QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: mol.106.030858v1 71/2/494    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Enquist, J. Articles by Leeb-Lundberg, L.M. F. Search for Related Content PubMed PubMed Citation Articles by Enquist, J. Articles by Leeb-Lundberg, L.M. F.

Kinins Promote B₂ Receptor Endocytosis and Delay Constitutive B₁ Receptor Endocytosis

Unit of Drug Target Discovery, Division of Cellular and Molecular Pharmacology, Department of Experimental Medical Science, Lund University, Lund, Sweden (J.E., C.S., L.M.F.L-L.); and Ernest Gallo Clinic and Research Center, University of California, San Francisco, California (J.L.W.)

Received September 13, 2006; accepted November 16, 2006

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Upon sustained insult, kinins are released and many kinin responses, such as inflammatory pain, adapt from a B₂ receptor (B₂R) type in the acute phase to a B₁ receptor (B₁R) type in the chronic phase. In this study, we show that kinins modulate receptor endocytosis to rapidly decrease B₂R and increase B₁R on the cell surface. B₂Rs, which require agonist for activity, are stable plasma membrane components without agonist but recruit -arrestin 2, internalize in a clathrin-dependent manner, and recycle rapidly upon agonist treatment. In contrast, B₁Rs, which are inducible and constitutively active, constitutively internalize without agonist via a clathrin-dependent pathway, do not recruit -arrestin 2, bind G protein-coupled receptor sorting protein, and target lysosomes for degradation. Agonist delays B₁R endocytosis, thus transiently stabilizing the receptor. Most of the receptor trafficking phenotypes are transplantable from one receptor to the other through exchange of the C-terminal receptor tails, indicating that the tails contain epitopes that are important for the binding of protein partners that participate in the endocytic and postendocytic receptor choices. It is noteworthy that the agonist delay of B₁R endocytosis is not transplanted to the B₂R via the B₁R tail, suggesting that this property of the B₁R requires another domain. These events provide a rapid kinin-dependent mechanism for 1) regulating the constitutive B₁R activity and 2) shifting the balance of accessible receptors in favor of B₁R.

GPCR signaling is tightly regulated at several steps in the receptor life cycle, ranging from receptor gene transcription to degradation of the mature receptor. At the cell surface, activation of the receptor by agonist is often followed by receptor phosphorylation via a G protein-coupled receptor kinase and subsequent arrestin binding to the receptor, which results in homologous desensitization (i.e., uncoupling of the receptor from the G protein and thereby from downstream signal transduction pathways) (Lefkowitz et al., 1998). Many GPCRs also subsequently undergo endocytosis through either clathrin-coated structures or lipid rafts, such as caveolae (Ferguson, 2001; Chini and Parenti, 2004). After endocytosis, GPCRs are either recycled back to the plasma membrane, leading to functional resensitization; remain internalized through storage, leading to desensitization; or are targeted to lysosomes for degradation, leading to functional down-regulation (von Zastrow, 2003). The postendocytic fate of a GPCR is determined via direct interactions of the receptor with various protein partners such as ERM-binding phosphoprotein 50/Na⁺-H⁺ exchanger regulatory factor, N-ethyl maleimide-sensitive factor, sorting nexin 1, GPCR-associated sorting protein (GASP), and lipid membrane components (Hall et al., 1998; Cao et al., 1999; Whistler et al., 2002; Xiang and Kobilka, 2003; Heydorn et al., 2004). The responsiveness of a GPCR signaling system is, to a large degree, determined by these regulatory events.

Kinin receptor adaptation is explained, at least in part, by the induction of B₁R expression by pro-inflammatory cytokines such as interleukin-1 (Bachvarov et al., 1998), which is observed in cellular systems (Phagoo et al., 1999; Bengtsson et al., 2006). This induction may function to convert kinin signaling from a transient one to a sustained one because B₁R is constitutively active (Leeb-Lundberg et al., 2001). Furthermore, B₁R is not phosphorylated either basally or in response to agonist (Blaukat et al., 1999) and desensitizes only marginally upon further stimulation by the agonist (Mathis et al., 1996). On the other hand, B₂R is subject to both basal and agonist-promoted phosphorylation (Blaukat et al., 1999, 2001) and desensitizes rapidly upon agonist stimulation (Mathis et al., 1996).

Kinins decrease the availability of cell surface B₂R and increase the availability of B₁R in some cell systems (Phagoo et al., 1999; Bengtsson et al., 2006), which may contribute to kinin receptor signal adaptation. The details of this regulation are not well understood, but some studies have suggested it may involve changes in receptor trafficking. Most trafficking studies of B₂R and B₁R have used bound radiolabeled agonist to monitor the receptors (Munoz and Leeb-Lundberg, 1992; Munoz et al., 1993; Faussner et al., 1998; Pizard et al., 1999; Lamb et al., 2001). In a few cases, receptors fused in their C-terminal domains to fluorescent proteins have also been used for this purpose (Bachvarov et al., 2001; Lamb et al., 2001; Sabourin et al., 2002). The former approach is restricted to monitoring only agonist-promoted changes, and is sensitive to changes in agonist affinity, whereas the latter approach requires receptor constructs drastically modified in their C-terminal domain, which can be critical for the postendocytic fate of the receptor (Cao et al., 1999; Finn and Whistler, 2001; Whistler et al., 2002; Gaborik and Hunyady, 2004; Heydorn et al., 2004; Bartlett et al., 2005). Nevertheless, these studies have proposed that B₂R is rapidly and almost completely internalized by the agonist, whereas B₁R is much less or differently affected by the agonist in this regard.

Our aim with this study was to use a representative cellular model with minimally modified receptors to determine the role of cellular trafficking in the rapid and inverse kinin regulation of B₂R and B₁R. HEK293 cells were chosen as a model system because this cell line is well characterized, easy to use and manipulate, has been shown to be a valuable tool in the trafficking studies of other model GPCR systems, and allows for the independent study of the trafficking of each receptor in the absence of regulation of de novo receptor synthesis.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Culture and DNA Constructs. IMR90 human embryonic lung fibroblast cells (American Type Culture Collection, Manassas, VA) were grown in minimum essential medium containing 200 U/ml penicillin, 200 µg/ml streptomycin, 2 mM L-glutamine (Invitrogen, Carlsbad, CA), and 10% fetal bovine serum (FBS) (Hyclone Laboratories, Logan, UT) in 5% CO₂ at 37°C. HEK293 cells (American Type Culture Collection) were grown in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen) supplemented with 10% FBS in 10% CO₂ at 37°C. The human B₂R and B₁R cDNA were subcloned into a pcDNA3.1 vector containing a Zeocin selection marker. An N-terminal artificial signal sequence, as described previously (Whistler et al., 2002), and the FLAG sequence tag were added in series to make the B₂R and B₁R constructs named SFB2 and SFB1, respectively. Receptor constructs with exchanged C-terminal tails starting from the first amino acid after the NPXXY motif (B1CB2 and B2CB1) and B₁R truncated after residue Phe³¹⁹ (B1Stop320), which is located seven residues beyond the NPXXY motif, were created by PCR. GFP--arrestin 2 was kindly provided by Dr. Marc Caron (Duke University Medical Center, Durham, NC) (Barak et al., 1997). The cells were transfected using the calcium phosphate precipitate method. Single colonies were then chosen and propagated in the presence of selection-containing media to generate clonal stable cell lines.

