitutive Phosphorylation and Internalization, but Not Signaling, of the Human B₂ Bradykinin Receptor^*

Irina Kalatskaya, Steffen Schüssler, Andree Blaukat, Werner Müller-Esterl¶, Marianne Jochum, David Proud||, and Alexander Faussner**

From the Abteilung für Klinische Chemie und Klinische Biochemie, Ludwig-Maximilians-Universität, Nussbaumstrasse 20, D-80336 München, Germany, the Institut für Pharmakologie, Universität Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Germany, the ^¶Institute for Biochemistry II, University of Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany, and the ^||Department of Physiology & Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Received for publication, February 18, 2004 , and in revised form, May 12, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Although the G protein-coupled receptors (GPCRs) share a similar seven-transmembrane domain structure, only a limited number of amino acid residues is conserved in their protein sequences. One of the most highly conserved sequences is the NPXXY motif located at the cytosolic end of the transmembrane region-7 of many GPCRs, particularly of those belonging to the family of the rhodopsin/-adrenergic-like receptors. Exchange of Tyr³⁰⁵ in the corresponding NPLVY sequence of the bradykinin B₂ receptor (B₂R) for Ala resulted in a mutant, termed Y305A, that internalized [³H]bradykinin (BK) almost as rapidly as the wild-type (wt) B₂R. However, receptor sequestration of the mutant after stimulation with BK was clearly reduced relative to the wt B₂R. Confocal fluorescence microscopy revealed that, in contrast to the B₂R-enhanced green fluorescent protein chimera, the Y305A-enhanced green fluorescent protein chimera was predominantly located intracellularly even in the absence of BK. Two-dimensional phosphopeptide analysis showed that the mutant Y305A constitutively exhibited a phosphorylation pattern similar to that of the BK-stimulated wt B₂R. Ligand-independent Y305A internalization was demonstrated by the uptake of rhodamine-labeled antibodies directed to a tag sequence at the N terminus of the mutant receptor. Co-immunoprecipitation revealed that Y305A is precoupled to G_q/11 without activating the G protein because the basal accumulation rate of inositol phosphate was unchanged as compared with wt B₂R. We conclude, therefore, that the Y305A mutation of B₂R induces a receptor conformation which is prone to ligand-independent phosphorylation and internalization. The mutated receptor binds to, but does not activate, its cognate heterotrimeric G protein G_q/11, thereby limiting the extent of ligand-independent receptor internalization.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
G protein-coupled receptors (GPCRs),¹ also known as seven-transmembrane domain receptors, represent one of the largest classes of membrane receptors in the mammalian genome (1). They are involved in all aspects of interaction with, and perception of, the environment, including sight, smell, and taste. Such receptors also play a vital role in the control of physiology and behavior, as evidenced by the immense chemical diversity of their endogenous and exogenous ligands. These receptors are named based on their ability to bind to and activate intracellular heterotrimeric G proteins when stimulated by an extracellular agonist. The A family of rhodopsin/-adrenergic-like receptors is the largest and most well studied of all GPCR families (2). Although the members of this family do not share a high overall sequence identity, they have a characteristic pattern of a few highly conserved residues and motifs in homologous positions (most of them located in the transmembrane domains) that are not present in the other GPCR families. Given that a high degree of conservation suggests that a residue or segment might play a pivotal structural, functional, or regulatory role in the receptor actions, these residues and motifs have been examined by mutagenesis studies in many GPCRs. One of the most highly conserved motifs, together with the E/DRY sequence at the cytosolic end of transmembrane domain-3, is the NPXXY sequence (where X usually represents a hydrophobic residue and N is rarely exchanged against D) located at the C terminus of transmembrane domain-7. The results of these mutagenesis studies implicate this motif in the signaling, sequestration, and internalization of many GPCRs (3–13).

A prominent member of the family A of GPCRs is the mammalian type-2 bradykinin receptor (B₂R), which mediates the effects of bradykinin (BK) and kallidin (14). These nona- and decapeptides, respectively, are released from their high molecular weight precursors, the kininogens, through the action of the kallikreins. Effects of kinins via the B₂R include vasodilatation, edema formation, and pain sensation (15, 16). As with any other highly potent peptides, the concentration of BK is strictly controlled by rapid degradation involving enzymes such as aminopeptidases, neutral endopeptidase, and angiotensin I converting enzymes. Inhibitors of the latter belong to the most important drugs in the treatment of heart disorders. Although the internalization and desensitization of B₂R and the involvement of serine/threonine residues in the C terminus and in other intracellular domains have been studied in great detail (17), nothing is known, thus far, about the role of its NPXXY motif in these processes.

Here we have used a complementary multi-assay approach measuring internalization of radiolabeled ligands as well as receptor sequestration (relative changes in the number of surface receptors after stimulation with unlabeled agonist), performing total phosphorylation and phosphopeptide mapping, using eGFP-receptor fusion proteins and fluorescent-labeled antibodies directed against a N-terminal receptor tag, to determine the role of the NPLVY sequence and in particular of the critical tyrosine residue in the regulation of wt B₂R.

