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Division of Molecular Neurobiology, Department of Physiological Sciences (D.S.K., C.G., L.M.F.L.-L.) and Division of Molecular Pathogenesis, Department of Cell and Molecular Biology (M.M.), Lund University, Lund, Sweden
Abstract
The human B1 bradykinin receptor is an inducible and constitutively active G protein-coupled receptor that is involved in the inflammatory and pain responses to injury. Here, we investigated the role of B1 receptor homo-oligomerization in cell surface receptor expression. B1 receptors tagged with either the FLAG or hemagglutinin epitope were monitored immunologically and by radio-ligand binding, biotinylation, and phosphoinositide hydrolysis in human embryonic kidney 293 cells. Selective immunoprecipitation, immunoblotting, and immunoelectron microscopy with epitope-specific antibodies together provided evidence for constitutively formed cell surface receptor homo-oligomers. Truncation of the receptor from the N- and C-terminal ends indicated that the epitope for oligomerization seems to be located between Leu26 on top of transmembrane helix 1 and Val71 at the bottom of helix 2. A receptor construct terminating at Asp134 at the bottom of helix 3, B1stop135, was expressed in the cell. It is interesting that this construct behaved as a dominant-negative mutant by competitively preventing formation of intact B1 receptor homo-oligomers, and redistributing B1 receptors from the cell surface to a common intracellular compartment. In contrast, expression of a construct containing the residues downstream of Asp134, B1del(2-134), was inactive in this regard. Together, these results are consistent with a mechanism where constitutive B1 receptor homooligomerization is required for expression of receptors on the cell surface and subsequent constitutive receptor signaling. This may be a novel mechanism by which the cell regulates the presentation of this constitutively highly active receptor at various stages of injury.
The functional significance of receptor homo-oligomerization has been extensively studied in the receptor tyrosine kinase family. Considerably less is known about oligomerization of G protein-coupled receptors (GPCRs). Nevertheless, recent data seriously challenge the 1:1:1 stoichiometry originally thought to exist in the agonist-GPCR-G protein interaction (Rios et al., 2001; Angers et al., 2002; George et al., 2002). The crystal structure of the metabotropic glutamate receptor mGluR1 N-terminal domain shows that the ligand binding pocket in this domain is a dimer (Kunishima et al., 2000). Furthermore, infrared laser atomic force microscopy of mouse retinal disc membranes illustrates that the native arrangement of rhodopsin is as a dimer and possibly higher order arrays (Fotiadis et al., 2003; Liang et al., 2003).
Coimmunoprecipitation and fluorescence or bioluminescence energy transfer of differentially tagged receptors expressed in transfected cell systems have been useful in identifying GPCR oligomerization (Rios et al., 2001; Angers et al., 2002; George et al., 2002). Some of these studies point toward the importance of receptor homo-oligomers in early receptor biosynthesis and in trafficking of receptors to the cell surface. On the other hand, little is known about how such oligomeric units are regulated structurally and functionally by agonists and other factors during the receptor life cycle.
In the model based on the rhodopsin arrays (Liang et al., 2003), it was concluded that intradimer contacts probably exist between transmembrane helices 4 and 5 with some interactions also provided by helices 1 and 2. Cross-linking studies with the D2 dopamine receptor have directly implicated a symmetric dimer interface in helix 4 (Guo et al., 2003). The same conclusion was drawn from cross-linking in the C5a receptor, which also inferred a symmetric interface in helix 1 and 2 (Klco et al., 2003). A role for helix 1 in receptor dimerization was also provided by fluorescence resonance energy transfer using the 1B-adrenergic receptor (Carrillo et al., 2003) and the yeast factor receptor (Overton et al., 2003).
Kinins are potent agonists who participate in the inflammatory and pain responses to insults by acting through two receptor subtypes named B1 and B2 (Couture et al., 2001). The B2 receptor is expressed constitutively and thought to mediate kinin responses under nonpathological and shortterm responses. Sustained insult leads to an induction of B1 receptors from essentially a null level, which is triggered by proinflammatory cytokines (Marceau et al., 1998) and important in the long-term and pathological response (Dray and Perkins, 1993; Pesquero et al., 2000). B1 receptor induction may be necessary to prolong the kinin response because it has been observed that the B2 receptor desensitizes rapidly, whereas the B1 receptor response is sustained (Mathis et al., 1996). Indeed, strict regulation of B1 receptor expression is probably critical because this receptor is constitutively highly active (i.e., active in the absence of agonist) (Leeb-Lundberg et al., 2001; Kang and Leeb-Lundberg, 2002).