Radioligand Binding Assay. Assays were performed on particulate preparations and on intact cells using [³H]BK and [³H]DAKD as described previously (Leeb et al., 1997; Phagoo et al., 1999). Pretreatment of intact cells with agonist at 37°C was followed by a 6-min wash with ice-cold 50 mM glycine-HCl, pH 3, and two washes in ice-cold phosphate-buffered saline (PBS) to remove the agonist.

Phosphoinositide Hydrolysis. Confluent cells were labeled with 0.4 µCi/well [myo-³H]inositol for 16 to 20 h in inositol-free DMEM containing 0.5% bovine serum albumin in 48-well dishes, washed in Leibovitz's L-15 medium, pH 7.4, containing 10 mM LiCl, and then stimulated with increasing concentrations of agonist for 30 min at 37°C. The cells were then lysed with 100 mM formic acid, and the lysate was mixed with anion exchange resin in a 48-well plate with a small hole in the bottom of each well. The resin was then washed sequentially with water and 60 mM ammonium formate by aspiration. Inositol phosphates were eluted from the resin with 1 M ammonium formate and counted for radioactivity in a scintillation counter (LS6000; Beckman Coulter, Fullerton, CA).

FACS Analysis. Confluent cells in 100-mm dishes were treated with and without agonist in DMEM and 10% FBS. The cells were then trypsinized and washed with ice-cold PBS plus Ca²⁺/Mg²⁺. Cells were then resuspended in 100 µl of ice-cold PBS plus Ca²⁺/Mg²⁺ with 50% FBS, 0.4 µg of M1 anti-FLAG antibody conjugated with APC using a commercial kit (Prozyme, San Leandro, CA), 1 µg of mouse IgG1 (MOPC 21), incubated for 20 min at 4°C, which was followed by two washes in PBS plus Ca²⁺/Mg²⁺. The cell pellet was finally resuspended in 1 ml of PBS plus Ca²⁺/Mg²⁺ with 50% FBS. Cells (20,000) were counted by FACS, and each receptor-positive cell line was gated against untransfected HEK293 cells to reduce background staining. Mean fluorescence was calculated for the gated signal-positive population of untreated and agonist-treated cells.

Biotinylation Protection Assay. Confluent cells were washed twice with ice-cold PBS and then incubated with 0.3 mg/ml disulfidecleavable sulfo-NHS-SS-biotin (Pierce) in PBS for 30 min at 4°C with gentle agitation. The cells were then washed twice with Tris-buffered saline (TBS) to quench the biotinylation reaction and returned to DMEM and 10% FBS for treatments. Cells were allowed to reequilibrate to growth conditions for 30 min at 37°C before further experimentation. Cells labeled Total and Strip in the figures were left on ice in TBS. At 37°C, cells were treated without (NT) or with 1 µM desArg¹⁰kallidin (DAKD) or 1 µM bradykinin (BK) (AG) for the indicated intervals and washed twice with ice-cold TBS. The remaining cell surface-biotinylated receptors on the cells together with those cells designated Strip were stripped with 50 mM glutathione, 0.3 M NaCl, 75 mM NaOH, and 1% FBS for 30 min at 4°C. The glutathione was then quenched using a 20-min wash with PBS containing 50 mM iodoacetamide and 1% bovine serum albumin. Cells were then extracted in 0.1% Triton X-100, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 25 mM KCl containing a complete protease inhibitor cocktail (Roche), and cell debris was removed by centrifugation at 10,000g for 10 min at 4°C. Receptors were immunoprecipitated in the extraction buffer by incubating in anti-FLAG M2 affinity resin (Sigma) overnight at 4°C. The precipitates were washed extensively and sequentially in the extraction buffer and in 10 mM Tris-HCl, pH 7.4. The receptors were then deglycosylated for 2 h at 37°C with PNGase F (New England Biolabs, Ipswich, MA). Proteins were denatured in SDS sample buffer without reducing agent, fractionated by SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and the membrane was blocked for at least 1 h in TBS containing 0.1% Tween 20 and 10% nonfat milk. Biotinylated proteins were visualized by incubating with the Vectastain avidin-biotinylated enzyme complex immuno-peroxidase reagent (Vector Laboratories, Burlingame, CA) followed by development with ECL reagents according to the manufacturer's instructions (GE Healthcare, Little Chalfont, Buckinghamshire, UK).

Biotinylation Degradation Assay. Confluent cells were washed twice with ice-cold PBS and then incubated with 0.3 mg/ml disulfidecleavable sulfo-NHS-SS-biotin in PBS for 30 min at 4°C with gentle agitation. The cells were then washed twice with TBS to quench the biotinylation reaction and returned to DMEM and 10% FBS. Cells were then left on ice (Total) or returned to 37°C for 30 min after which they were incubated without (NT) or with 1 µM DAKD or BK (AG) for 120 min at 37°C. The receptors were then extracted, immunoprecipitated, and detected as described above under Biotinylation Protection Assay.

Fluorescence Microscopy. Cells were propagated in growth media on glass coverslips to 50% confluence and then treated in one of three ways. For whole-cell receptor distribution experiments, cells were incubated without and with agonist or antagonist for 30 min at 37°C. For cell surface receptor redistribution experiments, cells were incubated in media containing 3.5 µg/ml primary mouse anti-FLAG M1 monoclonal antibody (Sigma) for 30 min at 37°C to label surface receptors. The cells were then incubated without agonist or antagonist or a combination of the two for an additional 30 min at 37°C. For receptor recycling experiments, cells were incubated as described immediately above followed by stripping of remaining cell surface M1 antibody by depleting the media of Ca²⁺ with PBS containing 0.1% EDTA at room temperature. The cells were then further incubated without and with agonist or antagonist for an additional 30 min at 37°C. In all experiments, cells were then fixed using 3.5% formaldehyde in PBS and permeabilized using 0.1% Triton X-100. For visualization of receptors, fixed specimens were incubated with Alexa488-labeled anti-mouse IgG2b antibody (Invitrogen) to detect FLAG M1 antibodies bound to the tagged receptors. For colocalization of FLAG-tagged receptors with LAMP 1 and LAMP 2, primary H4A3 and H4B4 mouse IgG1 (Developmental Studies Hybridoma Data Bank) antibodies were incubated with secondary Alexa568-labeled anti-mouse IgG₁ antibody (Invitrogen), to detect LAMP 1 and LAMP 2, along with secondary Alexa488-labeled antimouse IgG2b, to detect FLAG M1 antibodies bound to receptors. Images were collected by confocal microscopy using a Nikon Eclipse confocal microscope. Quantification of colocalized fluorescence was done using the Imaris software (Bitplane AG, Zurich, Switzerland).