Our results suggest that Tyr³⁰⁵ (Tyr 7.53 according to the generalized numbering scheme (Ref. 18)) plays an important role in keeping the receptor in an inactive uncoupled state, thereby preventing spontaneous phosphorylation and subsequent interaction with the internalization machinery.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Phosphate-free Dulbecco's modified Eagle's medium (DMEM), sodium orthovanadate, aprotinin, leupeptin, pepstatin A, and poly-D -lysine hydrobromide were from Sigma (Taufkirchen, Germany); myo-[2-³H]inositol (21 Ci/mmol), [2,3-prolyl-3,4-³H]bradykinin (108 Ci/mmol), and [³⁵S]GTPS (1250Ci/mmol) from PerkinElmer Life Sciences; bradykinin from Bachem (Heidelberg, Germany); [³²P]orthophosphate (500 mCi/ml) from ICN (Eschwege, Germany); okadaic acid from Calbiochem (Bad Soden, Germany); cellulose thin layer chromatography (TLC) plates, 1.10-phenanthroline, and Pefabloc SC from Merck (Germany); FuGENE 6, protein G-agarose, anti-HA-antibody, rhodamine-labeled anti-HA antibody, and anti-HA affinity matrix from Roche (Mannheim, Germany); AG 1-X8 anion exchange columns, protein markers, and nitrocellulose membranes from Bio-Rad (Muenchen, Germany); sequencing grade trypsin from Promega (Mannheim, Germany); peroxidase-labeled rabbit anti-rat antibody from DAKO (Hamburg, Germany); G_q/11 antibody from Santa Cruz Biotechnology (Heidelberg, Germany); and Western Blot Chemiluminescence Reagent Plus from PerkinElmer Life Sciences. PCR primers were synthesized by Invitrogen (Groningen, The Netherlands) and delivered desalted and lyophilized. JE049 was a generous gift from Jerini AG (Berlin, Germany).

Mutations, Expression System, and Cell Culture (19)—The wild-type (wt) B₂R gene and all other constructs started with the third encoded Met (20), which, until recently, was assumed to be the start codon. Site-directed mutagenesis of B₂R was performed with standard PCR methodology using appropriate primers and confirmed by sequence analysis (Medigenomix, Martinsried, Germany). Similar methods were used for the construction of chimera with eGFP (enhanced green fluorescent protein) joined to the C terminus of B₂R with omission of the stop codon. The Flp-In system from Invitrogen was used to generate stable clones of the constructs. All receptor coding sequences were cloned into the HindIII and the XhoI sites of the pcDNA5/FRT vector and were preceded at the N terminus by a single hemagglutinin (HA) tag of MGYPYDVPDYAGS, with the last two amino acids (GS) of the tag being generated by the insertion of a BamHI site. Transfection of Flp-In TREx-293 (HEK293) cells was performed using FuGENE (Roche) following the instructions of the manufacturer, i.e. 2 µg of plasmid(s) plus 5 µl of FuGENE/6-well dish. Single stably transfected clones were obtained after selection with 250 µg/ml hygromycin B. Clones exhibiting similar high binding activity were considered to represent a single insertion of the pCDNA5/FRT vector DNA containing the receptor gene at the recombinase target site (usually at least 3 of 6 clones). Cells with twice as much binding activity were presumed to have an additional insertion of the vector (rare); clones with less binding activity were assumed to be inhomogeneous and were reselected with hygromycin B or not further propagated. The cells were cultivated in DMEM supplemented with 10% fetal calf serum and penicillin/streptomycin. For experiments that required rinsing of the cells, multiwell plates were pretreated with 0.01% poly-D -lysine hydrobromide (molecular mass > 300 kDa) in PBS.

Internalization of [³H]Bradykinin—Monolayers on 12-well/24-well plates were rinsed three times with PBS and incubated with 0.3/0.15 ml of [³H]BK in incubation buffer (40 mM PIPES, 109 mM NaCl, 5 mM KCl, 0.1% glucose, 0.05% bovine serum albumin, 2 mM CaCl₂, 1 mM MgCl₂, pH 7 containing 2 mM bacitracin, 0.8 mM 1.10-phenanthroline, 100 µM captopril) for 90 min on ice to reach equilibrium binding. [³H]BK internalization was started by placing the plates in a water bath at 37 °C. The internalization process was stopped at the indicated times by putting the plates on ice and washing the cells four times with ice-cold PBS. Thereafter, the cell monolayers were treated with 0.2 ml of an ice-cold dissociation solution (0.2 M acetic acid, 0.5 M NaCl, pH 2.7) for 10 min on ice to induce dissociation of surface bound [³H]BK. The supernatant with formerly surface-bound [³H]BK was quantitatively transferred to a scintillation vial by rinsing the cells with another 0.2 ml of PBS. The remaining monolayer, containing the internalized [³H]BK, was lysed using 0.2 ml of 0.3 M NaOH and transferred with another 0.2 ml of water to a scintillation vial. The radioactivity of both samples was determined in a -counter after addition of scintillation fluid. Non-receptor-mediated [³H]BK internalization and surface binding was determined in the presence of 5 µM unlabeled BK and subtracted from total binding. Internalization was expressed as intracellular [³H]BK in percentage of the combined amounts of internalized and surface-bound [³H]BK.

[³H]Bradykinin Binding Studies—For the determination of the dissociation constant K_D and receptor number B_max at 4 °C, confluent monolayers on 24-well/48-well trays were rinsed three times with PBS and incubated with 0.3/0.2 ml of ice-cold incubation buffer containing increasing concentrations of [³H]BK (10 concentrations ranging from 0.01 to 30 nM) for at least 90 min on ice. The incubation was stopped by rinsing the cells four times with ice-cold PBS. The cell monolayer was then lysed in 0.2 ml of 0.3 M NaOH and transferred to a scintillation vial. To calculate specific binding, the nonspecific binding (usually less than 5% of total binding) was determined in the presence of 5 µM of unlabeled BK and subtracted from total binding being determined with [³H]BK alone.