The mechanism underlying B1 receptor induction lays in part at the level of mRNA synthesis (Menke et al., 1994). Changes in mRNA stability may also participate in receptor induction (Zhou et al., 1999). We hypothesized that B1 receptor induction may in part also involve changes in receptor maturation and trafficking to the cell surface. To begin to address this, we investigated receptor homo-oligomerization. Herein, we provide evidence that B1 receptors constitutively homo-oligomerize. Using a truncated receptor construct with dominant-negative behavior, we show that homo-oligomerization is apparently necessary for the expression of constitutively signaling B1 receptors on the cell surface. This may be a novel mechanism of regulating the cellular presentation of this constitutively highly active receptor.
Materials and Methods
Cell Culture, Mutation, and Transfection. HEK293 human embryonic kidney cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated horse serum in 10% CO2 at 37°C. B1 receptor cDNA in pcDNA3 were mutated using a polymerase chain reaction-ligation-polymerase chain reaction protocol as described previously (Kang and Leeb-Lundberg, 2002). The FLAG and hemagglutinin (HA) epitopes were inserted at the receptor N terminus immediately after the initial methionine using synthesized primers containing the epitope sequences. The cells were transiently transfected with varying amounts of DNA using the calcium phosphate precipitate method as described previously (Kang and Leeb-Lundberg, 2002). The total amount of cDNA transfected was always kept constant by supplementing with pcDNA3 vector.
Cell Fractionation. Cells were lysed in ice-cold hypotonic lysis buffer (20 mM HEPES, pH 7.4, 6 mM MgCl2, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 10 e leupeptin) using a Dounce homogenizer. The homogenate was then centrifuged at 1000g for 5 min at 4°C. Sucrose was then added to the supernatant to obtain a final concentration of 0.2 M, and the lysate (1 ml) was layered on top of a discontinuous sucrose step gradient containing 0.5, 0.9, 1.2, 1.35, 1.5, and 2.0 M, each in a volume of 1.5 ml. The gradient was centrifuged for 16 h at 29,000 rpm in a Beckman SW41 rotor (Beckman Coulter, Fullerton, CA). Fractions (0.5 ml) were collected from the bottom of the tube and then immunoblotted with anti-FLAG or anti-HA antibodies. Anti-Na/K-ATPase subunit antibodies (Sigma-Aldrich, St. Louis, MO) were used to identify the plasma membrane fraction.
Functional Receptor Assays. The B1 receptor activity was assayed by monitoring phosphoinositide (PI) hydrolysis in cells transfected with a series of receptor cDNA amounts and labeled with 1 e藽i/ml [myo-3H]inositol (PerkinElmer Life and Analytical Sciences, Boston, MA) as described previously (Kang and Leeb-Lundberg, 2002). The B1 receptor density was determined using saturating concentrations (2-3 nM) of [3H]desArg 10kallidin (PerkinElmer Life and Analytical Sciences). The slope factors of the increases in basal cellular PI hydrolysis and agonist-stimulated PI hydrolysis as a function of the level of receptor expression were used as parameters of constitutive and agonist-dependent receptor activity.
Cell Surface Biotinylation. Cells were biotinylated with 4 ml of 0.3 mg/ml nonpermeable polyethylene oxide-maleimide-activated biotin (Pierce Chemical, Rockford, IL) for 30 min at 4°C, washed two times with ice-cold phosphate-buffered saline (PBS), and quenched with 1 mM dithiothreitol in PBS for 10 min at 4°C. After washing three times with ice-cold PBS, cells were immunoprecipitated and electroblotted as described below. Biotinylated receptors were detected with streptavidin conjugated to horseradish peroxidase (Vector Laboratories, Burlingame, CA).
