Type-specific Sorting of G Protein-coupled Receptors after Endocytosis*

Analysis of Receptor Number in a Crude Membrane Fraction

Cell monolayers were lifted with PBS supplemented with 2 mm EDTA, washed twice with PBS by centrifugation (200 × g for 5 min), and lysed in 10 mm Tris-Cl, 2 mm EDTA, pH 7.4, containing a protease inhibitor mixture (leupeptin, aprotinin, pepstatin, and phenylmethylsulfonyl fluoride) followed by four passes using a tight-fitting Dounce homogenizer. Large particulates and nuclear material were removed by centrifugation at 500 × g for 5 min, and a crude membrane and cytosol fraction was isolated. Binding assays were conducted in 120 μl of 25 mm Tris-Cl, 1 mm EDTA, pH 7.4. Assay tubes contained 50–100 μg of the crude membrane preparation (determined by the method of Bradford et al. (33) using reagents from Bio-Rad) and 2 nm [³H]diprenorphine (opioid binding) or 10 nm [³H]alprenolol (adrenergic binding) and were incubated for 30 min at room temperature. Incubations were terminated by vacuum filtration through glass fiber filters (Packard Instruments) and repeated washes with ice-cold Tris-buffered saline, pH 7.4. Bound radioactivity was determined by scintillation counting (Scintiverse, Fisher) using a Beckman LS 6500 instrument. Bound counts represented ≤10% of input radioligand. Nonspecific binding, defined by assays conducted in the presence of 10 μm naloxone (opioid binding) or alprenolol (adrenergic binding), was ≤10% of total counts isolated on filters. All assays were conducted in triplicate with similar results. Results are expressed as mean picomoles of radioligand specifically bound per mg of crude membrane preparation assayed.

Assay of Receptor Down-regulation in Intact Cells

Agonist-induced down-regulation of receptors was assayed in intact cells using a previously described method (34). Briefly, monolayers of cells expressing FLAG-tagged B2AR (B2AR 3) or DOR (DOR 5) were incubated for 3 h at 37 °C in the absence or presence of 10 μm isoproterenol or 10 μm DADLE (Research Biochemicals), respectively. To ensure a saturating concentration of peptide agonist over the incubation period, monolayers incubated with DADLE were supplemented with fresh peptide every hour during the incubation. At the end of the incubation, cells were lifted with PBS supplemented with EDTA and washed four times by centrifugation with 10 ml of warm (37 °C) PBS. Then cells were washed once by centrifugation in 10 ml of Krebs-Ringer HEPES buffer (KHRB: 110 mm NaCl, 5 mm KCl, 1 mmMgCl₂, 1.8 mm CaCl₂, 25 mm glucose, 55 mm sucrose, 10 mmHEPES, pH 7.3). Radioligand binding was carried out in 120 μl of KHRB containing equal amounts of washed cells (50–100 μg of protein) and ligand concentrations as above. Incubations were carried out for 30 min at room temperature, and cells were harvested and washed using vacuum filtration on glass fiber filters as above. For all determinations, bound radioligand represented ≤10% of total radioligand present in the incubation, and nonspecific binding (defined as above) was ≤10% of counts isolated on glass fiber filters.

Surface Recovery Assay

Recycling of epitope-tagged receptors back to the plasma membrane was estimated by assaying the recovery of immunoreactive receptors accessible at the cell surface to monoclonal antibody recognizing the extracellular epitope tag. This assay is a variant of a previously described flow cytometric assay for estimating receptor internalization (35). Briefly, cell monolayers expressing FLAG-tagged B2AR or DOR were incubated in the presence of 10 μm of the appropriate agonist (isoproterenol or etorphine, respectively) for 30 min at 37 °C to drive agonist-induced internalization to steady-state levels (23, 29), then rinsed with ice-cold PBS, and subsequently incubated at 37 °C in the presence of the appropriate antagonist (10 μm alprenolol or naloxone (Research Biochemicals)) to block additional endocytosis of receptors. At the indicated time points, monolayers were chilled on ice to stop membrane trafficking, and cells were lifted with a protease-free Cell Dissociation Buffer (Life Technologies, Inc.). Resuspended cells were then incubated at 4 °C for 60 min in the presence of 10 μg/ml M1 anti-FLAG antibody (Eastman Kodak Co.) that had been conjugated with fluorescein isothiocyanate (Molecular Probes) using standard methods, and receptor immunoreactivity was quantitated by fluorescence flow cytometry (FACScan, Becton Dickinson, Palo Alto, CA). Fluorescence intensity of 10,000 cells was collected for each sample. Cellquest software (Becton Dickinson) was used to calculate the mean fluorescence intensity of single cells in each population. All experiments were conducted ≥3 times with similar results. The mean values for each experiment were averaged to obtain the overall mean fluorescence intensity and S.E. reported in the figure.