Measurement of Total Inositol Phosphate Release—Cell monolayers at 80% confluence in 12-well dishes were labeled for 18–24 h with 0.5 µCi of myo-[³H]inositol in 0.5 ml of DMEM containing fetal calf serum and penicillin/streptomycin. The monolayers were then placed on ice, rinsed three times with ice-cold PBS (pH 7.2), and preincubated with the indicated concentration of BK in the presence of 50 mM LiCl for at least 90 min. The stimulation was started by placing the tray in a water bath at 37 °C for 30 min and terminated by exchanging the buffer with 0.75 ml of ice-cold 20 mM formic acid, keeping the tray on ice for an additional 30 min. Subsequently, the supernatant was combined with another 0.75 ml of 20 mM formic acid and, after alkalization with 0.2 ml of 3% ammonium hydroxide solution, was applied to an AG 1-X8 anion exchange column. The column was washed with 1 ml of 1.8% ammonium hydroxide, followed by 9 ml of 5 mM tetraborate, 60 mM sodium formate. Total inositol phosphates (IP) were eluted in 3 ml of 4 M ammonium formate, 0.2 M formic acid. The radioactivity was determined in a -counter after addition of scintillation fluid.

Immunoprecipitation and Western Blotting—Cells were washed once with PBS and solubilized in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, pH 7.5) including 0.5 mM Pefabloc SC and 10 µM each 1.10-phenanthroline, aprotinin, leupeptin, and pepstatin A for 45 min at 4 °C with gentle rocking. The lysate was centrifuged at 6.240 x g for 20 min at 4 °C. The supernatant (0.5 ml with 1.5 mg of total protein) was incubated with 35 µl of protein G-agarose and 2.5 µl of antiserum AS346 (21, 22) for 3 h at 4 °C. The mixture was then washed twice with RIPA buffer and once with distilled water, resuspended in 30 µl of Laemmli buffer, and incubated 6 min at 95 °C. Following electrophoresis on 10% SDS-polyacrylamide gels, the fractionated proteins were electroblotted onto 0.45-µm nitrocellulose. The membranes were then incubated overnight with blocking buffer (0.25% gelatin in 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 0.05% Triton X-100, pH 7.5). The primary high affinity anti-HA antibody (0.1 µg/ml) was added for 2 h at room temperature in fresh blocking buffer. The membranes were washed twice for 10 min in Tris-buffered saline with 0.1% Tween 20 before adding the corresponding secondary peroxidase-labeled rabbit anti-rat Ig (1:1000) for 1 h at room temperature in blocking buffer. After washing in Tris-buffered saline with 0.1% Tween 20 three times each for 15 min, antibody binding was detected using Western Blot Chemiluminescence Reagent Plus.

Receptor Phosphorylation—Confluent cells on 6-well plates were washed twice with phosphate-free DMEM, incubated for 3 h at 37 °Cin the same medium, and labeled with 0.2 mCi/ml [³²P]orthophosphate for 10–12 h. After a 5-min exposure to 1 µM BK at 37 °C, cells were scraped into 0.5 ml of RIPA buffer containing protease inhibitors (see above) and phosphatase inhibitors (25 mM NaF, 1 mM sodium orthovanadate, 0.3 µM okadaic acid). Immunoprecipitation and separation on a 10% SDS-polyacrylamide gel were carried out as detailed above. The proteins of interest electroblotted onto nitrocellulose membranes were identified by autoradiography. Two-dimensional mapping of the phosphorylation sites was performed as described (17, 22) with minor modifications. Briefly, phosphoproteins were cut out from the membrane, blocked with 0.5% polyvinylpyrrolidone-40 in 0.6% acetic acid, and 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. After oxidation in performic acid, the obtained peptides were frozen, vacuum-dried, and digested again with 1 µg of trypsin in 50 µl of 50 mM (NH₄)HCO₃ for 12 h at 37 °C. The first dimension separation was performed on cellulose thin layer plates in formic acid/acetic acid/water buffer (46:156:1790 (v/v/v)), pH 1.8 at 2000 V for 30 min. The plates were air-dried and subjected to ascending chromatography in isobutyric acid/1-butanol/pyridine/acetic acid/water buffer (1250:38:96:58:558 (v/v/v/v/v)) until the solvent front reached the top of the plate. Plates were then extensively dried and exposed to Fuji X-ray film for 7–10 days at –80 °C.

Crude Membrane Preparation—Cell monolayers were washed three times with ice-cold PBS, scraped in ice-cold homogenization buffer (20 mM HEPES/Na-HEPES, pH 7.4, 10 mM EDTA), and homogenized using a Dounce homogenizer with five strokes at 1400 rpm. Thereafter, the cell lysate was centrifuged for 10 min at 20,000 x g. The resulting pellet was resuspended in the homogenization buffer and the full procedure repeated. The final crude membrane preparation was resuspended in storage buffer (20 mM HEPES/Na-HEPES, pH 7.4, 0.1 mM EDTA) at a concentration of 1 mg/ml protein and stored at –80 °C. Total protein was quantified with the Micro BCA protein assay reagent kit from Pierce using bovine serum albumin as standard.