Immunoprecipitation and Immunoblotting. Cells were subjected to immunoprecipitation and/or immunoblotting essentially as described previously (Kang and Leeb-Lundberg, 2002). In short, cells were solubilized in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 10 mM sodium phosphate, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 10 e/ml leupeptin, and 0.1 mM phenylmethylsulfonyl fluoride) for 30 min at 4°C. The lysate was centrifuged at 13,000g for 15 min at 4°C. For immunoprecipitation, the lysate was incubated 12 to 18 h at 4°C with anti-FLAG M2 antibody (1:300; Sigma-Aldrich), anti-HA antibody (1:300; Covance, Denver, PA), or polyclonal antibodies raised against the B1 receptor C-terminal tail residues 317 to 353 [anti-B1R(317-353); Lamb et al., 2002], followed by incubation with protein A-Sepharose beads with or without precoupled rabbit anti-mouse IgG for an additional 2 h at 4°C. The beads were then washed twice with 1 ml of lysis buffer and then with 1 ml of 10 mM Tris-HCl, pH 7.4. For immunoblotting, samples were dissolved in SDS sample buffer and subjected to SDS-PAGE under reducing conditions (6% -mercaptoethanol) on 12% gels, unless otherwise indicated, electroblotted onto nitrocellulose membranes, and then stained with anti-FLAG M2 antibody (1:1000) or anti-HA antibody (1:1000). Immunoreactive bands were visualized with a chemiluminescence immunodetection kit using peroxidase-labeled sheep anti-mouse antibody according to the procedure described by the supplier (PerkinElmer Life and Analytical Sciences). In some experiments, blots were sequentially stained with the FLAG and HA antibodies. Stripping of blots between antibody staining was done by washing in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, and 100 mM -mercaptoethanol for 45 min at 50°C. The blots were then washed three times for 30 min in Tris-buffered saline. Residual immunoreactivity was checked with immunodetection kits in the absence of primary antibody.
Enzymatic Deglycosylation. To determine the presence of N-glycosylation in the B1 receptor, anti-FLAG or anti-HA immunoprecipitates were treated with 2 units of peptide-N-glycosidase F (PNGase F; Roche Diagnostics, Indianapolis, IN) in 10 mM Tris-HCl, pH 7.4, for 2 h at 37°C. Partially deglycosylated immunoprecipitates were washed with 1 ml of 10 mM Tris-HCl, pH 7.4.
Immunoeletronmicroscopy. HA antibodies were labeled with 5-nm colloidal gold as described previously (Baschong and Wrigley, 1990). Cells expressing HA-tagged B1 receptor were incubated with the gold-labeled antibodies for 30 min at 4°C. The cells were fixed for 1 h at room temperature and then fixed overnight at 4°C in 2.5% glutaraldehyde in 0.15 M sodium cacodylate, pH 7.4 (cacodylate buffer). The fixed samples were washed with cacodylate buffer and postfixed for 1 h at room temperature in 1% osmium tetroxide in cacodylate buffer. The samples were then dehydrated in a graded series of ethanol and embedded in Epon 812 using acetone as intermediate solvent. Specimens were sectioned with a diamond knife into 50-nm-thick ultrathin sections on an LKB ultramicrotome. The sections were then stained with uranyl acetate and lead citrate. Specimens were observed in a Jeol JEM 1230 electron microscope operated at 80-kV accelerating voltage. Images were recorded with a Gatan Multiscan 791 charge-coupled device camera.
Data Analysis. Where indicated, data are presented as the mean ± S.E. and were compared using the Student's t test.