Loss of Internal Receptor Assay

Recycling of antibody-labeled receptors from the endocytic pathway was estimated using an alternate flow cytometric assay. FLAG-tagged B2AR present in the plasma membrane of stably transfected cells were specifically labeled with fluorescein-conjugated M1 anti-FLAG antibody (5 μg/ml), which binds the FLAG epitope in a calcium-dependent manner. Cell monolayers were incubated with 10 μm isoproterenol for 30 min at 37 °C to stimulate receptor internalization and then rinsed three times with calcium, magnesium-free PBS supplemented with 0.4% EDTA in order to elute antibody bound to residual receptors remaining in the plasma membrane and selectively label endocytosed receptors. At this point, different protocols were followed to measure recycling under different conditions. To measure recycling in the presence of antagonist, samples were incubated at 37 °C in DMEM in the presence of 10 μm alprenolol (to block additional endocytosis) and then chilled at the indicated time point to stop membrane trafficking. Cells were again rinsed three times at 4 °C with EDTA-supplemented PBS to elute antibody from antibody-labeled receptors that recycled to the cell surface during the incubation with antagonist. To measure recycling in the presence of agonist, samples were incubated at 37 °C in EDTA-supplemented PBS containing 10 μm isoproterenol. Under these conditions, antibody bound to receptors that recycle back to the plasma membrane was immediately eluted and was therefore not re-endocytosed in the presence of isoproterenol. Control experiments indicated that >95% of surface receptors were eluted within 1 min under these conditions. At the indicated time point, monolayers were again chilled to 4 °C, lifted, washed, and analyzed by flow cytometry (as above) to detect antibody bound to internalized receptors remaining within the cell.

Dual Staining of Permeabilized Cells

Colocalization of FLAG-tagged B2AR and HA-tagged DOR expressed in a single cell line was examined using a modification of a previously described protocol for dual fluorescence immunohistochemical staining (20). Briefly, cells grown on glass coverslips (Corning) were treated with 10 μm isoproterenol and 10 μm etorphine (Research Biochemicals) for 60 min, washed, fixed with a 4% formaldehyde solution in PBS, and permeabilized in 0.1% Triton X-100 in Blotto (3% dry milk in TBS + 1 mm CaCl₂). Specimens were incubated with anti-FLAG M1 antibody (IgG2b, 5 μg/ml) and mouse monoclonal anti-HA antibody (HA.11, IgG1, 5 μg/ml, Berkeley Antibody Co.) for 30 min, washed, incubated with subtype-specific rabbit anti-mouse IgG2b antibody (0.5 μg/ml, Zymed Laboratories Inc.) to label the M1 antibody, washed again, and treated with 0.1% Triton X-100 in Blotto. Finally, the B2AR (labeled with M1 monoclonal and rabbit anti-mouse IgG2b) and the DOR (labeled with anti-HA mouse monoclonal) were visualized by incubating with Texas Red donkey anti-rabbit (5 μg/ml, Jackson ImmunoResearch) and fluorescein isothiocyanate subtype-specific anti-mouse IgG1 (2 μg/ml, Roche Molecular Biochemicals). Stained specimens were examined by conventional epifluorescence microscopy using a Nikon Diaphot microscope equipped with a 60× NA1.4 objective and standard fluorescein/Texas Red dichroic filter sets. Confocal microscopy was performed using a Bio-Rad MRC1000 confocal microscope equipped with a Zeiss 100× NA1.3 objective. Negligible bleed through was confirmed in dual labeling experiments by imaging single-labeled control specimens. The estimated depth of optical sections under the confocal imaging conditions used was 0.5–1 μm.

Pulse-Chase of Receptors with Endocytosed Transferrin

An immunocytochemical “pulse-chase” assay was developed to estimate the degree to which a “pulse” of internalized B2AR or DOR was accessible to a subsequent “chase” of endocytosed transferrin. Briefly, stably transfected cells expressing either FLAG-tagged B2AR or DOR (grown on glass coverslips) were preincubated at 37 °C in serum-free DMEM; receptors were surface-labeled with 5 μg/ml M1 antibody, and cells were incubated with 10 μmisoproterenol or etorphine for 30 min to drive endocytosis of antibody-labeled receptors. Next, cells were chilled on ice, rinsed with EDTA-supplemented PBS to elute antibody bound to residual receptors remaining in the plasma membrane, rewarmed to 37 °C for 15 min in serum-free media lacking agonist but containing Texas Red-conjugated diferric transferrin (50 μg/ml, Molecular Probes), and coverslips fixed with 4% formaldehyde in PBS. Cells were permeabilized using 0.1% Triton X-100 in Blotto (3% dry milk in TBS + 1 mm CaCl₂), and antibody-labeled B2AR or DOR was detected using fluorescein-conjugated donkey anti-mouse antibody (2 μg/ml, Jackson ImmunoResearch). Dual-label fluorescence microscopy was performed as described above.