Co-immunoprecipitation of Receptor-G Protein Complexes—Crude membrane preparations were thawed on ice and solubilized by adding 20 mM CHAPS dissolved in 20 mM PIPES, pH 6.8, to give a final concentration of 4 mM CHAPS (23). After incubation for 1 h at 4 °C, the insoluble particles were removed by centrifugation at 10,000 x g for 5 min. The supernatant was incubated overnight with 20 µl of anti-HA affinity matrix (Roche) at 4 °C. The pellet obtained by centrifugation was washed as described above. After SDS-PAGE and electroblotting onto nitrocellulose, the membrane was incubated with the indicated anti-HA or anti-G_q/11 antibodies (1:1000).

Confocal Microscopy—Cells were plated on glass coverslips pretreated with 0.01% poly-D -lysine in PBS. For confocal microscopy, cells were washed with, and incubated for 2 h in, serum-free medium with 20 mM HEPES, pH 7.3, before addition of rhodamine-labeled anti-HA antibody or stimulation with BK. Images were taken halfway through most of the cells with a confocal microscope (Zeiss LSM 410) using a 63x/1.4 oil immersion objective with no zoom.

[³⁵S]GTPS Binding Assay—Aliquots of crude membrane preparations containing 20 µg of total protein each were incubated for 15 min on ice in 500 µl of 20 mM HEPES/Na-HEPES, pH 7.4, 100 mM NaCl, 10 mM MgCl₂, 1 µM GDP, containing 2 mM bacitracin, 0.8 mM 1.10-phenanthroline, 100 µM captopril including 10⁵ cpm [³⁵S]GTPS in the absence or presence of 1 µM BK. Incubation was continued for 30 min in a water bath at 30 °C. Free and protein-bound [³⁵S]GTPS were separated by rapid filtration of the preparation through glass fiber filters (Whatman GF/C) and thorough washing of the filters with 15 ml of ice-cold 10 mM sodium phosphate buffer, pH 7.4. The radioactivity retained by the filters was determined in a -counter using liquid scintillation mixture. Nonspecific binding was measured in the presence of 1 mM GTP and subtracted from all values to give specific binding.

Data Analysis—All data analysis was performed using GraphPad Prism for Macintosh, version 3.0a (GraphPad Software, Inc.).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mutation of Tyr³⁰⁵ Does Not Affect Ligand Affinity of B₂R— The NPLVY sequence located in the C-terminal portion of transmembrane domain-7 of B₂R (positions 301–305) represents the highly conserved NPXXY motif of family A-type GPCRs. To determine the structural and functional significance of this motif in B₂R, Tyr³⁰⁵ was replaced by Ala (mutant receptor Y305A) and more conservatively by Phe (Y305F). Both mutants and the wt B₂R were stably expressed in HEK293 cell using the Flp-In system. Because, in this expression system, the gene of interest is inserted in the identical position in the genome of the host cell through the transient action of a recombinase, we varied expression levels by using a vector with either the original cytomegalovirus (CMV) promoter for higher expression, or the minimal CMV promotor (P_minCMV) for lower expression levels. Maximal binding capacities of the stable clones were estimated by incubation on ice with a saturating concentration of [³H]BK (10 nM). Mutant receptor Y305A exhibited considerably less surface binding (2.1 pmol/mg of protein) than either wt B₂R (10.4 pmol/mg of protein) or mutant Y305F (12.5 pmol/mg of protein). Expression of the B₂R under the control of the P_minCMV promotor (4.4 pmol/mg of protein) was closer to the levels of the Y305A mutant expressed under the control of the CMV promotor. It should, however, be mentioned that even cells with a low expression profile produced copy numbers of B₂R that were consistently higher than those observed in native cells, e.g. human foreskin fibroblasts. The differences observed in surface expression among the constructs using the Flp-In system (despite using the identical promotor) are likely caused by differences in cellular trafficking and transportation of the newly synthesized proteins. The affinities of B₂R, whether expressed at high or low levels, and of the two mutant receptors did not differ markedly (see Table I), indicating that the mutations do not directly affect the ligand binding site.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Internalization of [3H]BK by wild-type and mutant B2R in HEK293 cells. Monolayers of stably transfected HEK293 cells were incubated with 1 nM [3H]BK for 90 min on ice. The internalization was started by placing the plates in a water bath at 37 °C. At the indicated times, the internalization process was stopped, and surface binding and internalized ligand were determined. The surface profile is depicted in the inset. , wild-type B2R; , Y305A; , Y305F. All values are the mean ± S.E. from at least three experiments performed in triplicate.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Sequestration of high and low expressing wild-type and mutant B2R. Cells were preincubated with 1 µM BK on ice for 90 min. Receptor internalization was started by warming the cells to 37 °C in a water bath. At the indicated times, cells were set back on ice and treated with an acidic acid solution to remove unlabeled BK. Remaining surface binding was determined with 2 nM [3H]BK at 4 °C. , low expressing wt B2R; , high expressing wt B2R; , mutant Y305A. All values are the mean ± S.E. from at least three experiments each performed in triplicate.

Wild-type and Mutant Receptors Show Similar Expression Levels—Immunoprecipitation of the epitope-tagged receptors followed by Western blot analysis revealed a major diffuse band in the region of 50–80 kDa for all constructs (Fig. 3). In addition, low molecular mass bands at 37 and 40 kDa and a high molecular mass band at 100–150 kDa were detected (data not shown). Despite the divergent levels of ligand surface binding in intact cells, the expression levels of wt B₂R and Y305A were comparable, suggesting that the mutant receptor is either not completely accessible for the ligand because of an intracellular localization or that some of the mutant receptors in the plasma membrane display very low affinity for [³H]BK.