-Arrestin Recruitment. Cells stably expressing GFP--arrestin 2 were transiently transfected with SFB1, SFB2, SFB1CB2, or SFB2CB1. Transfected cells were plated on polylysine-treated glass coverslips in six-well dishes for 24 h before assay. The cells were then incubated in growth media without and with agonist for 5 min at 37°C. Cells were then fixed in PBS containing 3.5% formaldehyde for 30 min followed by primary staining with M1 anti-FLAG antibodies and secondary staining with Alexa568-labeled anti-mouse IgG1 antibody. Images were then collected by confocal microscopy using a confocal microscope (Eclipse; Nikon, Tokyo, Japan).

In Vitro Affinity Assay of GST Fusion Proteins and GASP. B₁R and B₂R receptor C-terminal tails starting after the NPXXY motif were cloned into the pGEX4t1 vector and C-terminal tail-GST fusion proteins were produced and attached to glutathione-Sepharose (Sigma, St. Louis, MO). Full-length GASP was in vitro-translated using the TNT quick-coupled transcription/translation systems (Promega, Madison, WI) with ³⁵S-labeled methionine. Fusion proteins and radiolabeled probe were mixed in a wash solution containing 0.1% Triton X-100 and incubated for 1 h at room temperature. Glutathione resin was washed several times followed by denaturation by boiling in SDS sample buffer and proteins fractionated by SDS-polyacrylamide gel electrophoresis. Protein concentrations of receptor tails and controls were estimated by Coomassie staining of the gel, and pulled down probe was visualized by autoradiography.

Coimmunoprecipitation of Receptor and GASP. Confluent cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 10 mM NaF, and 10 mM Na₂HPO₄) with complete protease inhibitor cocktail (Roche, Indianapolis, IN). Lysates were cleared by centrifugation at 14,000g for 15 min at 4°C. An aliquot of the cleared lysates was withdrawn from each sample to compare the GASP concentration by immunoblotting as described below. The rest of the cleared lysate was immunoprecipitated using 15 µl of M2 anti-FLAG affinity resin (Sigma) for 1 h at 4°C, washed extensively with 0.1% Triton X-100, 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 25 mM KCl containing a complete protease inhibitor cocktail (Roche), and deglycosylated with PNGase F (New England Biolabs) for 2 h. Precipitates were resolved ona4to 20% gradient Tris-HCl precast gel (Bio-Rad Laboratories, Hercules, CA) and transferred to a polyvinylidene difluoride membrane. The blots were cut below the 75-kDa marker band to separately immunoblot for either receptor (lower blot) or GASP (upper blot). GASP blots were incubated for 2 h at room temperature with rabbit anti-GASP (1:1000) and for 1 h at room temperature with HRP-conjugated anti-rabbit antibody (New England Biolabs) (1:4000), then visualized with ECL Plus (GE Healthcare). Receptor blots were incubated for 2 h with biotinylated FLAG M2 antibody (1:250) (Sigma) and then visualized with streptavidin overlay (Vectastain ABC reagents; Vector Laboratories) and ECL Plus.

Data Analysis. Data were analyzed by the Student's t test as indicated.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Effect of Kinin Agonists on Cell Surface Availability of Kinin Receptors. Human embryonic lung fibroblast IMR90 cells express B₂R and B₁R at 1283 and 15 fmol/mg of protein, respectively (Phagoo et al., 1999). Treatment of these cells with a 0.1 µM concentration of the cognate B₂R ligand BK at 37°C led within 30 min to a 78% decrease in the amount of B₂R available on the cell surface, as determined by radioligand binding, and this effect was maintained at 120 min of treatment (Fig. 1A). In contrast, treatment with a 0.1 µM concentration of the cognate B₁R ligand DAKD led within the same time period to an 81% increase in the number of available B₁R, which was also maintained at 120 min of treatment (Fig. 1A). Thus, kinins rapidly and inversely regulate the cell surface availability of native B₂R and B₁Ras reported previously (Phagoo et al., 1999; Bengtsson et al., 2006).

View larger version (12K): [in this window] [in a new window]   Fig. 1. Effects of kinins on cell surface receptors expression. A, IMR90 cells were treated with 0.1 µM BK or 0.1 µM DAKD for 30 min and 120 min at 37°C, washed with low-pH buffer at 4°C, and then assayed for specific [3H]BK or [3H]DAKD binding at 4°C as described under Materials and Methods. The results are shown as percentage of basal, where 100% basal is the specific binding in the absence of agonist treatment. Data are presented as mean ± S.E. of at least five independent experiments. Comparison with the absence of agonist treatment: *, p < 0.05; ***, p < 0.001. B, HEK293 cells stably expressing B1R, B2R, B1CB2, and B2CB1 were treated with 1 µM BK (B2R, B2CB1) or 1 µM DAKD (B1R, B1CB2) for 30 min at 37°C. Cells (20,000) were then counted by FACS using APC-conjugated M1 anti-FLAG antibodies as described under Materials and Methods. The results are shown as percentage of basal, where 100% basal refers to the mean fluorescence intensity in the absence of agonist. Data are presented as mean ± S.E. of three independent experiments. Comparison with the absence of agonist treatment: *, p < 0.05; **, p < 0.01, ***, p < 0.001; NS, not significant. C, amino acid sequences of the C-terminal domains of the human B1R, B1stop320, B2R. Indicated is the predicted junction of the seventh transmembrane domain and the C-terminal domain (vertical line), conserved residues between the receptor subtypes (bold italics), and residues shown to be phosphorylated in vivo (Blaukat et al., 2001) (star).

Receptor-Mediated -Arrestin Recruitment. -Arrestin is a key GPCR regulatory protein that is rapidly recruited from the cytosol to the plasma membrane receptor upon agonist stimulation and involved in both receptor desensitization and internalization. Agonist stimulation of B₂R led to -arrestin 2 recruitment to the plasma membrane within 5 min (Fig. 2, B2 NT, AG). In contrast, no apparent effect on -arrestin 2 distribution was observed either without or with agonist stimulation of B₁R for the same time period (Fig. 2, B1 NT, AG). These results are consistent with the different agonist regulation of these receptors, where B₂R is rapidly phosphorylated at specific serines and threonines in the C-terminal tail (Fig. 1C) (Blaukat et al., 1999, 2001) and desensitized by the agonist (Mathis et al., 1996), whereas B₁R is apparently not phosphorylated (Fig. 1C) (Blaukat et al., 1999) and desensitizes very slowly in response to agonist (Mathis et al., 1996), as well as being constitutively active (Leeb-Lundberg et al., 2001).