Results
Identification of B1 Receptors. To identify the human B1 receptor (Fig. 1A), the FLAG or HA epitope was inserted at the N-terminal end of the receptor immediately after the initial methionine. The tagged receptors were expressed in HEK293 cells and monitored using epitope-specific antibodies and antibodies against the human B1 receptor C-terminal residues 317 to 353 [anti-B1R(317-353)]. Mock-transfected cells were always included to identify receptor-specific species. Epitope-tagged B1 receptors were expressed and functionally active, as described previously (Leeb-Lundberg et al., 2001; Kang and Leeb-Lundberg, 2002). Immunoprecipitation followed by immunoblotting revealed that both FLAG-tagged B1 receptor (FB1) (Fig. 1B, lane 1) and HA-tagged B1 receptor (HB1) (lane 2) expressed as 35-kDa species, which is close to the theoretical molecular mass of the receptor, and consequently thought to be the monomeric form of the receptor. However, HB1 seemed more heterogeneous than FB1 with additional major bands at about 39 to 45 kDa and a weak band at about 28 kDa (Fig. 1B). Treatment of anti-HA immunoprecipitates with 2 units of PNGase F for 2 h at 37°C resulted in the conversion of the 39- to 45-kDa HB1 species to an approximately 37-kDa rather than a 35-kDa species (Fig. 1C, lane 3). Thus, the complex HB1 pattern is caused to a large extent by receptor N-glycosylation. That the receptor species remained at 37 kDa is probably due either to incomplete N-deglycosylation during the relatively short time period of PNGase F treatment or to the presence of further covalent modification. This receptor contains several serines and threonines in the N-terminal domain and extracellular loops that may be subject to O-glycosylation. To further investigate the role of the N-terminal domain in receptor microheterogeneity, residues 2 to 25 in the B1 receptor were deleted to make B1del(2-25) (Fig. 1A). HB1del(2-25) migrated as a relatively homogeneous species at about the predicted theoretical molecular mass (Fig. 1C, lane 4). On the other hand, HB1stop320, in which the assumed nonglycosylated C-terminal residues 320 to 353 had been deleted in the B1 receptor (Fig. 1A), retained a significant amount of the microheterogeneity observed in HB1 (Fig. 1C, lane 5). We conclude from these results that N-glycosylation occurs in the N-terminal domain, and presumably on Asn14 and/or Asn22. Long exposure of FB1 immunoblots revealed that this receptor displayed microheterogeneity similar to HB1 but to a significantly lesser degree (data not shown). We have previously reported that this heterogeneity is not altered by PNGase F treatment, which also shows that the effect on HB1 was not simply caused by proteolytic degradation during the incubation with the enzyme (Kang et al., 2004). The reason for the difference in the degree or detection of N-glycosylation between the two epitope-tagged versions of the receptor is currently unknown. Possible explanations are that the two differentially tagged receptors become N-glycosylated to different degrees or that the two antibodies exhibit slightly different sensitivity to this type of N-glycosylation.
Constitutive B1 Receptor Homo-oligomers. In addition to a specific apparently monomeric FB1 receptor species at approximately 35 kDa, specific receptor species of relatively higher molecular mass were also present on SDS-PAGE gels (Fig. 2, lanes 4-6). Whereas these species may be receptors tightly associated with some accessory protein, their relative migration suggested that they may be multiples of 35 kDa, and therefore some higher order homo-oligomeric receptor forms, including, e.g., dimers, trimers, etc. Treatment of cells with iodoacetamide to block free sulfhydryl groups had no effect on the receptor migration pattern either in the absence (Fig. 2, lane 4) or presence (lane 5) of reducing agent. Thus, if these B1 receptor species represent homo-oligomeric complexes in the cell, their formation is disulfide-independent.
To further investigate B1 receptor homo-oligomerization, FB1 and HB1 were expressed in cells either individually or in combination and then immunoprecipitated and immunoblotted with epitope-specific antibodies. Figure 3A shows that FB1 and HB1 were selectively recognized by FLAG and HA antibodies, respectively, whether the constructs were expressed individually (lanes 2, top, and 5, bottom) or together (lanes 3, top, and 4, bottom). Immunoprecipitation of HB1 with HA antibodies yielded coprecipitation of FB1 (Fig. 3A, lane 4, top), and immunoprecipitation of FB1 with FLAG antibodies yielded coprecipitation of HB2 (lane 3, bottom). FLAG antibody blots of anti-HA immunoprecipitates always contained a band at about 25 kDa. Because this band was also present in samples of mock-transfected cells (Fig. 3A, lane 6), it was clearly nonspecific. The varying intensity of this band in different blots depended on the exposure time of the blot. Coprecipitation of the HB1 and FB1 constructs was cell-dependent because mixing lysates from cells expressing each tagged receptor individually did not yield any FB1 reactivity in the HB1 precipitate (Fig. 3B, lane 3). Incubation of cells with the B1 receptor agonist desArg 10kallidin (DK) at 0.1 e for various lengths of time had no apparent effect on the amount of FB1 coprecipitated with HB1 (Fig. 3C). These results are consistent with the constitutive formation of B1 receptor homo-oligomers.