View larger version (43K): [in this window] [in a new window]   FIG. 3. Immunoprecipitation of wild-type and mutant B2 R. Wild-type and mutant receptors containing an N-terminal HA tag were immunoprecipitated from the lysates of a confluent 10-cm cell culture dish, using antibody AS346 directed to the receptor C terminus. The precipitated proteins were separated by reducing 10% SDS-PAGE, transferred onto nitrocellulose, and immunoblotted with anti-HA. Relative molecular masses determined with standard proteins are indicated on the left. The blot shown is representative of three experiments with similar results.

View larger version (119K): [in this window] [in a new window]   FIG. 4. Cellular distribution of wild-type and mutant B2R fused to eGFP. HEK293 cells stably expressing constructs of eGFP fused to the C terminus of wt B2R, Y305F, or Y305A were incubated in the absence (–BK) or presence (+BK) of 5 µM BK for 30 min at 37 °C and examined by confocal laser scanning microscopy. The experiments were repeated three times. Similar results were obtained with constructs stably expressed in Chinese hamster ovary cells DUKXB1 (ATCC).

View larger version (48K): [in this window] [in a new window]   FIG. 5. Agonist-induced phosphorylation of wild-type and mutant B2R. Upper panel, HEK293 cells expressing wt B2R, Y305A, or Y305F were labeled for 10 h with [32P]orthophosphate before stimulation with 1 µM BK for 5 min. Cells were lysed, and proteins were solubilized, immunoprecipitated, and visualized by autoradiography. Molecular size markers are indicated to the left. Lower panel, protein phosphorylation, given as optical densities of the bands in the area between 50 and 85 kDa, are presented as means ± S.D. from five independent experiments; unstimulated wt B2R was set as 100%.

View larger version (34K): [in this window] [in a new window]   FIG. 6. Two-dimensional phosphopeptide maps of wild-type and mutant B2R. Cells expressing wild-type B2R (upper panels) or mutant Y305A (lower panels) cultured in the presence of [32P]orthophosphate were incubated for 5 min in the absence (left) or presence (right) of 1 µM BK. Receptors were immunoprecipitated by AS346 from the cell lysates, the 32P-labeled proteins were in situ digested with trypsin, and the resulting peptides were separated on thin layer chromatography plates as indicated. The sample application site is marked by x, and the polarity of the electrophoresis is indicated (+ and –). The phosphopeptide maps shown are representative for the results of six (B2R) and five (Y305A mutant) experiments.

View larger version (155K): [in this window] [in a new window]   FIG. 7. Internalization of rhodamine-labeled antibodies of wild-type and mutant B2R. Cells expressing HA-tagged wt B2R, Y305F, and Y305A were incubated with rhodamine-labeled antibody to HA in the absence (upper panels; lower panel, left) or presence of antagonist JE049/HOE140 (lower panel, right) at 37 °C for 1 h. Receptor distribution was monitored by confocal laser scanning microscopy. The experiment shown is representative of several independent experiments with identical results.

View larger version (45K): [in this window] [in a new window]   FIG. 8. Differential coupling of wild-type and mutant B2 R to Gq/11. Receptors present in crude membrane preparations of unstimulated cells expressing wt B2R or mutant Y305A were solubilized with 4 mM CHAPS and immunoprecipitated with anti-HA. The immunoprecipitates were subjected to SDS-PAGE and Western blotting using anti-HA (upper panel) or anti-Gq/11 antibodies (lower panel). The experiment shown was repeated twice with identical results.

View larger version (16K): [in this window] [in a new window]   FIG. 9. [35S]GTPS binding to crude membranes wild-type B2R and mutant Y305A. Aliquots of crude membrane preparations containing 20 µg of total protein each of high and low expressing wt B2R (left and center, respectively) and of mutant Y305A receptor (right) were incubated at 30 °C with 500 µl of assay buffer in the presence of 105 cpm [35S]GTPS in the presence (gray bars) or absence (open bars) of 1 µM BK. After 30 min specific binding was determined. Data shown are the means ± S.E. from three independent experiments each performed in triplicate and are presented as percentage of the basal binding obtained for high expressing wt B2R (100%).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

To monitor ligand-induced receptor translocation from the surface to the interior of a cell, two assays can be employed that both have their advantages and disadvantages. In the sequestration assay, cells are stimulated at 37 °C for various times with a saturating concentration of unlabeled agonist, and then a binding assay is performed at 4 °C with a radiolabeled, nonmembrane-permeable agonist or antagonist to assess the remaining surface receptor expression. In this assay, receptor sequestration is expressed as the amount of remaining receptors on the cell surface of treated cells as a percentage of that initially present on unexposed cells. The ligand internalization assay is based on the assumption that the receptor-bound ligand and the receptor itself share, at least for a certain initial time frame, the same fate, i.e. the same cellular localization. Consequently, cells are incubated immediately with radiolabeled ligand at 37 °C (optionally after reaching equilibrium binding at 4 °C). At various times the amounts of surface-bound, and of internalized, ligand are determined separately at low pH. In this approach, internalization is expressed as amount of internalized ligand as a percentage of the total bound ligand (the sum of internalized and surface-bound ligand). As previously reported, the usage of the sequestration assay for cells that highly overexpress B₂R may grossly underestimate "true" receptor sequestration, presumably because of an overload of the internalization machinery (19). The ligand internalization assay, in contrast, can be applied even under these conditions, provided appropriate low concentrations of radiolabeled agonist are used to avoid saturation of the internalization machinery. This method does not, however, differentiate between ligand internalization caused by ligand-promoted receptor translocation and that occurring via constitutive receptor translocation, because, in both cases, rapid ligand transport into intracellular compartments is observed. As our data with the mutant Y305A show, this is not just a theoretical problem.

