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First published on October 18, 2004; DOI: 10.1124/mol.104.003608

0026-895X/05/6701-288-297$20.00
Mol Pharmacol 67:288-297, 2005

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The Composition of the {beta}-2 Adrenergic Receptor Oligomer Affects Its Membrane Trafficking after Ligand-Induced Endocytosis

Tracy T. Cao, Anne Brelot, and Mark von Zastrow1

Departments of Biochemistry and Biophysics (T.T.C.), Psychiatry (A.B., M.v.Z.), and Cellular and Molecular Pharmacology (M.v.Z.), Program in Cell Biology, University of California, San Francisco, San Francisco, California

Received June 4, 2004; accepted October 15, 2004


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The {beta}-2 adrenergic receptor (B2AR) is well known to form oligomeric complexes in vivo, but the functional significance of this process is not fully understood. The present results identify an effect of oligomerization of the human B2AR on the membrane trafficking of receptors after agonist-induced endocytosis in stably transfected human embryonic kidney 293 cells. A sequence present in the cytoplasmic tail of the B2AR has been shown previously to be required for efficient recycling of internalized receptors. Mutation of this sequence was observed to inhibit recycling not only of the receptor containing the mutation but also of the coexpressed wild-type B2AR. Coexpression of recycling-defective mutant B2ARs also enhanced proteolytic degradation of the wild-type B2AR after agonist-induced endocytosis, consistent with trafficking of both receptors to lysosomes in an oligomeric complex. Coexpression of the {delta} opioid receptor (DOR) at similar levels produced a much smaller effect on endocytic trafficking of the B2AR, even though DOR traverses a similar membrane pathway as recycling-defective mutant B2ARs. Biochemical studies confirmed that B2AR/B2AR-ala homomeric complexes form more readily than DOR/B2AR heteromers in expression-matched cell clones and support the hypothesis that B2AR/B2AR-ala complexes are not disrupted by agonist. These results suggest that a significant fraction of B2ARs exists in oligomeric complexes after ligand-induced endocytosis and that the composition of the oligomeric complex influences the sorting of endocytosed receptors between functionally distinct recycling and degradative membrane pathways.

There is a growing appreciation that many G protein-coupled receptors (GPCRs) exist in oligomeric complexes (Zeng and Wess, 2000Go; Bouvier, 2001Go; Milligan, 2001Go). For some GPCRs, notably, family C receptors such as the GABA-B receptor (White et al., 1998Go; Kuner et al., 1999Go) but also for certain family A GPCRs (Lee et al., 2000Go; Salahpour et al., 2004Go), oligomerization is critical for surface expression of functionally active receptors. For other GPCRs, oligomerization is not known to be essential for functional activity, and particularly for many family A GPCRs, the physiological significance of this process is not clear.

The {beta}-2 adrenergic receptor (B2AR) is a family A GPCR that has been shown, in extensive biochemical and biophysical studies, to form homodimers and larger oligomers in biological membranes and to do so at physiologically relevant levels of expression (Angers et al., 2000Go; Mercier et al., 2002Go; Salahpour et al., 2003Go). Chemical disruption of B2AR oligomers inhibits receptor activity in an isolated membrane fraction (Hebert et al., 1996Go), overexpression of the wild-type B2AR can rescue activity of a functionally impaired mutant receptor (Hebert et al., 1998Go), and oligomerization is required for efficient delivery of newly synthesized receptors from the biosynthetic pathway to plasma membrane (Salahpour et al., 2004Go). To our knowledge, no functional consequence of oligomerization has been demonstrated at later stages in the life cycle of the B2AR.

The B2AR undergoes rapid endocytosis via clathrin-coated pits after ligand-induced activation in the plasma membrane (von Zastrow and Kobilka, 1994Go; Goodman et al., 1996Go; Zhang et al., 1996Go). This process can mediate distinct functional effects, which are determined in large part by the specificity with which receptors are sorted between divergent downstream membrane pathways after endocytosis. Recycling of B2ARs to the plasma membrane promotes functional resensitization of receptor signaling, whereas trafficking of internalized receptors to lysosomes is one mechanism promoting proteolytic down-regulation (Pippig et al., 1995Go; Gagnon et al., 1998Go; Lefkowitz et al., 1998Go; Tsao and von Zastrow, 2000aGo). The sorting of internalized receptors between these distinct membrane pathways is controlled by a sequence present in the carboxyl-terminal cytoplasmic domain of the B2AR. Mutation of this sequence does not detectably affect receptor-mediated activation of heterotrimeric G proteins or agonist-induced endocytosis of receptors, but it specifically disrupts protein interaction(s) with the cytoplasmic tail that control the postendocytic sorting mechanism (Cao et al., 1999Go; Cong et al., 2001Go; Gage et al., 2001Go).

Despite considerable progress in identifying specific posttranslational modifications and cytoplasmic protein interactions that determine the endocytic trafficking fate of the B2AR (Ferguson et al., 1998Go; Goodman et al., 1998Go; Shenoy et al., 2001Go; Whistler et al., 2002Go), to our knowledge the possibility that interactions between receptors themselves might influence later stage(s) of endocytic membrane traffic has not been investigated. Nevertheless, there is considerable evidence that various GPCRs can endocytose in homo- or hetero-oligomeric complexes (Overton and Blumer, 2000Go; Yesilaltay and Jenness, 2000Go; Jordan et al., 2001Go; He et al., 2002Go; Terrillon et al., 2004Go), and biophysical studies suggest that B2AR homo-oligomers are not disrupted after agonist activation (Mercier et al., 2002Go; Salahpour et al., 2003Go). The present results provide cell biological evidence for the existence of B2AR oligomers in the endocytic pathway, and they suggest that the composition of B2AR oligomers influences the sorting of internalized receptors between distinct recycling and degradative fates.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
cDNA Constructs, Cell Culture, and Transfection. Amino-terminally HA and FLAG-tagged versions of the human B2AR (Kobilka et al., 1987Go) and mouse {delta} opioid receptor (DOP-1 or DOR; Evans et al., 1992Go) were described previously (von Zastrow and Kobilka, 1992Go; Chu et al., 1997Go; Cao et al., 1998Go). Mutant versions of the B2AR in which the distal carboxyl-terminal cytoplasmic domain was truncated after Gly 364 (B2AR-T364) or in which a single alanine residue or a 10-residue EE-epitope tag sequence were added to the full-length carboxy terminus (B2AR-ala and B2AR-EE, respectively) have been described previously (Cao et al., 1999Go). All receptor cDNAs were cloned into pcDNA 3.0 or 3.1 (Invitrogen, Carlsbad, CA) for mammalian expression. Human embryonic kidney (HEK) 293 cells (American Type Culture Collection, Manassas, VA) were grown in Dulbecco's modified Eagle's media supplemented with 10% fetal bovine serum (University of California, San Francisco Cell Culture Facility). Stably transfected cells expressing epitope-tagged B2AR and/or DOR were generated by calcium phosphate coprecipitation of the appropriate constructs (Okayama and Berg, 1983Go) followed by culture of cells for several weeks at limiting dilution in 200 µg/ml G418 (Geneticin; Invitrogen) and/or 40 µg/ml Zeocin (Invitrogen). Cell clones were isolated and amplified in separate flasks.