Immunoelectronmicroscopy was used as a detergent-free technique to provide additional evidence in support of B1 receptor homo-oligomeric complexes in intact cells. To this end, intact cells expressing HB1 were first incubated with 5-nm gold-labeled HA antibodies at 4°C to selectively detect cell surface receptors and then fixed and prepared for microscopy. As shown in Fig. 4, surface B1 receptors were readily detected by this method. Furthermore, clusters of labeling were clearly visible, including dimers and trimers. Higher order structures were also apparent but not represented in the figure. Because the specimens were cut only in one plane, additional apparently invisible or only partially visible particles may be present in some clusters. Closer inspection of some single particles revealed that they were not perfectly round, suggesting additional particles in proximity. These results provide evidence that constitutively formed higher order B1 receptor complexes are present on the cell surface.
Mapping of the B1 Receptor Homo-oligomerization Epitope. Truncating the B1 receptor by 8 and 34 residues from the C-terminal end to make B1stop346 and B1stop320, respectively, (Fig. 1A), followed by tagging with either FLAG or HA, did not significantly interfere with receptor expression as determined by both radioligand binding (Kang and Leeb-Lundberg, 2002) and immunoblotting (Fig. 5A). Figure 5A further shows that FB1stop346 coprecipitated with its HA-tagged counterpart HB1stop346 (lane 5), and FB1stop320 coprecipitated with its HA-tagged counterpart HB1stop320 (lane 6), when the two epitope-tagged versions were expressed together. Furthermore, the degrees of coprecipitation of the truncated constructs were very similar to that observed with the intact receptors (compare Fig. 5A, lanes 4-6). In addition, all the truncated constructs readily "hetero"-oligomerized with FB1 and HB1 (data not shown).
The same approach was used to determine the role of the N-terminal tail of the receptor. Deletion of the 25 N-terminal residues to make FB1del25 and HB1del25 had no apparent effect on receptor expression as determined by both radioligand binding (data not shown) and immunoblotting (Fig. 5B, lanes 2, top and 5, bottom). As discussed above, truncation of the N-terminal end erased the majority of the difference in the banding patterns of the two tagged receptors. HB1del25 coprecipitated with its FLAG-tagged counterpart FB1del25 (Fig. 5B, lanes 3 and 4) as well as with FB1 (data not shown). In addition, incubation of cells coexpressing FB1 and HB1 with 1.25 mM of a peptide comprised of N-terminal residues 2 to 37 for 2 h had no effect on the ability of these constructs to coprecipitate (Fig. 5C, lanes 3 and 4). These results reveal that, as determined by coimmunoprecipitation, neither the C-terminal nor the N-terminal tails are critical for B1 receptor homo-oligomerization.
To further search for the epitope(s) involved in B1 receptor homo-oligomerization, the receptor was divided in two parts after Arg134, which is located in the receptor DRY sequence at the bottom of transmembrane helix 3, to make B1stop135 and B1del(2-134) (Fig. 6A). HA-tagged versions of both constructs were readily expressed in cells as determined both by anti-HA immunoprecipitation of HB1stop135 (Fig. 6B, lane 5, bottom) and HB1del(2-134) (Fig. 6C, lane 1). Considering that HB1del(2-134) contains an intact C-terminal domain, this construct was also detected by immunoprecipitation with anti-B1R(317-353) (Fig. 6C, lane 2). No immunoreactivity was detected in lysates of mock-transfected cells (Fig. 6C, lane 3). HB1del(2-134) expressed as two species, at about 22 and 26 kDa, that did not seem to be multiples of each other. The reason for this is currently unknown. HB1stop135, of which the monomer migrated at <15 kDa, readily formed detergent-resistant higher order structures (Fig. 6B, lane 5, bottom). This construct also readily coimmunoprecipitated with FB1 (Fig. 6B, lanes 3, bottom, and 4, top). In contrast, HB1del(2-134) did not seem to form higher order structures (Fig. 6C, lanes 2 and 3) or coimmunoprecipitate with FB1 (Fig. 6D, lane 4). HB1stop135 was further truncated after Val71 at the C-terminal end of intracellular loop 1 to make HB1stop72. Monomeric HB1stop72 migrated at <15 kDa and readily formed higher order structures (Fig. 6E, lane 5, bottom). Again, this construct also coimmunoprecipitated with FB1 (Fig. 6E, lanes 3, bottom, and 4, top). Together, these results show that, as determined by coimmunoprecipitation, the epitope for B1 receptor homo-oligomerization seems to reside somewhere between residues 25 and 71.