View larger version (32K): [in this window] [in a new window]   Fig. 2. Effects of kinins on receptor-mediated -arrestin recruitment. Cells stably expressing GFP--arrestin 2 and transiently transfected to express B1R, B2R, B1CB2, and B2CB1 were incubated in the absence (NT) and presence of 1 µM receptor-specific agonist (AG) for 5 min at 37°C as indicated. Cells were then stained with primary mouse M1 anti-FLAG antibodies and secondary ALEXA488-labeled anti-mouse antibody as described under Materials and Methods. Individual images of receptors (REC) and -arrestin (ARR) and their merged images (MERGE) were collected with a Nikon Eclipse confocal microscope, 60x objective, 50 µm zoom. Bar, 10 µm.

Whole-Cell Distribution of Receptors. We next examined the distribution of the receptors and the tail swaps in the presence and absence of ligand. The whole-cell distribution of each receptor was analyzed by confocal fluorescence microscopy of formaldehyde-fixed and permeabilized cells. Under these conditions, the vast majority of B₂R were located in the plasma membrane in the absence of agonist (Fig. 3A, B2 NT). On the other hand, treatment with 1 µM BK (B2 AG) for 30 min at 37°C promoted the redistribution of receptors to intracellular compartments, although some plasma membrane staining was still observed. Treatment with a 1 µM concentration of the B₂R-specific antagonist icatibant alone had little effect on B₂R distribution (data not shown). In contrast to B₂R, a disperse pattern of B₁R distribution was observed even in the absence of agonist, with receptors localized primarily intracellularly, in both a tubular network and in distinct puncta, and to a lesser degree in the plasma membrane (Fig. 3A, B1 NT). No obvious change in receptor distribution was observed after treatment with 1 µM DAKD (B1 AG) or the B₁R-specific antagonist des-Arg¹⁰-icatibant (data not shown). Thus, the steady-state distribution of B₂R and B₁R are different, with a much greater intracellular pool of B₁R. The tubular network may represent slow B₁R maturation, whereas the distinct puncta may represent constitutive B₁R endocytosis (see below). B1CB2 exhibited both basal and agonist-promoted distribution patterns (Fig. 3A, B1CB2 NT, AG) that were very similar to the B₂R. Likewise, the B2CB1 distribution patterns in both the presence and absence of agonist (B2CB1 NT, AG) were very similar to B₁R. Thus, the receptor C-terminal tails seem to be involved in both receptor maturation and endocytosis.

View larger version (61K): [in this window] [in a new window]   Fig. 3. Whole-cell receptor distribution and constitutive and kinin-dependent receptor endocytosis as determined using confocal microscopy. A, cells stably expressing B2R, B1R, B2CB1, and B1CB2 were incubated in the absence (NT) or presence of 1 µM receptor-specific agonist (AG) for 30 min at 37°C as indicated. Cells were then fixed and permeabilized before incubation with primary mouse M1 anti-FLAG antibody and secondary ALEXA488-labeled anti-mouse antibody as described under Materials and Methods. B, cells stably expressing B2R, B1R, B2CB1, and B1CB2 were incubated with primary mouse M1 anti-FLAG antibody followed by incubation in the absence (NT) or presence of 1 µM receptor-specific agonist (AG) for 30 min at 37°C as indicated. Cells were then fixed, permeabilized, and stained with secondary ALEXA488-coupled anti-mouse antibody as described under Materials and Methods. In both A and B, images were collected using a Nikon Eclipse confocal microscope, 60x objective, 50 µm zoom. Bar, 10 µm.

Constitutive and Kinin-Dependent Endocytosis of Receptors. To specifically address receptor endocytosis without the interference of receptor maturation, we used two assays that specifically monitor the fate of the plasma membrane pools of receptors. The first assay was an immunofluorescence confocal microscopy assay in which live cells are "fed" primary antibody to label surface receptors. The second assay was a quantitative biochemical assay, the biotinylation protection assay (BPA). The immunofluorescence assay takes advantage of the extracellular FLAG epitope tag on the receptors to monitor selectively the movement of cell surface receptors. Live cells were first incubated with M1 anti-FLAG antibody in the absence (NT) or presence of agonist (AG) for 30 min at 37°C. The cells were then fixed, permeabilized, and incubated with secondary antibody to detect receptor-antibody complexes. In the absence of agonist, B₂R were located almost exclusively in the plasma membrane (Fig. 3B, B2 NT). Incubation with agonist at 1 µM promoted endocytosis of the B₂R-antibody complexes (B2 AG), which was blocked by coincubation with a 5-fold excess (5 µM) of antagonist (data not shown). On the other hand, spontaneous agonist-independent endocytosis of the B₁R-antibody complexes was found to occur (Fig. 3B, B1 NT). No apparent change in B₁R distribution was observed when cells were treated with the agonist at 1 µM either without (B1 AG) or with a 5-fold excess (5 µM) of antagonist (data not shown). It is noteworthy that the tubular distribution of B₁R that was seen in Fig. 3A was absent using this protocol, which strongly argues that the intracellular pool of B₁R is a combination of both slow receptor maturation and constitutive receptor endocytosis.

In the BPA, cell surface receptors were first selectively labeled with a membrane-impermeable biotin analog. Internalized receptors were then selectively monitored by their protection from stripping by a membrane-impermeable reducing agent. This assay revealed internalization of B₂R only in the presence of agonist (Fig. 4A, compare B2 NT 30 min and B2 AG 30 min), whereas B₁R internalization occurred both in the absence and presence of agonist (Fig. 4A, compare B1 NT 30 min and B1 AG 30 min). These data confirm the results obtained using the immunofluorescence microscopy assay (Fig. 3). It is noteworthy that detailed densitometric analysis of the BPA revealed that significantly less B₁R (43%, p = 0.02) was internalized in the presence of agonist than in the absence of agonist at 30 min (Fig. 4B). Thus, the B₁R is subject to constitutive endocytosis but the agonist delays this event. In contrast, the B₂R resides in the plasma membrane until it is exposed to agonist, at which point it rapidly internalizes. The relatively low amount of internalized B₂R recovered in this assay may be explained in part by the extraction conditions used. However, other factors are probably also involved, because B1CB2, which behaved similarly to B₂R, was significantly easier to extract.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Constitutive and kinin-dependent receptor endocytosis as determined by BPA. A, cells stably expressing B2R, B1R, and B1CB2 were first labeled at 4°C with a biotin reagent containing a reducible linker (TOTAL). The cells were then either stripped directly of remaining surface biotin (STRIP) or incubated in absence (NT) or presence of 1 µM receptor-specific agonist (AG) for 30 min at 37°C as indicated and then stripped. The receptors were then immunoprecipitated as described for biotinylation protection assay under Materials and Methods. Blots were visualized by chemiluminescence in a Fluoromax Image Analyzer using Quantity One software (Bio-Rad Laboratories). B, densitometric analysis of the results in A. The results are shown as percentage of total where 100% total refers to the density of biotinylated cell surface receptors at 4°C in the absence of agonist (TOTAL). Data are presented as mean ± S.E. from three independent experiments. Comparison between the absence and presence of agonist: *, p < 0.05; **, p < 0.01; ***, p < 0.005.