At first sight, Y305A displays properties similar to those of wt B₂R; it binds [³H]BK with the same affinity, it internalizes the agonist at a similar rate, and it induces cellular responses at similar EC₅₀ (see Table I). By contrast, despite its relatively low surface expression, Y305A shows markedly reduced sequestration, a property previously noted to an even greater extent (i.e. no sequestration at all) for the corresponding ₂-adrenergic receptor mutant (13). This dichotomous behavior, that is rapid ligand internalization and attenuated receptor sequestration, provided the first evidence that the Y305A mutant undergoes constitutive internalization. This conclusion was underlined by the demonstration of similar expression levels of wt B₂R and Y305A despite markedly lower surface binding capacity of the latter. Hence a majority of mutant receptors were either not accessible to the hydrophilic ligand, because of an intracellular location, or a major fraction of Y305A had very low affinity for the ligand. Usage of chimeric constructs showed that Y305A-eGFP fusion protein was, indeed, predominantly located inside the cell, even in the absence of a stimulus. Final proof that Y305A spontaneously internalized came from the finding that rhodamine-labeled antibodies to the N-terminally tagged receptor were rapidly internalized by Y305A, but not by wt B₂R. Two antagonists blocked this ongoing internalization, i.e. Y305A-mediated uptake of rhodamine-labeled antibody was markedly halted by JE049/HOE140, and there was no significant internalization of the radiolabeled antagonist [³H]NPC17731 by Y305A (data not shown). This suggests that these ligands act as inverse agonists reducing the spontaneous internalization of Y305A.

Our initial studies of agonist-induced phosphorylation of Y305A suggested that this mutant is phosphorylation-resistant, as has previously been reported for the corresponding ₂-adrenergic receptor mutant (11). Phosphopeptide analysis, however, suggested that this apparent resistance might be because the accessible mutant receptors on the plasma membrane are already phosphorylated on Ser/Thr residues in their basal state. This ligand-independent phosphorylation would also explain the enhanced spontaneous internalization of this mutant. Further support for the theory that the spontaneous internalization is indeed caused by ligand-independent phosphorylation is provided by the lack of spontaneous as well as agonist-induced internalization of a Y305A mutant in which the critical Ser/Thr residues had been replaced by Ala (results not shown). Hence the NPXXY sequence appears not to serve as an endocytotic motif interacting directly with the internalization apparatus, but, rather, is involved in the phosphorylation of Ser/Thr residues of the receptor tail.

We considered whether our data could be explained in the context of the two-state model in which an equilibrium exists between the receptor in its inactive and its active state (27). In the absence of agonist, one would expect this equilibrium to be in favor of the inactive state of wt B₂R, whereas it would be shifted markedly in favor of the active state in the Y305A mutant, explaining the enhanced internalization. Binding of an antagonist also would promote the inactive state, attenuating internalization, as observed in our experiments. This model, however, is inconsistent with the lack of constitutive basal IP accumulation and of constitutive guanine nucleotide exchange. We, therefore, propose an alternative model in which the NPXXY sequence plays an important role in the interaction of the receptor with the G protein. We postulate that mutation Y305A induces a semi-active receptor conformation that is prone to phosphorylation and consequently to internalization, and that is also able to couple to the G protein, without, however, significantly activating it. Accordingly, the affinity of Y305A for G_q/11 and/or the probability of a spontaneous ligand-independent activation of bound G_q/11 would determine whether the internalization machinery gets access to the (already phosphorylated) C terminus resulting in the internalization of the receptor. Our hypothetical model predicts that there would be little or no increased basal IP accumulation, but stimulation with an agonist would nevertheless lead to robust phospholipase C activation and rapid receptor internalization. The fact that Y305A gives a stronger ligand-induced IP response than wt B₂R may be explained in our model by assuming that, following ligand-dependent receptor activation and G_q/11 dissociation, the precoupled semi-active Y305A conformation is more rapidly restored than the corresponding wt B₂R form, allowing for an increased G protein turnover. Additionally, a reduced desensitization rate and/or a higher recycling rate of Y305A may contribute to the observed increase in IP accumulation. Inverse agonists could exert their inhibiting effect on the constitutive internalization either by converting Y305A back to an inactive conformation or, alternatively, through stabilization of the complex of Y305A with inactive G_q/11. Our finding that mutant Y305A, unlike wt B₂R, is coupled to G_q/11 even in the absence of a stimulus is consistent with this latter model.