Selection of Stably Transfected Cell Clones by Radioligand Binding and Fluorescence Flow Cytometry. Initial screening of antibiotic-resistant cell clones was conducted by single-point radioligand binding in whole cells using 5 nM [3H]dihydroalprenolol or diprenorphine (Amersham Biosciences, Piscataway, NJ) to detect adrenergic or opioid receptors, respectively, and 1 µM alprenolol or naloxone (Sigma/RBI, Natick, MA) to define nonspecific binding, as described previously (Tsao and von Zastrow, 2000bGo). Singly transfected cell clones, expressing either FLAG-B2AR or DOR constructs separately, were selected for further study whether they bound 0.5 to 1 pmol of the appropriate radioligand per milligram of cell protein in this assay. Doubly transfected FLAG-B2AR/HA-DOR clones were selected in a similar manner, except that specific binding of 0.5 to 1 pmol/mg was required for both radioligands.

Doubly transfected cell clones coexpressing differentially epitope-tagged adrenergic receptor mutants (FLAG-B2AR/HA-B2AR-ala, FLAG-B2AR/HA-B2AR-T364, FLAG-B2AR/HA-B2AR-EE, and FLAG-B2AR-EE/HA-B2AR) were selected by a two-stage process. This was done because the HA- and FLAG-epitope tags used do not detectably affect B2AR ligand binding properties (Guan et al., 1992Go; von Zastrow and Kobilka, 1992Go); therefore, differentially tagged receptors cannot be distinguished by radioligand binding assay. Antibiotic-resistant cell clones were first selected for total [3H]dihydroalprenolol binding (regardless of epitope tag) of ≤3 pmol/mg. Clones that satisfied this criterion were subjected to secondary screening using a previously described flow cytometric assay to specifically detect either FLAG- or HA-tagged receptors using specific monoclonal antibodies (von Zastrow and Kobilka, 1992Go; Chu et al., 1997Go; Keith et al., 1998Go). Singly transfected cell clones (expressing HA-B2AR or FLAG-B2AR separately at 0.5 to 1 pmol/mg, as estimated by saturation radioligand binding) were analyzed alongside doubly transfected cell clones to provide an internal reference for the desired expression level of the corresponding tagged receptors. Doubly transfected cell clones that expressed surface FLAG and HA immunoreactivity within 30% of that detected (in the same experiment) from the corresponding FLAG- and HA-B2AR singly transfected reference cells were selected for further study.

Total B2AR and/or DOR expression level was then reassayed in selected cell clones by multipoint saturation radioligand binding assay (Tsao and von Zastrow, 2000bGo) and confirmed to be <3 pmol/mg in all cases. Actual receptor number per cell was estimated for each cell clone by dividing Bmax determinations derived from radioligand binding assay by cell number determined using a hema-cytometer (Fisher Scientific Co., Pittsburgh, PA), assuming one radioligand binding site per receptor. Total B2AR expression estimates (from [3H]dihydroalprenolol Bmax determinations) ranged from 42,000 ± 3700 receptors/cell to 139,000 ± 3900 receptors per cell across B2AR-transfected cell clones. Total DOR expression (from [3H]diprenorphine Bmax determinations) ranged from 61,000 ± 4300 to 210,000 ± 14,000 receptors per cell across DOR-transfected cell clones. Endogenous [3H]dihydroalprenolol binding in untransfected HEK293 cells was estimated at ≤1000 receptors/cell, and [3H]diprenorphine binding was undetectable.

Cell Surface Biotinylation and Assay of Agonist-Induced Proteolysis. Cells were grown on poly-L-lysine (Sigma-Aldrich, St. Louis, MO)-coated 6-cm tissue culture dishes (Falcon Plastics, Oxnard, CA), and cell surface biotinylation was conducted by incubating monolayers with 300 µg/ml sulfo-NHS-biotin (Pierce Chemical, Rockford, IL) in phosphate-buffered saline (PBS), pH 7.4, at 4°C for 30 min. Unreacted sulfo-NHS-biotin was removed by three washes with ice-cold Tris-buffered saline (TBS), pH 7.5. One dish was left at 4°C to determine total surface biotinylated proteins, and the other dishes were incubated with normal culture media at 37°C for 4 h in the absence or presence of a saturating concentration of agonist (10 µM isoproterenol for B2AR singly transfected and B2AR/B2AR-ala cotransfected cells, or 10 µM isoproterenol + 10 µM etorphine for B2AR/DOR cotransfected cells). Cells were then chilled on ice, washed three times with PBS, and extracted with 1 ml/dish of ice-cold TX-100 extraction buffer [0.5% (v/v) TX-100, 10 mM Tris-HCl, pH 7.5, 120 mM NaCl, 25 mM KCl, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin, 2 µg/ml phenylmethylsulfonyl fluoride). Extracts were clarified by centrifugation in a microcentrifuge (12,000g for 10 min) before immunoprecipitation of receptors.