B1CB2 was located exclusively in the plasma membrane in the absence of agonist, whereas incubation with agonist led to rapid endocytosis of the construct as evidenced by both the immunofluorescence assay (Fig. 3B, B1CB2 NT, AG) and BPA (Fig. 4, A and B). Thus, B1CB2 gains the endocytic features of B₂R, which is consistent with introduction of both agonist-promoted -arrestin recruitment (Fig. 2) and agonist-promoted decrease in the cell surface receptors (Fig. 1B). The immunofluorescence assay revealed that B2CB1 is constitutively internalized (Fig. 3B). This constitutive endocytosis, like that for the B₁R, apparently proceeds by a -arrestin 2-independent mechanism, because this receptor does not recruit -arrestin 2 in either the presence or the absence of agonist (Fig. 2). The low level of expression of this construct in the stable cell lines prohibited BPA analysis. We were thus unable to determine whether agonist decreased receptor endocytosis of the B2CB1, as it does for the B₁R (Fig. 4). However, the failure of BK to increase the number of accessible cell surface B2CB1 (Fig. 1B) suggests that the agonist does not delay the endocytosis of this receptor.

Inhibition of Receptor Endocytosis by Hyperosmotic Sucrose Treatment. Treatment of cells with hyperosmotic sucrose treatment has been shown to yield abnormal clathrin polymerization resulting in empty microcages in the membrane (Heuser and Anderson, 1989). Pretreatment of cells stably expressing B₁R with 0.4 M sucrose for 60 min led to a 31% increase in the number of available B₁R on the cell surface (Fig. 5A). Confocal microscopy of cells incubated with primary antibody during the last 30 min of the sucrose treatment showed that the increase in B₁R was due to inhibition of constitutive receptor endocytosis (Fig. 5B). On the other hand, treatment with 5 mM methyl--cyclodextrin, which depletes the cells of cholesterol and perturbs lipid rafts, had no apparent effect on B₁R endocytosis (data not shown). Hyperosmotic sucrose treatment had no effect on the number of cell surface B₂R (Fig. 5A) or their localization in the plasma membrane (Fig. 5B). However, sucrose completely inhibited the agonist-promoted B₂R endocytosis (Fig. 5B). Thus, both constitutive B₁R and agonist-promoted B₂Rendocytosis proceed through a clathrin-dependent pathway.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Inhibition of receptor endocytosis by hyperosmotic sucrose treatment. A, HEK293 cells stably expressing B1RorB2R were treated without or with 0.4 M sucrose in DMEM for 60 min at 37°C and then assayed for specific [3H]DAKD or [3H]BK binding at 4°C as described under Materials and Methods. The results are shown as percentage of Basal where 100% Basal is the specific binding in the absence of sucrose treatment. Data are presented as mean ± S.E. of at least three independent experiments. Comparison with the absence of sucrose treatment: *, p < 0.05. B, cells stably expressing B2R and B1R were treated with 0.4 M sucrose in DMEM for 60 min at 37°C. During the last 30 min of the 60-min incubation, the cells were also incubated with primary mouse M1 anti-FLAG antibody without (NT) and with 1 µM receptor-specific agonist (AG). Cells were then fixed, permeabilized, and stained with secondary ALEXA488-coupled anti-mouse antibody as described under Materials and Methods. In both A and B, images were collected using a Nikon Eclipse confocal microscope, 60x objective, 50 µm zoom. Bar, 10 µm.

View larger version (43K): [in this window] [in a new window]   Fig. 6. Postendocytic sorting of receptors as determined by confocal microscopy. Cells stably expressing B2R, B1R, B1CB2, B2CB1, and B1Stop320 were incubated with primary mouse M1 anti-FLAG antibody for 30 min followed by further incubation in the absence (NT) or presence of 1 µM receptor-specific agonist (AG) for 30 min at 37°C, upon which they were washed with EDTA-containing medium (NT STRIP, AG STRIP) to strip the surface of remaining antibody. The cells treated with agonist and stripped were then incubated in medium for 30 min (AG STRIP NT). Cells were then fixed, permeabilized, and stained with secondary ALEXA488-labeled anti-mouse antibody as described under Materials and Methods. Images were collected using a Nikon Eclipse confocal microscope, 60x objective, 50 µm zoom. Bar, 10 µm.

To determine whether B₁R enters a degrading pathway, we first used the BPA, which selectively monitors the stability of internalized receptors. Clearly, the internalized B₁R were significantly degraded between the 30- and 120-min time points in both the absence (Fig. 7, NT) (p < 0.05) and presence of agonist (AG) (p < 0.01). The degrading phenotype of B₁R was further investigated by colocalization of receptors with LAMP 1 and LAMP 2, which are markers for the endstage compartments of the degrading pathway. B₁R showed an overlap with LAMP 1 and LAMP 2 at 120 min both without (Fig. 8, B1 NT) (29%, n 25 cells) and with agonist treatment (B1 AG) (25%, n 25 cells) that was much higher than for B₂R without (B2 NT) (0.01%, n 25 cells, p < 0.001) and with agonist (B2 AG) (0.66%, n 25 cells, p < 0.001) at this time point. Together these data demonstrate that the B₂R is targeted to a recycling pathway, whereas the B₁Ris targeted to a degrading pathway.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Postendocytic degradation of intracellular B1 receptors. A, cells stably expressing B1R were first labeled at 4°C with a biotin reagent (TOTAL). The cells were then either stripped directly of remaining surface biotin (STRIP) or incubated in absence (NT) or presence of 1 µM DAKD (AG) for 30 and 120 min at 37°C as indicated as indicated and then stripped. The receptors were then immunoprecipitated as described for biotinylation protection assay under Materials and Methods. Blots were visualized by chemiluminescence in a Fluoromax Image Analyzer using Quantity One software (Bio-Rad Laboratories). B, densitometric analysis of the results in A. The results are shown as percentage of Total where 100% total refers to the density of biotinylated cell surface receptors at 4°C in the absence of agonist (TOTAL). Data are presented as mean ± S.E. from three independent experiments. Comparison between the absence and presence of agonist: *, p < 0.05; **, p < 0.01.

View larger version (18K): [in this window] [in a new window]   Fig. 8. Colocalization of receptors with lysosomal markers as determined by confocal microscopy. Cells stably expressing B2R, B1R, B2CB1, and B1CB2 were incubated with primary mouse M1 anti-FLAG antibody in absence (NT) or presence of 1 µM receptor-specific agonist (AG) for 60 min at 37°C as indicated. Cells were then fixed, permeabilized, and stained with primary mouse anti-lamp 1 and anti-lamp 2 antibodies and then specifically stained with secondary ALEXA568-labeled anti-mouse antibody for LAMP 1 and LAMP 2 (LAMP 1 LAMP 2) and secondary ALEXA488-labeled anti-mouse antibody for receptors (REC). Images collected using a Nikon Eclipse confocal microscope, 60x objective, 50 µm zoom. Bar, 10 µm. B, quantification of colocalized receptors with LAMP 1 and 2 (MERGE). The results are shown as percentage of total receptors. Data are presented as mean ± S.E. from at least 25 different cells.