B1stop135 Exhibits Dominant-Negative Behavior on B1 Receptor Cell Surface Expression. Because the B1stop135 fragment apparently oligomerize with intact B1 receptor, we next determined the effect of this fragment on B1 receptor homo-oligomers. Expression of FB1 and HB1 together with different concentrations of tag-less B1stop135 led to a concentration-dependent decrease in the amount of FB1 that coprecipitated with intact HB1 (Fig. 7, A and B). This may be caused by the ability of B1stop135 to disrupt B1 receptor homo-oligomers and form hetero-oligomers with the intact receptor. If so, this behavior may be used to determine the role of homo-oligomers in receptor expression and signaling.
To study the effect of B1stop135 on B1 receptor expression, we first used a membrane-impermeable maleimide-activated biotin derivative that reacts only with extracellular cysteines, which are present on both constructs. Incubation of the cells with the biotin derivative resulted in the detection of 35-kDa FB1 (Fig. 8A, lane 2). On the other hand, no specific biotinylated band was present in lysates expressing FB1stop135 (Fig. 8A, lane 4). Furthermore, and as expected, expression of B1stop135 did not introduce specific cellular [3H]DK binding (data not shown). Thus, it is seems that FB1 is expressed on the cell surface, whereas FB1stop135 is not. On the other hand, FB1stop135 may express on the surface but with cysteines not available for reaction with the biotin reagent. It is interesting that B1stop135 decreased the amount of 35-kDa FB1 available for biotinylation (Fig. 8A, lane 3, and C). On the other hand, B1stop135 did not decrease significantly the expression of the 35-kDa FB1 species (compare Fig. 8B, lanes 2 and 3; C). An increase in the 35-kDa FB1 product could have occurred and existed in higher order complexes. However, this was difficult to assess because such complexes would have been hidden by the immunoglobulin bands present on the immunoblot at >50 kDa. That B1stop135 decreased the number of intact cell surface B1 receptors was also evident by the dose-dependent decrease in the total cellular binding of the peptide agonist [3H]DK assayed at 4°C, which selectively measures cell surface receptors (Fig. 9A). In addition, B1stop135, which did not exhibit any intrinsic signaling activity on its own, dose dependently decreased expression of constitutive B1 receptor activity as measured by receptor-dependent and agonist-independent basal PI hydrolysis (Fig. 9B). In contrast, the C-terminal half of the receptor B1del(2-134), which was expressed but apparently did not hetero-oligomerize with intact B1 receptor (Fig. 6D), also did not influence either [3H]DK binding (Fig. 9A) or the expression of constitutive B1 receptor activity (Fig. 9B).
To investigate in more detail whether B1stop135 interfered with the plasma membrane targeting of the B1 receptor or simply reduced the total B1 receptor expression, cells expressing HB1 and FB1stop135 either individually or in combination were lysed hypotonically and fractionated on a sucrose density gradient. The fractions were then directly immunoblotted with either HA or FLAG antibodies. Figure 10A shows that HB1 immunoreactivity migrated primarily in the plasma membrane fractions (fraction 14-16). On the other hand, the distribution of FB1stop135 was broad with immunoreactivity in fractions 14 to 20. Fractions 17 and 18 contained a <15-kDa species, which is presumed to be the monomer of this construct, whereas fractions 18 and 19 contained an approximately 20-kDa species and an approximately 30-kDa species. Fractions 14 and 15, the plasma membrane fractions, contained a 40-kDa species and some higher order forms. Thus, the various oligomeric forms of B1stop135 seem to be targeted to different intracellular compartments as well as the plasma membranes. Coexpression of HB1 and FB1stop135 resulted in the redistribution of HB1 from fractions 14 to 16 to fractions 17 to 19. The nature of the compartment to which B1stop135 redistributed HB1 is unknown other than that it is of less density than the plasma membrane fraction and also targeted by some forms of B1stop135. Thus, B1stop135 may accomplish its dominantnegative behavior by forming hetero-oligomers, which are unable to properly target to cell surface.