Receptors were immunoprecipitated from cell extracts using 3 µg of anti-HA (HA.11) or 3 µg of anti-FLAG (M2) monoclonal antibodies (Sigma-Aldrich), 6 µg of rabbit anti-mouse linker antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) and 30 µl of protein A-Sepharose beads (Amersham Biosciences) as described previously (Cao et al., 1998Go). To minimize nonspecific binding, immunoprecipitates were spun through a 1 M sucrose cushion in a high-stringency wash buffer (0.1% SDS, 0.5% TX-100, 20 mM Tris-HCl, pH 7.5, 120 mM NaCl, 25 mM NaCl, 25 mM KCl) (Mays et al., 1995Go), and then washed sequentially three times with 1 M NaCl in high-stringency wash buffer followed by three washes with a low-salt wash buffer (0.5% TX-100 in 10 mM Tris-HCl, pH 7.5). Washed beads were extracted with SDS sample buffer containing 20 mM dithiothreitol and eluted proteins were resolved by SDS-PAGE. Resolved proteins were transferred to nitrocellulose membranes (GE Osmonics, Inc.) and blocked for 30 min in 5% dry milk and 0.1% Tween 20 in TBS. Biotinylated proteins were detected using horseradish peroxidase (VectaStain ABC; Vector Laboratories, Burlingame, CA) followed by enzyme-linked chemiluminescence (ECL system; Amersham Biosciences). Band intensities were quantified by densitometry (Alpha Innotech, San Leandro, CA) of films (Kodak XAR; Eastman Kodak, Rochester, NY) exposed in the linear range.

Visualization of Receptor Trafficking by Fluorescence Microscopy. Cells were grown on poly-L-lysine-coated coverslips and incubated with 10 µg/ml anti-HA (HA.11) and 10 µg/ml anti-FLAG (M1) monoclonal antibodies (Sigma-Aldrich), and 10 µM isoproterenol at 37°C for 30 min to drive internalization of labeled B2ARs. Coverslips were then washed with ice-cold PBS to remove residual agonist and either immediately fixed, to determine total internalized receptors, or incubated for the indicated time periods at 37°C with culture media containing 10 µM of the adrenergic antagonist alprenolol (Sigma-Aldrich). At the indicated time point, cells were then fixed in 4% formaldehyde (Sigma-Aldrich) dissolved freshly in PBS, pH 7.4, for 10 min, and then quenched with three washes of TBS + 1 mM CaCl2. Specimens were permeabilized with 0.1% TX-100, 3% bovine serum albumin (Sigma-Aldrich) in TBS + 1 mM CaCl2, and incubated with 1:1000 rabbit anti-mouse IgG2b antibody (Zymed Laboratories, South San Francisco, CA) to specifically label M1-bound FLAG-B2AR. A fluorescein-conjugated goat anti-mouse IgG1 (Roche Diagnostics, Indianapolis, IN) followed by Texas Red-conjugated goat anti-rabbit (1:1000 dilution; Jackson ImmunoResearch Laboratories) was used to specifically detect labeled HA-B2AR-ala and FLAG-B2AR, respectively, as described previously for specific detection of coexpressed dopamine receptors (Vickery and von Zastrow, 1999Go). Epifluorescence microscopy was carried out using an inverted Nikon microscope, a Nikon 60x, 1.4 numerical aperture objective and standard filter sets (Omega Optical Inc., Brattleboro, VT) that have been previously established to prevent detectable bleedthrough between fluorescein and Texas Red channels (Vickery and von Zastrow, 1999Go). Images were collected using a cooled (-10°C) charge-coupled device camera (Princeton Scientific Instruments, Monmouth Junction, NJ). Illumination intensity and exposure time were adjusted to prevent pixel saturation, and 12-bit images were collected and processed uniformly using IPLab Spectrum software (Scanalytics, Fairfax, VA) running on a Macintosh computer.

Fluorescence Flow Cytometric Assay of Receptor Recycling. Cells were grown on six-well tissue culture dishes (not coated with poly-L-lysine) and incubated with 5 µg/ml M1 antibody for 30 min to label FLAG-tagged receptors present in the plasma membrane, then incubated in the absence (untreated condition) or presence of 10 µM isoproterenol for 30 min at 37°C. Cells were chilled to 4°C, and untreated cells were washed with PBS containing 1 mM CaCl2, whereas agonist-treated cells were washed with calcium-free PBS supplemented with 1 mM EDTA to dissociate antibodies from receptors remaining in the plasma membrane. Agonist-treated dishes were either left at 4°C (iso condition) or washed and incubated in media containing excess (10 µM) of the adrenergic antagonist alprenolol (iso->alp condition) for 15, 30, or 60 min at 37°C. Cells were collected from dishes by gentle mechanical dissociation at 4°C, and approximately 2 x 105 cells were incubated in suspension for 1 h at 4°C in 100 µl of phosphate-buffered saline, 2% fetal bovine serum containing a phycoerythrin-conjugated goat anti-mouse secondary antibody (1:70; DakoCytomation California Inc., Carpinteria, CA), and then fixed in 1.5% paraformaldehyde freshly dissolved in PBS. Cells were analyzed for PE fluorescence using a FACScan flow cytometer (BD Biosciences, Franklin Lakes, NJ). Mean fluorescence intensities were determined from fluorescence histograms (representing analysis of 10,000 cells per data point) using CellQuest software. Mean fluorescence values were used to compute the fractional recycling of antibody-labeled receptors after agonist washout: % recycling = ([mean PE (iso->alp) - mean PE (iso)]/[mean PE (untreated) - mean PE (iso)]) x 100.