Interaction of Receptors with GASP. GASP is one candidate molecule responsible for preferentially targeting B₁R to the lysosomal degrading pathway as opposed to the plasma membrane recycling pathway. We have previously established that this protein specifically interacts with the intracellular C-terminal tail of some GPCRs targeted for destruction by lysosomal proteolysis (Whistler et al., 2002; Bartlett et al., 2005). Because the B₁R was degraded after its endocytosis whereas the B₂R was not, we examined whether GASP binds specifically to the former receptor.

A difference in GASP1 binding to B₁R and B₂R was first evaluated using in vitro affinity assays with in vitro-translated GASP1 and GST fusion proteins corresponding to the C-terminal tails of B₂R and B₁R beyond the NPXXY motif. GASP1 showed at least a 5-fold higher affinity for the B₁R tail than for the B₂R tail (Fig. 9A). Furthermore, the binding of GASP1 to the B₁R tail was directly dependent on the concentration of the tail in the in vitro affinity assays. The binding preference of GASP1 for B₁R was also evaluated in vivo in HEK293 cells stably expressing each receptor. Immunoprecipitation of receptors with M1 anti-FLAG antibodies revealed that endogenous GASP1 bound to the B₁R but not the B₂R (Fig. 9B). Thus, GASP1 exhibits a clear preference for B₁R over B₂R. Substituting the B₂R C-terminal tail in the B1CB2 receptor decreased the binding of GASP1 by 50% (Fig. 9B). Thus, GASP1 binding to B₁R involves the C-terminal tail of the receptor, but another epitope(s) also seems to be involved. Such multiepitopal binding seems to common for several GPCR effector proteins, including -arrestins.

View larger version (29K): [in this window] [in a new window]   Fig. 9. Binding of GASP to receptors. A, in vitro-translated and radioactively labeled GASP1 was incubated with GST-fusion constructs of the B1R and B2R C-terminal tails as described under Materials and Methods. The receptor tails were quantified by Coomassie staining and GASP1 by immunoblotting. The relative amounts of pulled-down GASP1 by the receptor tails were quantified by densitometric analysis. B, cells stably expressing B1R, B2R, and B1CB2 were immunoprecipitated with M2 anti-FLAG affinity resin as described under Materials and Methods. Nontransfected HEK293 were used as negative control. The receptors and GASP1 were quantified by immunoblotting. The relative amounts of pulled-down GASP1 by the receptors were quantified by densitometric analysis. In A and B, the results are presented as percentage of B1 where 100% B1 refers to the amount of GASP1 pulled down with the B1R. In B, the results were normalized to the amount of GASP1 in the lysate. Data are presented as mean ± S.E. from three independent experiments.

Effect of Kinins on Total Receptor Stability. The above results suggest that kinins increase available cell surface B₁R by delaying constitutive receptor endocytosis and consequently delay their degradation. In contrast, kinins acutely decrease available B₂R by promoting endocytosis, but they do not promote down-regulation of this receptor because they enter an endocytic recycling pathway. Thus, kinins would be expected to increase the stability of the total cell surface pool of B₁R. To examine this hypothesis, a biotin degradation assay was used, which monitors the stability of the total pool of mature cell surface receptors. At 120 min, 29 ± 4% of the B₁R originating on the plasma membrane remained intact (Fig. 10, NT120min), whereas the presence of agonist significantly increased this amount to 56 ± 14% at this time point (p = 0.03) (AG120min). B1CB2 was more stable than B₁R at 120 min, and agonist did not have any significant effect on the B1CB2 receptor stability. The stability of B2CB1 at 120 min was similar to B₁R but agonist did not have any effect on the B2CB1 receptor stability, which is consistent with the failure of the B₁R C-terminal tail to transplant the agonist delay of constitutive endocytosis to B₂R. Agonist was unable to delay the degradation of B1Stop320. On the other hand, truncation of the B₁R C-terminal tail led to a significant decrease in the degradation of the receptor. Thus, the B₁R tail contains an epitope that participates in targeting the receptor for degradation but additional epitopes are likely to be involved as well.

View larger version (32K): [in this window] [in a new window]   Fig. 10. Postendocytic degradation of total cell surface receptors. A, cells stably expressing B1R, B1stop320, B1CB2, or B2CB1 were first labeled at 4°C with a biotin reagent containing a reducible linker (TOTAL). The cells were then incubated in absence (NT) or presence of 1 µM receptor-specific agonist (AG) for 120 min at 37°C as indicated. The receptors were then immunoprecipitated as described for biotinylation degradation assay under Materials and Methods. Blots were visualized by chemiluminescence in a Fluoromax Image Analyzer using Quantity One software (Bio-Rad Laboratories). B, densitometric analysis of the results in A. The results are shown as percentage of total, where 100% total refers to the density of biotinylated cell surface receptors at 4°C in the absence of agonist (TOTAL). Data are presented as mean ± S.E. from three independent experiments. Comparison between the absence and presence of agonist: *, p < 0.05; NS, not significant.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Here, we used a HEK293 cell model to elucidate the trafficking mechanism underlying the relatively rapid kininpromoted decrease of B₂R and increase of B₁R on the cell surface seen in native cells. In our model, B₂R are relatively stable cell surface receptor components in the absence of agonist but translocate rapidly and in a clathrin-dependent manner to intracellular endocytic vesicles in response to agonist stimulation with the cognate agonist ligand BK and recycle back to the plasma membrane. In contrast, B₁R are subject to constitutive endocytosis, also via a clathrin-dependent pathway, and target lysosomes for degradation. Binding of the cognate B₁R agonist DAKD inhibits constitutive B₁R endocytosis thereby decreasing the rate of spontaneous B₁R clearance from the cell surface and delaying degradation. This regulation is also consistent with the agonist-dependent and constitutive activities of these receptors and the pattern of inflammatory kinin signaling during sustained pathological insult in vivo, which adapts from a B₂-type to a B₁-type during the course of the insult. Furthermore, delaying spontaneous endocytosis to temporarily increase the steady-state level of receptors on the cell surface presents a novel mechanism by which some agonists may act to promote GPCR signaling.

B₂R is expressed almost exclusively in the plasma membrane, whereas B₁R is retained to a large degree intracellularly. The tubular shape of the receptor-retaining intracellular structures suggests a membrane-bound localization of the receptor rather than a diffuse cytosolic localization, which presumably reflects the biosynthetic pathway. PNGase treatment of biotinylated cell surface B₁R led to the conversion of higher molecular mass receptor species to a relatively homogeneous species of approximately 35 kDa consistent with the idea that the cell surface receptor is glycosylated as suggested previously (Fortin et al., 2006). The difference in distribution of B₁R and B₂R was apparently determined entirely by the C-terminal tail. Thus, the B₂R C-terminal tail may be equipped with a plasma membrane targeting motif or the B₁R tail with an intracellular retention motif. Whether or not the intracellularly retained B₁R species can be recruited to the plasma membrane is unclear. However, considering the rapid spontaneous endocytosis of the receptor once it reaches the cell surface and the postendocytic targeting of the receptor for destruction described here, the retained receptor pool could possibly be viewed as a deposit to ensure that a steady flow of receptors reach maturation once expression of the receptor has been induced by a pro-inflammatory stimuli.