B2AR and DOR Differ in Agonist-induced Down-regulation

As an initial step toward comparing the effects of agonist on numbers of B2AR and DOR present in cells, we used an established radioligand binding assay (34) to measure down-regulation of receptor binding activity following incubation of cells in the presence of agonist for 3 h. This time point was chosen because, whereas both B2AR and DOR have been previously shown to undergo substantial down-regulation after incubation of cells for 18–24 h in the continuous presence of agonist, down-regulation has also been observed for certain GPCRs at much shorter times (e.g. Refs. 5, 16, and 17). Consistent with previous studies (36), incubation of stably transfected cells expressing the FLAG-tagged B2AR for 3 h with a saturating concentration (10 μm) of the adrenergic agonist isoproterenol caused little (<10%) down-regulation of receptor sites measured in whole cells (Fig.1 A). In contrast, incubation of cells expressing the FLAG-tagged DOR for 3 h in the presence of the opioid agonist DADLE revealed a substantial (greater than 50%) decrease in diprenorphine-binding sites detected under these conditions (Fig. 1 A). The diprenorphine concentration used in this assay was near saturation, suggesting that this decrease reflects a change in the number of DOR-binding sites (34). Saturation binding analysis confirmed that DADLE pretreatment caused a profound decrease in B _max (Fig. 1 B). A similar amount of down-regulation was observed with radioligand binding on membrane preparations of DOR-expressing cells treated with [d-Pen^2,5]enkephalin.2Substantial down-regulation of functional DOR has been observed previously under similar conditions in studies conducted in neuroblastoma cells, where this process has been associated with proteolytic degradation of receptors in lysosomes (4, 37).

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 1

Agonist promotes substantial loss of DOR functional ligand-binding sites and total receptor protein. Loss of ligand-binding sites and total receptor protein was measured by intact cell ligand binding assays and immunoblotting, respectively, performed on stably transfected cells expressing FLAG-tagged B2AR or DOR as described under “Experimental Procedures.” A,cells were incubated in the absence (untreated, ut) or presence of 10 μm agonist (isoproterenol, iso, or DADLE) for 3 h. Ligand-binding sites were measured as bound tritium counts and expressed as picomoles of ligand specifically bound/mg of total cell protein. Results are plotted as percentage of total bound specific radioactivity in untreated cells. Error bars represent S.E. (n = 3 assays performed in triplicate). B, Scatchard analysis of saturation ligand binding to DOR-expressing cells incubated in the absence (open circles) or presence (filled squares) of DADLE for 3 h. Each data point represents the mean of triplicate determinations. C, immunoreactive bands resolving at the appropriate molecular mass of the complex-glycosylated B2AR and DOR were detectable in extracts from cells expressing epitope-tagged receptors but not from untransfected cells. The mobility of molecular mass standards (in kDa) is indicated to the left. D, receptor protein was detected on immunoblots loaded with equal amounts of extract. Cells were incubated in the absence (untreated, ut) or presence of 10 μm agonist (isoproterenol, iso, or etorphine, et) for 3 h. Studies using lysosomal protease inhibitors (leupeptin (leu), chloroquine (cq), or ammonium chloride (AC)) were performed by preincubation with inhibitors for 1 h at 37 °C and then treatment with agonist for 3 h in the continued presence of inhibitors. E, immunoblots were quantitated by densitometric scanning from multiple experiments (n = 3). Results are plotted as percentage of total receptor in untreated cells. Error bars represent S.D. of three experiments.