Analysis of Receptor Recycling Using Fluorescence Ratio Imaging. Recycling of FLAG antibody-labeled mutant B2ARs was quantified in individual cells by a previously established method using fluorescence ratio imaging (Tanowitz and von Zastrow, 2003Go). In brief, stably transfected cell clones plated on glass coverslips were incubated with 5 µg/ml Alexa488-conjugated M1 anti-FLAG antibody (prepared by standard methods using Alexa-fluor 488 N-hydroxysuccinimide ester; Molecular Probes, Eugene, OR) to selectively label FLAG-tagged receptors present in the plasma membrane at the beginning of the experiment. Then, cells were incubated (at 37°C for 30 min) in the presence of 10 µM isoproterenol to promote internalization of receptors. Cells were then quickly washed three times at 4°C in PBS lacking Ca2+ or Mg2+ and supplemented with 0.04% EDTA, to dissociate FLAG antibody bound to residual surface receptors remaining in the plasma membrane, thereby leaving antibody bound only to the internalized pool of receptors. EDTA-stripped cells were then incubated (at 37°C for 45 min) in the presence of 10 µM alprenolol to prevent receptor activation by possible residual agonist, then cells were fixed without permeabilizing by exposure to 4% paraformaldehyde freshly dissolved in PBS at 4°C for 20 min. Cells were washed in PBS, blocked using PBS containing 3% BSA (without detergent), and surface-accessible FLAG antibody was detected by incubation at 4°C for 30 min with 5 µg/ml Cy3-conjugated goat anti-mouse antibody (Jackson ImmunoResearch Laboratories) followed by three washes in PBS to remove unbound antibody. In each experiment, and for each receptor construct examined, two parallel control coverslips were included: one in which cells were fixed after a 30-min incubation in the absence of agonist and without an EDTA stripping step (100% surface control), and one in which cells were fixed immediately after the EDTA-mediated stripping step (0% surface control). Cells were examined by epifluorescence microscopy using appropriate filter sets to selectively detect Alexa488 or Cy3, images were collected under nonsaturating conditions using the 12-bit charge-coupled device camera, and staining intensities of each fluorochrome in individual cells were integrated using IPLab software. The percentage of FLAG-labeled receptors recycled in individual cells (20-30 per coverslip) after agonist washout was then calculated from the red/green ratios determined from the control conditions: (E - Z)/(C - Z) x 100, where E is the mean ratio for the experimental coverslip, Z is the mean ratio for the 0% surface control, and C is the mean ratio for the 100% surface control.

Coimmunoprecipitation of Receptors. Cell monolayers maintained in 6-cm tissue culture dishes (60-80% confluence) were incubated for 15 min at 37°C in the absence or presence of 10 µM isoproterenol (together with 10 µM etorphine for cells coexpressing B2AR and DOR) and then rapidly chilled to 4°C and washed 3 times in ice-cold PBS. Monolayers were extracted with 1.0 ml/dish buffer A [0.3% n-dodecyl-{beta}-D-maltoside (Sigma-Aldrich) in TBS + 1 mM CaCl2, supplemented with 1 µg/ml leupeptin, 1 µg/ml pepstatin, 2 µg/ml aprotinin, 2 µg/ml freshly prepared phenylmethylsulfonyl fluoride, and supplemented with 5 mM freshly prepared iodoacetamide]. Extracts were clarified by centrifugation, and receptors were immunoprecipitated by adding 0.3 µg of HA.11 or 0.3 µg of M1 antibody and 30 µl of protein A-Sepharose. HA.11 immunoprecipitations were supplemented with 0.4 µg of rabbit anti-mouse linker antibody (Jackson ImmunoResearch Laboratories). Antibody incubations were conducted for 3 h at 4°C with slow inversion of tubes. Immunoprecipitates were collected by sedimentation using a microcentrifuge, and then resuspended and washed five times with 1.0 ml of buffer A (without added iodoacetamide). Washed beads were eluted with SDS sample buffer under nonreducing conditions (to mimimize dissociation of IgG chains in the gel) and resolved by SDS-PAGE using 10% acrylamide (Bio-Rad, Hercules, CA). Proteins were transferred to nitrocellulose membrane, blocked with 5% dry milk, 0.1% Tween 20 in TBS supplemented with 1 mM CaCl2, and probed with 3 µg/ml HA.11 (anti-HA) or 3 µg/ml M1 (anti-FLAG). In some experiments, the wild-type B2AR was also detected using a 1:1000 dilution of a previously described antiserum (86.72) generated against a peptide corresponding to the extreme C terminus of the receptor (von Zastrow and Kobilka, 1992Go). Because this antibody recognizes the wild-type B2AR tail better than the B2AR-ala mutant tail (not shown), this reagent was used only for detection of receptors containing the wild-type B2AR tail. Blots were washed and incubated with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (1:1000; Jackson ImmunoResearch Laboratories), and immunoreactive receptors were detected by enzyme-linked chemiluminescence.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
A FLAG epitope-tagged version of the wild-type B2AR (FLAG-B2AR) was not detectably proteolyzed after exposure of stably transfected 293 cells to a saturating concentration of agonist (10 µM isoproterenol) for 4 h after surface biotinylation in stably transfected HEK293 cells (Fig. 1A, left), whereas adding an alanine residue to the carboxy terminus resulted in extensive proteolysis of receptors under these conditions (Fig. 1A, right). These results are consistent with previous studies indicating that the wild-type B2AR can endocytose and recycle repeatedly in the continuous presence of agonist (Kurz and Perkins, 1992Go; von Zastrow and Kobilka, 1992Go; Tsao and von Zastrow, 2000bGo) and that disruption of a specific recycling signal present in distal cytoplasmic tail inhibits recycling and promotes proteolytic degradation of internalized receptors (Cao et al., 1999Go; Cong et al., 2001Go; Gage et al., 2001Go). Significantly different results were obtained in doubly transfected cells coexpressing FLAG-tagged B2AR and HA-tagged B2AR-ala mutant receptors together. In these cells, both the wild-type FLAG-B2AR and recycling-defective HA-B2AR-ala mutant receptors were extensively proteolyzed within 4 h after agonist addition to the culture medium (Fig. 1B, left and right). Quantification of these results confirmed, in multiple experiments conducted on independently isolated cell clones (n = 3), that coexpression of the HA-B2AR-ala mutant receptor significantly enhanced agonist-induced proteolysis of the FLAG-B2AR (Fig. 1C). Agonist-induced proteolysis of both receptors was strongly (~75%) inhibited when agonist incubations were conducted (as described previously; Tsao and von Zastrow, 2000bGo) with chloroquine (200 µM) or ammonium chloride (50 mM) added to the culture medium (not shown), consistent with previous studies demonstrating lysosomal trafficking of wild-type B2AR and B2AR-ala mutant receptors in HEK293 cells (Gagnon et al., 1998Go; Cao et al., 1999Go). Together, these observations suggested that coexpression of the B2AR-ala mutant receptor may affect membrane trafficking of the wild type B2AR after agonist-induced endocytosis.