Discussion
Together, the results from this study provide evidence for B1 receptor homo-oligomerization and the importance of this mechanism in the expression of the receptor on the cell surface of HEK293 cells. This mechanism directly translates into the presentation of a constitutive mechanism for cellular signaling through Gq/11-mediated phospholipase C activity. Such homo-oligomers may be a regulatory point in B1 receptor maturation, and their disruption could provide a unique way for silencing signaling through this and other B1 receptor-stimulated pathways during inflammation.
Multiple methods were used to investigate whether B1 receptors form homo-oligomers in HEK293 cells. Although neither of these methods alone is sufficient to prove oligomerization, together they are consistent with such a mechanism. Higher order oligomeric receptor forms were readily apparent on immunoblots of SDS-PAGE gels. Such complexes were also indicated by coimmunoprecipitation of differentially tagged receptors. Immunoelectron microscopy of intact cells using gold-labeled antibodies enabled the visualization of oligomeric structures on the cell surface. Finally, the redistribution of the B1 receptor after coexpression with the C-terminally truncated construct B1stop135 from the plasma membrane to an apparently common intracellular compartment as determined by sucrose-density gradient fractionation of hypotonically lysed cells is supportive of complex formation between these two receptor constructs.
Although various methods were used to provide evidence for B1 receptor homo-oligomerization, it is not absolutely clear whether the oligomeric complexes detected by each method are the same. For example, complexes detected after detergent treatment may not strictly reflect those observed in the absence of detergent. In this regard, it was interesting to observe that the various detergent-resistant forms of B1stop135 fractionated differently on a sucrose-density gradient. The B1stop135 monomer and some lower order detergent-resistant forms were present in relatively lighter fractions, possibly containing Golgi structures, whereas higher order forms were present in the plasma membrane fractions. The fractionation pattern of B1stop135 suggests that it is capable of targeting the plasma membrane but only in certain higher order homo-oligomeric form. The B1 receptor apparently also exists as a higher order structure in the plasma membrane fraction, which is in agreement with the immunoelectronmicroscopy results.
Analysis of the crystallographic rhodopsin structure and the rhodopsin dimer rows in rod outer segments identified by atomic force laser microscopy has led to a model suggesting the involvement of multiple GPCR interfaces in different domains in receptor oligomerization (Liang et al., 2003). Mutagenesis studies in several different GPCR systems also suggest multiple points of receptor contacts. Truncation of the B1 receptor revealed that homo-oligomerization of this receptor, as determined by coimmunoprecipitation of differentially tagged receptors, is dependent on an epitope(s) located between residue Leu26 on top of transmembrane helix 1 and Val71 at the C-terminal end of intracellular loop 1. This epitope may be similar to that in the yeast -factor receptor (Overton et al., 2003), C5a receptor (Klco et al., 2003), and CCR2 receptor (Mellado et al., 2001), which apparently require helix 1 for homo-oligomerization, or the CXCR2 receptor, which requires helix 3 (Trettel et al., 2003). On the other hand, the dopamine D2 receptor forms a symmetric dimer interface involving helix 4 (Guo et al., 2003), and the B2 bradykinin receptor subtype apparently requires an epitope in the N-terminal domain (AbdAlla et al., 1999). B1 and B2 bradykinin receptors heterodimerize (Kang et al., 2004), suggesting that these receptor subtypes do indeed contain multiple epitopes through which they interact with GPCR.