The various phenotypes observed in the corresponding Tyr mutants of other GPCRs could be explained, according to our model, on the basis either of different affinities of these mutants for their cognate G proteins and/or by different degrees of spontaneous activation of these mutants, e.g. Y305A mutation of the neurokinin 1 receptor generated a mutant receptor, NK1-Y305A, that was primarily located intracellularly, even in its unstimulated state, whereas only minimal amounts of mutant were found on the cell surface (9). Moreover, the relative distribution (intracellular versus cell surface) of NK1-Y305A did not vary, regardless of whether it was incubated with agonist or not, implying deficient receptor sequestration. The mutant receptor, however, internalized radiolabeled ligand at a rate similar to that of the corresponding wt receptor. These data could be reconciled, according to our model, by assuming that NK1-Y305A forms a less stable complex with G_q/11 than, e.g., the Y305A mutant of B₂R, making the phosphorylated C terminus of NK1-Y305A more accessible to the internalization machinery and thereby promoting constitutive internalization. Assumption of a high affinity of the respective ₂-adrenergic receptor mutant Y326A for G_s, combined with the observed inability of this mutant to activate the G protein, would explain the lack of sequestration and resistance to phosphorylation, as well as the fact that this mutant is located preferentially in the plasma membrane (10, 11, 13). As the binding of the G protein should nevertheless be reversible, the reported slightly increased intracellular localization (10) as well as the fact that overexpression of GPCR kinases or -arrestin were able to rescue sequestration of this mutant (11) would fit our model.

In summary, our results demonstrate that the tyrosine residue of the NPXXY motif present in the human bradykinin B₂ receptor plays a decisive role in the regulation of activation, phosphorylation, internalization, and thus sequestration of the receptor, presumably by controlling the affinity and activation capacity for the cognate G protein and by controlling access of the phosphorylation as well as internalization machinery.

	FOOTNOTES

 
^* This work was supported by Deutsche Forschungsgemeinschaft Grant FA 288/3-1 (to A. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Ludwig-Maximilians-Universitaet Muenchen, Abt. Klinische Chemie und Klinische Biochemie, Nussbaumstr. 20, D-80336 Muenchen, Germany. Tel.: 49-89-5160-2602; Fax: 49-89-5160-4740; E-mail: alexander.faussner{at}med.uni-muenchen.de.

¹ The abbreviations used are: GPCR, G protein-coupled receptor; B₂R, bradykinin B₂ receptor; BK, bradykinin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CMV, cytomegalovirus; DMEM, Dulbecco's modified Eagle's medium; eGFP, enhanced green fluorescent protein; GTPS, guanosine 5'-3-O-(thio)triphosphate; HA, hemagglutinin; IP, inositol phosphates; PBS, phosphate-buffered saline; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); RIPA, radio-immunoprecipitation assay; Ser(P), phosphorylated serine; Thr(P), phosphorylated threonine; wt, wild-type.

	ACKNOWLEDGMENTS

 
We give our thanks to Drs. Petra Kameritsch and Ulrich Pohl for their support with confocal microscopy.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Gether, U. (2000) Endrocr. Rev. 21, 90–113[Abstract/Free Full Text]
2. Frederikson, R., Lagerström, M. C., Lundin, L.-G., and Schiöth, H. B. (2003) Mol. Pharmacol. 63, 1256–1272[Abstract/Free Full Text]
3. Fritze, O., Filipek, S., Kuksa, V., Palczewski, K., Hofmann, K. P., and Ernst, O. P. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2290–2295[Abstract/Free Full Text]
4. Bouley, R., Sun, T.-X., Chenard, M., McLaughlin, M., McKee, M., Lin, H. Y., Brown, D., and Ausiello, D. A. (2003) Am. J. Physiol. Cell Physiol. 285, C750–C762[Abstract/Free Full Text]
5. Prioleau, C., Visiers, I., Ebersole, B. J., Weinstein, H., and Sealfon, S. C. (2002) J. Biol. Chem. 277, 36577–36584[Abstract/Free Full Text]
6. Rosendorff, A., Ebersole, B. J., and Sealfon, S. C. (2000) Mol. Brain Res. 84, 90–96[Medline] [Order article via Infotrieve]
7. Hukovic, N., Panetta, R., Kumar, U., Rocheville, M., and Patel, Y. C. (1998) J. Biol. Chem. 273, 21416–21422[Abstract/Free Full Text]
8. Wang, J., Zheng, J., Anderson, J. L., and Toews, M. L. (1997) Mol. Pharmacol. 52, 306–313[Abstract/Free Full Text]
9. Böhm, S. K., Khitin, L. M., Smeekens, S. P., Grady, E. F., Payan, D. G., and Bunnett, N. W. (1997) J. Biol. Chem. 272, 2363–2372[Abstract/Free Full Text]
10. Gabilondo, A. M., Krasel, C., and Lohse, M. J. (1996) Eur. J. Pharmacol. 307, 243–250[CrossRef][Medline] [Order article via Infotrieve]
11. Ferguson, S. S. G., Menard, L., Barak, L. S., Koch, W. J., Colapietro, A. M., and Caron, M. G. (1995) J. Biol. Chem. 270, 24782–24789[Abstract/Free Full Text]
12. Hunyady, L., Bor, M., Baukal, A. J., Balla, T., and Catt, K. J. (1995) J. Biol. Chem. 270, 16602–16609[Abstract/Free Full Text]
13. Barak, L. S., Tiberi, M., Freedman, N. J., Kwatra, M. M., Lefkowitz, R. J., and Caron, M. G. (1994) J. Biol. Chem. 269, 2790–2795[Abstract/Free Full Text]
14. Regoli, D., and Barabe, L. (1988) Methods Enzymol. 163, 210–230[Medline] [Order article via Infotrieve]
15. Proud, D., and Kaplan, A.P. (1988) Annu. Rev. Immunol. 6, 4–83
16. Bhoola, K. D., Figueroa, C. D., and Worthy, K. (1992) Pharmacol. Rev. 44, 1–80[Medline] [Order article via Infotrieve]
17. Blaukat, A., Pizard, A., Breit A., Wernstedt, C., Alhenc-Gelas, F., Müller-Esterl, W., and Dikic, I. (2001) J. Biol. Chem. 276, 40431–40440[Abstract/Free Full Text]
18. Ballesteros, J. A., and Weinstein, H. (1995) Methods Neurosci. 25, 366–428
19. Faussner, A., Bauer, A., Kalatskaya, I., Jochum, M., and Fritz, H. (2003) Am. J. Physiol. 284, H1909–H1916
20. Hess, J. F., Borkowski, J. A., Young, G. S., Strader, C. D., and Ransom, R. W. (1992) Biochem. Biophys. Res. Commun. 184, 260–268[CrossRef][Medline] [Order article via Infotrieve]
21. Blaukat, A., AbdAlla, S., Lohse, M. J., and Müller-Esterl, W. (1996) J. Biol. Chem. 271, 32366–32374[Abstract/Free Full Text]
22. Boyle, W. J., van der Geer, P., and Hunter, T. (1991) Methods Enzymol. 201, 110–149[Medline] [Order article via Infotrieve]
23. Faussner, A., Heinz-Erian, P., Klier, C., and Roscher, A. A. (1991) J. Biol. Chem. 266, 9442–9446[Abstract/Free Full Text]
24. Sabourin, T., Bastien, L., Bachvarov, D. R., and Marceau, F. (2002) Mol. Pharmacol. 61, 546–553[Abstract/Free Full Text]
25. Marchese, A., Chen, C., Kim, Y. M., and Benovic, J. L. (2003) Trends Biochem. Sci. 28, 369–376[CrossRef][Medline] [Order article via Infotrieve]
26. Trowbridge, I. S., Collawn, J. F., and Hopkins, C. R. (1993) Annu. Rev. Cell Biol. 9, 129–161[CrossRef][Medline] [Order article via Infotrieve]
27. Samama, P., Cotecchia, S., Costa, T., and Lefkowitz, R. J. (1993) J. Biol. Chem. 268, 4625–4638[Abstract/Free Full Text]
28. Milligan G. (2003) Trends Pharmacol. Sci. 24, 87–90[CrossRef][Medline] [Order article via Infotrieve]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				L. Zhang, H. Zhao, Y. Qiu, H. H. Loh, and P.-Y. Law Src Phosphorylation of {micro}-Receptor Is Responsible for the Receptor Switching from an Inhibitory to a Stimulatory Signal J. Biol. Chem., January 23, 2009; 284(4): 1990 - 2000. [Abstract] [Full Text] [PDF]