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  		Fig. 1. Effect of B2AR-ala mutant receptor coexpression on agonist-induced proteolysis of the wild-type B2AR. A, cells that were singly transfected with FLAG-B2AR (left) or HA-B2AR-ala were analyzed. Total indicates biotinylated receptors recovered from cell extracts that were maintained on ice immediately after biotinylation. iso indicates recovery from cells that were biotinylated and then incubated for 37°C for 4 h in the absence of agonist before lysis. +iso indicates recovery from biotinylated cells after incubation in the presence of 10 µM isoproterenol for 4 h before lysis. B, equivalent experiment conducted using FLAG-B2AR/HA-B2AR-ala coexpressing cells. Results shown are representative of three independent cell clones for each receptor combination. C, recovery of biotinylated receptors after agonist exposure of cells for 4 h was estimated across multiple experiments by scanning densitometry of streptavidin-horseradish peroxidase blots. Bars 1 to 3 (from left) show recovery of the indicated biotinylated receptors isolated from singly transfected cells, and bars 4 to 5 show receptor recovery from doubly transfected (FLAG-B2AR/HA-B2AR-ala) cells. Error bars represent standard deviation among individual determinations (three cell clones each). Coexpression of HA-B2AR-ala produced a significant reduction in FLAG-B2AR (compare bar 4 to bar 1, * indicates p < 0.01 by nonpaired Student's t test).

 

To further investigate this possibility, fluorescence microscopy was used to visualize the distribution of antibody-labeled receptors in stably transfected HEK293 cells expressing these receptors separately or in combination. In singly transfected cells (Fig. 2A, top), the FLAG-B2AR was observed to redistribute from the plasma membrane to numerous endocytic vesicles within 30 min after addition of agonist (10 µM isoproterenol) to the culture medium (a and b). A nearly complete return of receptor immunoreactivity to the plasma membrane was evident within ~30 min after agonist washout (c-e), consistent with efficient recycling of this receptor shown previously in HEK293 cells under similar conditions (Cao et al., 1999Go; Tsao and von Zastrow, 2000bGo). The FLAG-B2AR-ala mutant receptor (Fig. 2A, bottom) also internalized rapidly after agonist addition (f and g), but, consistent with inhibited recycling shown previously (Cao et al., 1999Go), remained in numerous endocytic vesicles after agonist washout (j). In doubly transfected cells (Fig. 2B), in which FLAG-B2AR and HA-B2AR-ala mutant receptors were expressed together at similar levels (see Materials and Methods), both receptors colocalized in the plasma membrane of untreated cells and translocated to an overlapping population of endocytic vesicles after agonist addition to the culture medium (b and g), consistent with previous studies indicating that both receptors undergo agonist-induced endocytosis via a similar membrane pathway (Cao et al., 1999Go). However, in marked contrast to results obtained in singly transfected cells, FLAG-B2AR did not recycle completely to the plasma membrane after agonist washout in doubly transfected cells. Numerous endocytic membrane structures containing antibody-labeled FLAG-B2AR were observed in cells at all time points after agonist washout (compare Fig. 2, B and A, c-e), and internalized FLAG-B2AR colocalized extensively with HA-B2AR-ala mutant receptors in these intracellular membranes (Fig. 2B, e and j).



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  		Fig. 2. Effect on B2AR endocytic trafficking visualized by fluorescence microscopy. Cells expressing differentially epitope-tagged B2ARs were labeled at the cell surface with the appropriate anti-HA or FLAG monoclonal, and receptor localization was visualized under the indicated conditions by epifluorescence microscopy. A, receptor localization in singly transfected cells. B, receptor localization in FLAG-B2AR/HA-B2AR-ala doubly transfected cells. The results shown are representative of five experiments using three clones of each singly and doubly transfected cell population.

 

We next examined the effects of HA-B2AR-ala coexpression on FLAG-B2AR recycling using a flow cytometric assay in which recycling of antibody-labeled receptors was detected by accessibility to a fluorochrome-conjugated secondary antibody in nonpermeabilized cells (see Materials and Methods). In singly transfected cells, FLAG-B2AR recycled efficiently within 60 min after agonist washout, as indicated by the nearly complete recovery of antibody-labeled receptors accessible at the surface of nonpermeabilized cells (Fig. 3A, left histogram). As expected, the FLAG-tagged B2AR-ala mutant receptor recycled to a much smaller extent, indicated by a pronounced left-shift in the histogram representing recycled receptors (Fig. 3A, middle; compare distributions indicated by arrows in left and middle). In cells cotransfected with FLAG-B2AR and HA-B2AR-ala, the histogram representing recycled FLAG-B2AR (Fig. 3A, right, arrow) was significantly left-shifted relative to that characteristic of the FLAG-B2AR in singly transfected cells (left), although recycling of wild-type receptors in doubly transfected cells still seemed to be greater than that of the recycling-defective FLAG-B2AR-ala mutant receptor observed either in singly transfected cells (middle) or doubly transfected cells (see below). These observations were verified across multiple experiments, and fractional recovery of surface receptor immunoreactivity was calculated from mean fluorescence intensity determinations (see Materials and Methods) at several time points after agonist washout. This analysis confirmed that coexpression of the HA-B2AR-ala mutant receptor significantly inhibited recycling of FLAG-B2AR in doubly transfected cells. However, the observed inhibition was not complete and was most pronounced at later time points after agonist washout (Fig. 3B). This inhibitory effect on FLAG-B2AR recycling was further verified using a different recycling assay (Tanowitz and von Zastrow, 2003Go) based on quantitative fluorescence microscopy (Fig. 3C, bars 1-3 from left). Two other recycling-defective mutant versions of the B2AR, in which the recycling signal was either removed by truncation (HA-B2AR-T364) or disrupted by fusion of a carboxyl-terminal epitope tag sequence (HA-B2AR-EE) (Cao et al., 1999Go), were also tested and found to produce significant inhibition of FLAG-B2AR recycling as the B2AR-ala mutant receptor (Fig. 3C, bars 4 and 5 from left). To investigate the converse possibility, that the wild-type B2AR may enhance recycling of a recycling-defective mutant receptor, we generated expression-matched stably transfected HEK293 cells expressing a FLAG-tagged version of the recycling-defective B2AR-EE mutant receptor by itself, or together with an HA-tagged version of wild-type B2AR. As expected the FLAG-B2AR-EE recycled poorly when examined in singly transfected HEK293 cells (Fig. 3C, bar 6 from left). This mutant receptor also recycled poorly in doubly transfected cells coexpressing an HA-tagged wild-type receptor (Fig. 3C, bar 7). Examination of mean recycling values averaged over multiple experiments (Fig. 3 C, compare bars 6 and 7) failed to detect a statistically significant enhancement of FLAG-B2AR-EE recycling by coexpression of the (recycling-competent) HA-B2AR, in contrast to the highly significant converse effect detected by the same assay. Thus, the major effect observed in doubly transfected cells was inhibited recycling of the wild-type B2AR. These results are consistent with the previous data (Fig. 1), indicating that both wild-type and tail-mutant receptors exhibit pronounced agonist-induced proteolysis when coexpressed.