It is interesting that coexpression of B1stop135 with the B1 receptor led to the redistribution of the intact receptor from the plasma membrane fraction to a lighter density fraction containing the B1stop135 monomer and some lower order detergent-resistant homo-oligomeric B1stop135 forms. Because B1stop135 and the B1 receptor coimmunoprecipitate, one possibility is that this redistribution is caused by the hetero-oligomerization of the B1 receptor with some form of B1stop135, possibly monomer, which traps the B1 receptor in some compartment in the receptor maturation pathway. This explanation would be indicative of the formation of receptor oligomers early during receptor maturation. The best support for such a mechanism is provided by the GABAB receptor system, where both the type 1 and 2 are required for proper trafficking of a functional hetero-oligomerized receptor to the cell surface (Jones et al., 1998; Kaufmann et al., 1998; White et al., 1998). Furthermore, coexpression of splice variants of the dopamine D3 receptor, gonadotropin-releasing hormone receptor, and V2 vasopressin receptor, or truncated V2 and CCR5 receptor mutants with their wild-type counterparts resulted in mislocalization of the wild-type receptor in the cell (Benkirane et al., 1997; Grosse et al., 1997; Zhu and Wess, 1998; Karpa et al., 2000). Our results show that receptor fragments can indeed have pronounced effects on receptor expression. It is interesting that bradykinin receptor fragments can be generated in vivo as a direct consequence of oligomerization. Formation of B1 and B2 receptor heterodimers is associated with partial degradation of the participating B2 receptor, with the N-terminal fragment remaining associated with the B1 receptor (Kang et al., 2004). Depending on where this fragmentation occurs, it could have dramatic effects on receptor expression and localization.
Because B1stop72 and B1stop135 readily homo-oligomerized and hetero-oligomerized with the intact receptor, oligomerization does not seem to be dependent on either agonist or G protein binding. In this regard, B1 receptor oligomers may be similar to, for example, CCR5 and CXCR4 receptor oligomers, which were reported to form constitutively and not to change in response to agonist treatment as determined by BRET (Issafras et al., 2002; Babcock et al., 2003), but different from constitutively formed 2-adrenergic receptor oligomers, which increased in response to agonist treatment as determined by both coimmunoprecipitation (Hebert et al., 1996) and BRET (Angers et al., 2002), and -opioid receptor oligomers, which decreased in response to agonist treatment as determined by coimmunoprecipitation (Cvejic and Devi, 1997). Although these results may reflect inherent differences in oligomerization of these receptors, differences in the techniques used to measure the agonist effect may also be the reason. Coimmunoprecipitation is a relatively insensitive method and requires detergent, which may partially perturb the native agonist-sensitive structure. Considering the likely presence of multiple interfaces, coprecipitation may also rely on a receptor interface that is not agonist sensitive, or agonists may modulate the availability of critical immunoreactive epitopes. For fluorescence resonance energy transfer and BRET, receptors are often fused to relatively large donor and acceptor fluorophore proteins that themselves may dimerize independently of the receptor. Furthermore, the energy transfer between the interacting fluorophores is highly sensitive to the distance between them and their orientation. Thus, agonist-induced conformational changes may occur that moves these fluorophores without disrupting the dimer. The immunoelectron microscopy protocol used here is an attractive additional method that involves minimal cellular perturbation and monitors the receptors in their natural state. On the other hand, this method does not provide direct proof for a physical interaction and cannot be used to dynamically detect rapid agonist-promoted effects.
The best evidence for a functional role of oligomers comes from the crystal structure of the N-terminal domain of mGluR1 showing that the ligand binding pocket of this receptor is a dimer (Kunishima et al., 2000). In the 2-adrenergic receptor, a peptide derived from transmembrane helix 6 that inhibited agonist-stimulated receptor dimerization also inhibited receptor activity (Hebert et al., 1996). On the other hand, in the B2 bradykinin receptor a peptide derived from the N-terminal domain that inhibited agonist-stimulated receptor dimerization had no effect on receptor activity (AbdAlla et al., 1999). However, this peptide attenuated agonist-promoted receptor internalization. A considerable amount of research is clearly still needed to understand the functional consequence of GPCR monomer-monomer and higher order interactions.
In conclusion, our results present a scenario in which B1 receptor oligomers form early in the biosynthesis or maturation of the B1 receptor and their existence is required for the presentation of a functional receptor in the plasma membrane. Induction of this receptor by proinflammatory stimuli involves changes in mRNA synthesis (Menke et al., 1994) and possibly mRNA stability (Zhou et al., 1999). Regulation at the level of receptor homo-oligomerization and maturation may be an additional more rapid mechanism by which the expression of this receptor is regulated by some stimuli.
Acknowledgements
We thank M. Ingemarsson for technical assistance with part of this study.
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