				R. Schroder, N. Merten, J. M. Mathiesen, L. Martini, A. Kruljac-Letunic, F. Krop, A. Blaukat, Y. Fang, E. Tran, T. Ulven, et al. The C-terminal Tail of CRTH2 Is a Key Molecular Determinant That Constrains G{alpha}i and Downstream Signaling Cascade Activation J. Biol. Chem., January 9, 2009; 284(2): 1324 - 1336. [Abstract] [Full Text] [PDF]

				B. W. Jones and P. M. Hinkle Arrestin Binds to Different Phosphorylated Regions of the Thyrotropin-Releasing Hormone Receptor with Distinct Functional Consequences Mol. Pharmacol., July 1, 2008; 74(1): 195 - 202. [Abstract] [Full Text] [PDF]

				L. Oliveira, C. M. Costa-Neto, C. R. Nakaie, S. Schreier, S. I. Shimuta, and A. C. M. Paiva The Angiotensin II AT1 Receptor Structure-Activity Correlations in the Light of Rhodopsin Structure Physiol Rev, April 1, 2007; 87(2): 565 - 592. [Abstract] [Full Text] [PDF]

				C. Dong and G. Wu Regulation of Anterograde Transport of {alpha}2-Adrenergic Receptors by the N Termini at Multiple Intracellular Compartments J. Biol. Chem., December 15, 2006; 281(50): 38543 - 38554. [Abstract] [Full Text] [PDF]

				A. C. Conner, J. Simms, S. G. Howitt, M. Wheatley, and D. R. Poyner The Second Intracellular Loop of the Calcitonin Gene-related Peptide Receptor Provides Molecular Determinants for Signal Transduction and Cell Surface Expression J. Biol. Chem., January 20, 2006; 281(3): 1644 - 1651. [Abstract] [Full Text] [PDF]

				M. Clement, S. S. Martin, M.-E. Beaulieu, C. Chamberland, P. Lavigne, R. Leduc, G. Guillemette, and E. Escher Determining the Environment of the Ligand Binding Pocket of the Human Angiotensin II Type I (hAT1) Receptor Using the Methionine Proximity Assay J. Biol. Chem., July 22, 2005; 280(29): 27121 - 27129. [Abstract] [Full Text] [PDF]

				Y. Namkung and D. R. Sibley Protein Kinase C Mediates Phosphorylation, Desensitization, and Trafficking of the D2 Dopamine Receptor J. Biol. Chem., November 19, 2004; 279(47): 49533 - 49541. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 279/30/31268    most recent M401796200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kalatskaya, I. Articles by Faussner, A. Search for Related Content PubMed PubMed Citation Articles by Kalatskaya, I. Articles by Faussner, A. Social Bookmarking                      What's this?