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  		Fig. 3. Quantitative analysis of recycling effects. A, fluorescence histograms representing surface-accessible FLAG-tagged receptor immunoreactivity in singly transfected cells expressing FLAG-B2AR (left), singly transfected cells expressing FLAG-B2AR-ala (middle), and doubly transfected cells coexpressing FLAG-B2AR and HA-B2AR-ala (right). Results are representative of three clones. Total B2AR expression levels in the example shown were 47,300 ± 3900 receptors/cell (FLAG-B2AR); 64,200 ± 2100 receptors/cell (FLAG-B2AR-ala); and 116,000 ± 2800 receptors/cell (FLAG-B2AR/HA-B2AR-ala). B, fractional recovery of antibody-labeled FLAG-B2AR to the plasma membrane after agonist washout calculated from flow cytometric data. Points represent mean surface receptor recovery at the indicated time points after agonist washout, calculated from mean fluorescence histograms (10,000 cells each, n = 3 experiments). Error bars represent S.E.M. values across these independent determinations. C, quantification of FLAG-tagged B2AR recycling by fluorescence ratio imaging performed 45 min after agonist washout. Bars represent the mean percentage of FLAG-tagged receptor recycling determined in three independent experiments. The transfected receptor complement is indicated below each bar. Error bars represent the S.E.M. across the experiments (n = 3-5 for each combination).

 

To investigate the specificity of the observed in trans effects on endocytic trafficking, we focused on the DOR, a distinct GPCR that internalizes by an indistinguishable early endocytic pathway as the B2AR (Keith et al., 1996Go; Zhang et al., 1999Go; Tsao and von Zastrow, 2000bGo). Unlike the wild-type B2AR, but similar to the B2AR-ala mutant receptor, DOR recycles inefficiently after agonist-induced endocytosis in HEK293 cells and traffics preferentially to lysosomes (Tsao and von Zastrow, 2000bGo). DOR is capable of forming mixed oligomers with the B2AR and this process has been reported to influence agonist-induced endocytosis of receptors (Jordan et al., 2001Go), although relatively high levels of receptor expression (>250,000 receptors/cell) are required for detectable B2AR/DOR hetero-oligomer formation in HEK293 cells (Ramsay et al., 2002Go). Other studies, using significantly lower levels of receptor expression, observed differential agonist regulation of coexpressed adrenergic and opioid receptor endocytosis in the same (HEK293) cell background (Tsao and von Zastrow, 2000bGo; Klein et al., 2001Go; He et al., 2002Go). Thus, we generated stably transfected cell clones expressing epitope-tagged DOR at levels closely similar to those of mutant B2AR constructs examined above. We confirmed by fluorescence microscopy that adrenergic (10 µM isoproterenol) and opioid (10 µM etorphine) agonist selectively promoted endocytosis of FLAG-B2AR or HA-DOR, respectively (not shown), as observed previously (Tsao and von Zastrow, 2000bGo). Then, the surface biotinylation assay was used to assay proteolysis of differentially tagged B2AR and DOR constructs in singly and doubly transfected cells in the presence of both agonists, a condition that is sufficient to promote rapid endocytosis of both receptors (Tsao and von Zastrow, 2000bGo; Klein et al., 2001Go). Surface biotinylated FLAG-B2AR was not detectably proteolyzed in doubly transfected cells over a 4-h time course, as indicated by the uniform recovery in anti-FLAG immunoprecipitates (Fig. 4A, left), whereas HA-DOR was proteolyzed almost completely in the same cells (Fig. 4A, right). These results are fully consistent with the differential endocytic trafficking of receptors established previously in singly transfected cells (Tsao and von Zastrow, 2000bGo) and are in marked contrast to the pronounced in trans enhancement of FLAG-B2AR proteolysis produced by coexpression of HA-B2AR-ala (Fig. 1). Together, these observations suggest that DOR has a significantly less pronounced effect on endocytic trafficking of the wild-type B2AR than do recycling-defective mutant B2ARs, at least when evaluated at similar moderate levels of receptor expression.



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  		Fig. 4. Examination of B2AR and DOR trafficking in doubly transfected cells and detection of receptor oligomers by coimmunoprecipitation. A, doubly transfected HEK293 cells coexpressing FLAG-B2AR and HA-DOR were surface biotinylated, and receptor recovery in cell extracts was determined at the indicated time points after addition of 10 µM isoproterenol and 10 µM etorphine to the culture medium. The results shown are representative of three separate experiments. B, coimmunoprecipitation analysis of FLAG-B2AR/HA-B2AR-ala doubly transfected cells. Left, HA-B2AR-ala mutant receptors were immunoprecipitated from FLAG-B2AR/HA-B2AR-ala doubly transfected cells using HA.11 anti-HA monoclonal antibody for immunoprecipitation and blotting for FLAG-B2AR. - and + refer to preincubation (15 min) with 10 µM isoproterenol before cell lysis. Con represents the nonspecific signal obtained from immunoprecipitates prepared from singly transfected cells that express FLAG-B2AR but not HA-B2AR-ala. Right, converse experiment using anti-FLAG immunoprecipitation followed by anti-HA immunoblot. Con represents the nonspecific signal detected in the control immunoprecipitate prepared from HA-B2AR-ala singly transfected cells. Lysate represents the signal of the blotted receptor (FLAG-B2AR or HA-B2AR-ala, respectively) detected in cell extracts corresponding to 5% of the input to the immunoprecipitation. C, coimmunoprecipitation analysis of FLAG-B2AR/HA-DOR doubly transfected cells. Left, expression-matched FLAG-B2AR/HA-DOR doubly transfected cells were immunoprecipitated with anti-HA monoclonal antibody and blotted for FLAG-B2AR. - and + refer to preincubation (15 min) with both 10 µM isoproterenol and 10 µM etorphine before cell lysis. Con represents negative control signal from FLAG-B2AR singly transfected cells. The additional band resolving near the top of the gel is a cross-reacting immunoglobulin species not related to the receptor (present also in the Con lane). Right, corresponding analysis performed using anti-FLAG immunoprecipitation followed by anti-HA immunoblot. Lysate represents the signal of the blotted receptor (FLAG-B2AR or HA-DOR, respectively) detected in cell extracts corresponding to 5% of the input to the immunoprecipitation.