The rapid agonist-promoted recruitment of -arrestin by B₂R, clathrin-dependent endocytosis, and the ability of this receptor to recycle after agonist-promoted endocytosis are consistent with previous studies. Indeed, agonist exposure of the B₂R leads, within minutes, to internalization of the receptor protein, which has been determined by immunoelectron microscopy (Haasemann et al., 1998), fluorescence microscopy (Bachvarov et al., 2001), the appearance of acidresistant radiolabeled agonist binding (Munoz and Leeb-Lundberg, 1992), and the reduction in the number of cell surface binding sites, which rapidly reappear on the cell surface upon agonist removal (Munoz et al., 1993). The fact that the internalized agonist reaches a steady-state level over time that is higher than can be explained by the total number of cell surface binding sites (Lamb et al., 2001) further supports our finding that the internalized B₂R recycles to the plasma membrane.

Unlike the B₂R, the plasma membrane B₁R is unstable in that it is subject to high spontaneous endocytosis and degradation. This observation is consistent with the inducible expression and high agonist-independent activity reported for this receptor (Leeb-Lundberg et al., 2001). In other words, the functional properties of this receptor necessitate a tight regulation at every level from transcription, through maturation and signaling, and ending in lysosomal targeting and destruction. Our results are also consistent with the rapid and spontaneous clearance of the B₁R response in rabbit aortic rings (Fortin et al., 2003). On the other hand, our observation apparently contradicts the results from the same group using a C-terminally GFP-tagged B₁R, which showed neither spontaneous nor agonist-promoted internalization, even though the receptor was reported to relocate on the plasma membrane in response to agonist (Sabourin et al., 2002). It is important to realize that the C-terminal tail of the receptor has been shown to be crucial for the trafficking properties of a number of GPCR (von Zastrow, 2003; Heydorn et al., 2004; Terrillon et al., 2004), which may be particularly apparent with spontaneous endocytosis (Waldhoer et al., 2003).

Previous studies have shown that the kinetics and amount of agonist internalization of the B₁R, as determined by the appearance of acid-resistant DAKD binding, is very slow and low, which is opposite to the B₂R, through which acid-resistant BK binding occurs very rapidly (Faussner et al., 1998; Lamb et al., 2001). This, at first, would seem to support a model of a noninternalizing receptor. However, as we present here, agonist binding to the B₁R in fact retards the rapid spontaneous internalization of this receptor, thereby temporarily stabilizing the receptor on the cell surface. The sucrose sensitivity of both constitutive and agonist-dependent B₁R endocytosis suggests a common endocytic pathway under the two conditions but that the agonist promotes a receptor conformation that has lower affinity for the endocytic machinery. Thus, the combined effect of spontaneous surface clearance of the B₁R and the retardation of the clearance by the agonist may then be viewed as a coincidence detector that would only allow optimal receptor signal transduction at the precise right moment.

The B₂R phenotype of agonist-dependent -arrestin 2 recruitment and sucrose-sensitive internalization support an internalization pathway well in line with a majority of the GPCR studied to date. On the other hand, the B₁R still lacks an identified adaptor protein that links it to the internalization machinery. Several receptors, such as GluR1 (Dale et al., 2001) and PAR-1 (Paing et al., 2002) have been reported to internalize by a clathrin-dependent but -arrestin-independent mechanism. The mechanisms underlying B₁R internalization, therefore, need further investigation for us to understand the regulation of receptor level and hence tissue responsiveness to receptor agonists.

Because most of the trafficking phenotypes of B₂R and B₁R were transplantable by C-terminal tail exchange, this domain must be important for the unique trafficking protein interactions. Substitution of the B₁R tail in B₂R yielded a loss of -arrestin 2 binding, which may be explained by the apparent absence of phosphorylation of this tail (Blaukat et al., 1999), in contrast to the B₂R tail, which is phosphorylated (Blaukat et al., 2001). The importance of the B₂R C-terminal tail for agonist-promoted endocytosis was confirmed when substituted in B₁R. In contrast to B₂R, the B₁R tail conferred constitutive endocytosis to B₂R, a process that must then be -arrestin 2-independent. The B₁R tail was unable to transplant the agonist delay in constitutive endocytosis to the B₂R. However, B₁R tail truncation also lost the agonist delay suggesting that this delay in endocytosis involves a fine interplay between the C-terminal tail and other domains in the receptor.

The receptor tails are also important for the postendocytic choices of the receptors. Lysosomal B₁R targeting may depend in part on GASP binding, which showed higher affinity for the B₁R tail than the B₂R tail both in vitro and in vivo. These results are consistent with the fact that substitution of the B₁R tail in B₂R prevented the receptor from recycling and promoted degradation. Furthermore, truncation of the B₁R tail decreased the rate of receptor degradation but did not abolish it, indicating that additional domains are involved. Therefore, the B₂R tail either contains an epitope important for recycling or is able to mask an epitope in B1R that is important for degradation.

The combined events of kinin-promoted rapid B₂R desensitization and internalization, together with the recruiting effect of kinins and their limited desensitizing potential on the induced B₁R, provide a mechanism for the inverse kinin regulation of these receptors observed in cultured cells and agree well with the prevailing paradigm of an acute inflammatory B₂-type response that over time shifts toward a chronic B₁-type response. Chronic phase B₁R signaling has attracted a lot of attention as a drug target for chronic pain management. Compounds have been, and are being, screened for their ability to block the ligand-independent signal transduction of this receptor, so called inverse agonists, but no candidate drugs are of yet available. Our data in the present study favor an increased rate of B₁R clearance as a means to reduce B₁R activity. Hence, we suggest screening for endocytic agonist properties rather than signal transduction inverse agonist properties as a novel approach to evaluate "antagonism" at B₁R.

	Acknowledgements

 
We thank Margareta Pusch and Joanna Daszkiewicz-Nilsson for expert technical assistance, and Dr. Marc Caron for kindly providing GFP--arrestin 2.

	Footnotes

 
This work was supported in part by funds from the Swedish Research Council grant 15057, AlfredÖsterlunds Stiftelse, Torsten och Ragnar Söderbergs Stiftelser, the Faculty of Medicine at Lund University in part through the Vascular Wall and Blood and Defense prioritized programs, National Institutes of Health grant GM41659 (to L.M.F.L.-L.), and funds provided from the State of California for medical research on alcohol and substance abuse through the University of California San Francisco (to J.L.W.).