 

We next investigated whether the selective effect of B2AR-ala relative to DOR on B2AR membrane traffic was associated with a biochemical difference in the ability of these receptors to form oligomeric complexes in intact cells. To accomplish this, we immunoprecipitated receptors, from the same doubly transfected cell clones as used for trafficking assays, and assayed for coimmunoprecipitation of the other receptor by immunoblotting. FLAG-B2AR/HA-B2AR-ala complexes were readily detected using either anti-FLAG or anti-HA immunoprecipitation (Fig. 4B). In most experiments, a major fraction (>50%) of coimmunoprecipitated receptor immunoreactivity resolved after solubilization in sample buffer with an electrophoretic mobility corresponding to the complex-glycosylated monomeric receptor (~55-75 kDa, indicated by the bracket marked B2AR in Fig. 4B). In some experiments, receptor species resolving at higher apparent molecular mass were also observed (not shown), consistent with previous studies indicating that HA-B2AR-ala and FLAG-B2AR may associate in these cells both in dimeric and higher order oligomeric complexes (Salahpour et al., 2003Go). Amounts of HA-B2AR-ala/FLAG-B2AR complexes recovered from extracts were not significantly affected by agonist pretreatment of cells (compare lanes marked - and +), consistent with previous studies indicating that B2AR dimers and oligomers form constitutively and are not disrupted by agonist activation (Mercier et al., 2002Go; Salahpour et al., 2003Go). In contrast HA-DOR/FLAG-B2AR complexes were detected at much lower levels, similar to nonspecific background levels, at the electrophoretic mobility corresponding to SDS-solubilized monomeric or larger forms (see legend), using identical conditions of anti-HA or anti-FLAG immunoprecipitation from the respective (expression-matched) doubly transfected cell clones and after incubation of cells either in the absence (-) or presence (+) of 10 µM each of isoproterenol and etorphine (Fig. 4C, left and right). These results are consistent with previous biophysical studies regarding B2AR/DOR hetero-oligomer formation in vivo (Ramsay et al., 2002Go) and demonstrate preferential formation of B2AR/B2AR-ala receptor complexes in the same cells in which a specific effect on endocytic trafficking of receptors was observed.


   Discussion
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 Abstract
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 Results
 Discussion
References
 
The present studies demonstrate that recycling-defective mutant versions of the B2AR, when coexpressed at similar moderate levels with the wild-type B2AR, function in trans to inhibit recycling of the wild-type allele. Inhibited recycling of the B2AR was associated with increased agonist-induced proteolysis after surface biotinylation. This proteolysis was sensitive to inhibitors of lysosome function and the internalized pool of B2AR colocalized extensively with the recycling-defective B2AR-ala mutant receptor, which has been shown previously to traffic rapidly to lysosomes in HEK293 cells (Cao et al., 1999Go). Coexpression of DOR at comparable levels produced a less pronounced effect on B2AR postendocytic trafficking, even though DOR traffics to lysosomes efficiently and via a similar endocytic pathway as the recycling-defective mutant versions of the B2AR studied (Cao et al., 1999Go; Tsao and von Zastrow, 2000bGo; Gage et al., 2001Go). B2AR/B2AR-ala complexes were readily detected by coimmunoprecipitation in cells in which postendocytic trafficking effects were observed, whereas B2AR/DOR complexes were not detected in comparable amount using the same methods in expression-matched cell clones. Together, these results indicate that recycling-defective mutant versions of the B2AR produce a trans-dominant effect on postendocytic membrane trafficking of the wild-type B2AR and suggest that this effect is likely to represent a specific functional consequence B2AR oligomer formation occurring in intact cells. Despite compelling evidence supporting the existence of B2AR dimers and oligomers (Hebert et al., 1996Go; Mercier et al., 2002Go; Ramsay et al., 2002Go; Salahpour et al., 2003Go) and recent data indicating that oligomer formation is important for receptor export from the biosynthetic pathway (Salahpour et al., 2004Go), we believe the present results are the first to identify an effect of B2AR oligomerization at later stages of receptor membrane traffic.

The presence of B2AR oligomers in the endocytic pathway is consistent with previous studies of several other GPCRs (Overton and Blumer, 2000Go; Yesilaltay and Jenness, 2000Go; Jordan et al., 2001Go; He et al., 2002Go; Terrillon et al., 2004Go) and with the ability of B2AR oligomers to remain intact after agonist activation (Mercier et al., 2002Go; Salahpour et al., 2003Go) (also evident in Fig. 4B). It is interesting to note that hetero-dimerization of V1 and V2 vasopressin receptors was shown recently to produce in trans effects on receptor endocytosis and recycling, although possible effect(s) on lysosomal traffic of receptor proteolysis were not investigated (Terrillon et al., 2004Go). In the case of heterodimerization of vasopressin receptors, the observed differences in recycling were proposed to be mediated by differences in endosome localization of arrestins elicited by activation of these distinct class A and class B GPCRs (Oakley et al., 2000Go; Terrillon et al., 2004Go). To our knowledge, the present results are the first to provide evidence for a significant role of homo- (as opposed to hetero-) oligomerization in determining the postendocytic membrane trafficking (as opposed to initial endocytosis) of any GPCR. We also note that the B2AR, as well as the B2AR-ala mutant receptor (not shown), does not promote detectable endosome recruitment of arrestins, in contrast to the V2 vasopressin receptor (Oakley et al., 2000Go; Klein et al., 2001Go; Terrillon et al., 2004Go). Thus, we also believe that the present results provide the first evidence for in trans postendocytic sorting effects that are not associated with differential endosome association of arrestins.

A limitation of the present studies is that they were conducted using heterologous expression of recombinant receptors. Although we have taken care to examine receptor trafficking effects in cells expressing receptors at moderate levels, which are significantly below levels at which nonspecific receptor interactions have been observed previously using energy transfer assays (Mercier et al., 2002Go; Ramsay et al., 2002Go; Salahpour et al., 2003Go), these levels of receptor expression may still exceed those occurring in native tissues (Mercier et al., 2002Go). A second caveat is that because we have focused entirely on trafficking of the B2AR and the specificity of endocytic sorting effects in expression-matched cell clones, we do not presently know to what degree the present results can be generalized to other GPCRs. Nevertheless, it is increasingly evident that many GPCRs can form dimers or larger oligomeric complexes when expressed at moderate levels (Bouvier, 2001Go; Milligan, 2001Go). Thus, together with the recent functional studies of vasopressin receptors (Terrillon et al., 2004Go), it seems likely that specific homo- and hetero-oligomerization events could be of rather widespread importance in determining the postendocytic fate of GPCRs. A third limitation is that we have focused entirely on the novel question of oligomerization effects on postendocytic membrane traffic of the B2AR, in contrast to effects of oligomerization on initial endocytosis as shown previously for a number of other GPCRs (Overton and Blumer, 2000Go; Yesilaltay and Jenness, 2000Go; Jordan et al., 2001Go; He et al., 2002Go). This could be addressed in future studies using endocytosis-defective mutant receptors, as was done previously to demonstrate endocytosis of GPCR oligomers in yeast (Overton and Blumer, 2000Go; Yesilaltay and Jenness, 2000Go). A fourth caveat is that our results do not define the stoichiometry of B2AR oligomerization relevant to postendocytic membrane traffic. Although our coimmunoprecipitation experiments readily detected complexes containing both FLAG-B2AR and HA-B2AR-ala mutant receptors, they do not define the stoichiometry. Recycling assays indicated that the inhibitory effect of B2AR-ala on recycling of coexpressed B2AR was not complete (Fig. 2), and recycling at the earliest time point was not detectably affected (Fig. 2B). The existence of B2AR in multiple forms, including monomers and various oligomeric complexes, is consistent with previous biochemical and biophysical studies (Mercier et al., 2002Go; Salahpour et al., 2003Go). It will be interesting in future experiments to define the specific oligomeric form(s) of the receptor protein relevant to postendocytic sorting.

Our results also provide cell biological support for the existence of considerable specificity in GPCR oligomer formation. Although previous studies indicate that B2AR/DOR complexes can form in intact cells (Jordan et al., 2001Go; Ramsay et al., 2002Go) and influence receptor endocytosis (Jordan et al., 2001Go), we observed relatively little effect of DOR coexpression on B2AR endocytic trafficking in the present work. A clue to this apparent discrepancy is that biophysical studies indicate that detectable B2AR/DOR hetero-oligomer formation requires expression levels in excess of 250,000 receptors/cell for both receptors (Ramsay et al., 2002Go), much higher than required to detect B2AR/B2AR homo-oligomers (Mercier et al., 2002Go; Ramsay et al., 2002Go) and also substantially higher than expression levels achieved in any of our doubly transfected cell clones (see Materials and Methods). Thus, we believe that although it is possible to generate both B2AR/B2AR and B2AR/DOR complexes in intact cells at sufficiently high levels of receptor expression, B2AR/B2AR homo-oligomers are highly favored at moderate levels that are more likely to be physiologically relevant (Mercier et al., 2002Go). This hypothesis is consistent with previous studies of differential receptor endocytosis (Chu et al., 1997Go; Tsao and von Zastrow, 2000bGo; He et al., 2002Go) and with the present coimmunoprecipitation studies. So far, we have been unable to generate stably cotransfected cell clones expressing both FLAG-B2AR and HA-DOR at >250,000 receptors/cell using the present system, but we have observed evidence for impaired recycling of FLAG-B2AR (in immunocytochemical experiments similar to those shown in Fig. 2) when HA-DOR was expressed by transient transfection at very high levels (≥5-fold over those tested in the present stably transfected cells, as estimated by relative fluorescence intensity), suggesting that B2AR/DOR hetero-oligomers can indeed influence endocytic traffic under some conditions (not shown). We also note that although DOR undergoes rapid proteolysis in HEK293 cells and certain neuronal cell types when activated either by peptide or alkaloid agonists (Law and Loh, 1999Go; Tsao and von Zastrow, 2000bGo), a recent study suggests that postendocytic sorting of this receptor can be differentially regulated by distinct agonists in SK-N-BE neuroblastoma cells (Marie et al., 2003Go). It will be interesting to investigate this phenomenon in future studies and to determine whether this additional complexity of GPCR regulation observed in certain cell types may reflect the existence of ligand-specific conformational states or oligomeric complexes.

In conclusion, the present results provide evidence that the composition of the B2AR oligomer influences the sorting of internalized receptors between distinct, and functionally important, downstream membrane pathways. Although our studies are presently limited to a single type of GPCR and to a transfected cell model system, they provide strong evidence for a role of oligomerization in determining the postendocytic membrane trafficking of a specific GPCR, and they identify a novel functional consequence of B2AR oligomerization occurring in intact cells.


   Acknowledgements
 
We thank Catherine de Coupade for conducting some of the radioligand binding assays, Paul Dazin and Nigel Killeen for advice and assistance with flow cytometry assays, Heather Deacon for advice on statistical analysis, and Michael Tanowitz for advice on quantitative fluorescence microscopy.


   Footnotes
 
This study was supported by research grants from the National Institutes of Health (to M.v.Z.). T.T.C. was supported for part of this study by a National Institutes of Health Institutional Predoctoral Training grant and was awarded the University of California San Francisco Chancellor's Fund fellowship. A.B. was awarded a European Molecular Biology Organization postdoctoral fellowship.

T.T.C. and A.B. contributed equally to this work.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi: 10.1124/mol.104.003608.

ABBREVIATIONS: GPCR, G protein-coupled receptor; B2AR, {beta}-2 adrenergic receptor; HA, hemagglutinin; DOR, {delta}-opioid receptor (DOP-1); HEK, human embryonic kidney; PBS, phosphate-buffered saline; PE, phycoerythrin; TBS, Tris-buffered saline; TX, Triton X-100.

1 Current address: Departement de Biologie Cellulaire, Institut Cochin, U567, Paris, France. Back

Address correspondence to: Dr. Mark von Zastrow, Genentech Hall, Rm N212E, 600 16th St., University of California, San Francisco, San Francisco, CA 94143-2140. E-mail zastrow{at}itsa.ucsf.edu


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