ABBREVIATIONS: GPCR, G protein-coupled receptors; B₁R, B₁ receptor; B₂R, B₂ receptor; GASP, GPCR-associated sorting protein; HEK, human embryonic kidney; FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; APC, allophycocyanin; TBS, Tris-buffered saline; DAKD, desArg¹⁰kallidin; BK, bradykinin; GST, glutathione transferase; BPA, biotinylation protection assay; PNGase, peptide N-glycosidase F.

Address correspondence to: L.M. Fredrik Leeb-Lundberg, Department of Experimental Medical Science, Lund University, BMC, A12, S-22184 Lund, Sweden. E-mail: fredrik.leeb-lundberg{at}med.lu.se

	References

Top Abstract Materials and Methods Results Discussion References

 
Bachvarov D, Hess JF, and Marceau F (1998) The B1 receptors for kinins. Pharmacol Rev 50: 357-386.[Abstract/Free Full Text]

Bachvarov DR, Houle S, Bachvarova M, Bouithillier J, Adam A, and Marceau F (2001) Bradykinin B2 receptor endocytosis, recycling, and down-regulation assessed using green fluorescent protein conjugates. J Pharmacol Exp Therap 297: 19-26.[Abstract/Free Full Text]

Barak LS, Ferguson SSG, Zhang J, and Caron MG (1997) A beta-arrestin/green fluorescent protein biosensor for detecting G protein-coupled receptor activation. J Biol Chem 272: 27497-27500.[Abstract/Free Full Text]

Bartlett SE, Enquist J, Hopf FW, Lee JH, Gladher F, Kharazia V, Waldhoer M, Mailliard WS, Armstrong R, Bonci A, and Whistler JL (2005) Dopamine responsiveness is regulated by targeted sorting of D2 receptors. Proc Natl Acad Sci USA 102: 11521-11526.[Abstract/Free Full Text]

Bengtsson S, Phagoo SB, Norrby-Teglund A, Påhlman L, Zuraw BL, Leeb-Lundberg LMF, and Herwald H (2006) Kinin receptor expression during Staphylococcus aureus infection. Blood 108: 2055-2063.[Abstract/Free Full Text]

Blaukat A, Herzer K, Schroeder C, Bachmann M, Nash N, and Müller-Esterl W (1999) Overexpression and functional characterization of kinin receptors reveal subtype-specific phosphorylation. Biochemistry 38: 1300-1309.[CrossRef][Medline]

Blaukat A, Pizard A, Breit A, Wernstedt C, Ahlenc-Gelas F, Muller Esterl W, and Dikic I (2001) Determination of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function. J Biol Chem 276: 40431-40440.[Abstract/Free Full Text]

Cao TT, Deacon HW, Reczek D, Bretscher A, and von Zastrow M (1999) A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor. Nature (Lond) 401: 286-290.[CrossRef][Medline]

Chini B and Parenti M (2004) G-protein coupled receptors in lipid rafts and caveolae: how, when and why do they go there? J Mol Endocrinol 32: 325-3381.[Abstract]

Dale LB, Bhattacharya M, Seachrist JL, Anborgh PH, and Ferguson SSG (2001) Agonist-stimulated and tonic internalization of metabotropic glutamate receptor 1a in human embryonic kidney 293 cells: agonist-stimulated endocytosis is -arrestin1 isoform-specific. Mol Pharmacol 60: 1243-1253.[Abstract/Free Full Text]

Dray A and Perkins M (1993) Bradykinin and inflammatory pain. Trends Neurosci 16: 99-104.[CrossRef][Medline]

Faussner A, Proud D, Towns M, and Bathon JM (1998) Influence of the cytosolic carboxyl termini of human B1 and B2 kinin receptors on receptor sequestration, ligand internalization, and signal transduction. J Biol Chem 273: 2617-2623.[Abstract/Free Full Text]

Ferguson SS (2001) Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 53: 1-24.[Abstract/Free Full Text]

Finn AK and Whistler JL (2001) Endocytosis of the mu opioid receptor reduces tolerance and a cellular hallmark of opiate withdrawal. Neuron 32: 829-839.[CrossRef][Medline]

Fortin J-P, Bouthillier J, and Marceau F (2003) High agonist-independent clearance of rabbit kinin B1 receptors in cultured cells. Am J Physiol 284: H1647-H1654.

Fortin J-P, Dziadulewicz EK, Gera L, and Marceau F (2006) A nonpeptide antagonist reveals a highly glycosylated state of the rabbit kinin B1 receptor. Mol Pharmacol 69: 1146-1157.[Abstract/Free Full Text]

Gaborik Z and Hunyady L (2004) Intracellular trafficking of hormone receptors. Trends Endocrinol Metab 15: 286-293.[CrossRef][Medline]

Haasemann M, Cartaud J, Muller-Esterl W, and Dunia I (1998) Agonist-induced redistribution of bradykinin B₂ receptor in caveolae. J Cell Sci 111: 917-928.[Abstract]

Hall RA, Premont RT, Chow C, Blitzer JT, Pitcher JA, Claing A, Stoffel RH, Barak LS, Shenolikar S, Weinman EJ, Grinstein S, and Lefkowitz RJ (1998) The beta2-adrenergic receptor interacts with the Na⁺/H⁺-exchanger regulatory factor to control Na⁺/H⁺ exchange. Nature (Lond) 392: 626-630.[CrossRef][Medline]

Heuser JE and Anderson RG (1989) Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J Cell Biol 108: 389-400.[Abstract/Free Full Text]

Heydorn A, Sondergaard BP, Ersboll B, Holst B, Nielsen FC, Haft CR, Whistler JL, and Schwartz TW (2004) A library of 7TM receptor C-terminal tails. Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP). J Biol Chem 279: 54291-54303.[Abstract/Free Full Text]

Kang DS and Leeb-Lundberg LMF (2002) Negative and positive regulatory epitopes in the C-terminal domains of the human B1 and B2 bradykinin receptor subtypes determine receptor coupling efficacy to G_q-mediated phospholipase C activity. Mol Pharmacol 62: 281-288.[Abstract/Free Full Text]

Lamb ME, de Weerd WFC, and Leeb-Lundberg LMF (2001) Agonist-promoted trafficking of human bradykinin receptors: arrestin- and dynamin-independent sequestration of the B2 receptor in HEK293 cells. Biochem J 355: 741-750.[Medline]

Leeb T, Mathis SA, and Leeb-Lundberg LMF (1997) The sixth transmembrane domains of the human B1 and B2 bradykinin receptors are structurally compatible and involved in discriminating between subtype-selective agonists. J Biol Chem 272: 311-317.[Abstract/Free Full Text]

Leeb-Lundberg LMF, Kang DS, Lamb ME, Mathis SA, and Fathy DB (2001) The human B1 bradykinin receptor exhibits high ligand-independent, constitutive activity: roles of residues in the fourth intracellular and third transmembrane domains. J Biol Chem 276: 8785-8792.[Abstract/Free Full Text]<