To determine whether the down-regulation of DOR detected by radioligand-binding sites was associated with proteolytic degradation of the receptor, whole-cell extracts were resolved by SDS-PAGE and immunoblotted using anti-FLAG monoclonal antibody recognizing the epitope-tagged receptor. Both the B2AR and DOR were specifically detected as strong immunoreactive bands in stably transfected cells (Fig. 1 C, 1st and 3rd lanes). The specificity of this detection was confirmed by the negligible background immunoreactivity detected in untransfected cells not expressing epitope-tagged receptors (Fig. 1 C, 2nd lane). Whereas both B2AR and DOR primarily resolved in these reducing gels at an apparent molecular mass consistent with a monomeric complex-glycosylated species (Fig. 1 C), as described previously (29, 38), additional immunoreactive species were also observed in some experiments at higher apparent molecular mass, consistent with previously described oligomers of B2AR and DOR (39, 40). Incubation of cells with isoproterenol for 3 h caused little or no detectable change in amounts of B2AR protein detected by immunoblotting (Fig. 1 D, 1st and2nd lanes). Consistent with the down-regulation observed by radioligand binding, there was also a pronounced decrease in the amount of immunoreactive DOR protein following DADLE treatment (Fig. 1 D, 3rd and 4th lanes). Interestingly, quantitation of multiple experiments by densitometric scanning indicates that the loss of immunoreactive DOR protein was even greater than the down-regulation of functional receptors detected by radioligand binding (Fig.1 E). This may reflect the existence of proteolytic receptor intermediates at this time point which bind ligand yet lack the FLAG epitope. Significant agonist-induced reduction in immunoreactive DOR was observed for the major band corresponding to the monomeric receptor protein (Fig. 1 D), as well as for minor species of detectable DOR (not shown). Importantly, these differences between agonist-induced reduction in immunoreactive receptor protein were observed in cells expressing closely similar receptor numbers, including cells expressing DOR at lower numbers than B2AR (see “Experimental Procedures”). Further studies indicated that the agonist-induced reduction in immunoreactive DOR protein was highly sensitive to inhibitors of lysosomal proteolysis (Fig. 1 D), confirming that this reduction represents proteolytic degradation of DOR and supporting pharmacological and immunocytochemical studies suggesting that internalized DOR traffic to lysosomes in neuroblastoma and neuro2a cells (34, 37). Taken together, these observations strongly suggest that internalized DOR are selectively targeted for relatively rapid degradation in lysosomes in HEK293 cells, in contrast to the much slower rate at which internalized B2AR are targeted to lysosomes in this cell type (3, 41).

B2AR and DOR Differ in Subcellular Localization after Endocytosis

If internalized B2AR and DOR do indeed differ in endocytic trafficking, one would expect significant differences in the subcellular localization of receptors at some point after internalization. As an initial step toward testing this hypothesis, fluorescence microscopy was used to compare the subcellular localization of B2AR and DOR when coexpressed in the same stably transfected HEK293 cells (clone B2DOR 1, see “Experimental Procedures”). Previous studies have established that both B2AR and DOR internalize in HEK293 cells with a t <10 min (23, 29). In addition, both receptors colocalize extensively with internalized transferrin receptors immediately (within 10–15 min) after endocytosis (12, 21-24). To compare receptor localization after more prolonged incubation with agonist, we performed confocal microscopy on cotransfected cells (B2DOR 1, see “Experimental Procedures”) fixed after incubation with both agonists for 60 min. This time point was chosen because it is relatively long compared with the rate of receptor endocytosis and corresponds to the time at which proteolytic degradation of DOR is first detected biochemically (see below and Fig. 3 E). Under these conditions, substantial differences were observed in the subcellular distribution of immunoreactive B2AR and DOR (Fig. 2,A and B, respectively). Whereas vesicles containing comparable amounts of immunoreactive B2AR and DOR were still observed at this time point (examples of colocalized structures are indicated by solid arrows in Fig. 2), we also observed numerous endocytic vesicles that were selectively enriched in DOR and contained little or no detectable B2AR (e.g. Fig. 2,open arrows). These differences are emphasized in the merged color image (Fig. 2 C), in which colocalized structures appear yellow and membranes selectively enriched in DOR appear green.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 3

Agonist promotes rapid degradation of surface DOR but not B2AR. Proteolytic degradation of surface-biotinylated receptors was measured as described under “Experimental Procedures.” A, biotinylated protein bands resolving at the appropriate molecular mass of the complex-glycosylated B2AR and DOR were specifically recovered in immunoprecipitates prepared from cells expressing the epitope-tagged receptors but not from untransfected cells. The mobility of molecular mass standards (in kDa) is indicated to the left. B and C display biotinylated protein recovered from equal amounts of cells prepared after incubation of cells in the absence (untreated) or presence of the appropriate agonist for the indicated time. B, under all conditions examined, B2AR was recovered in similar amounts as present in cells lysed immediately after biotinylation (hrs). Comparable amounts of B2AR were also observed after enzymatic deglycosylation (PNGase F, lower lanes). C, in cells incubated in the absence of agonist, DOR was recovered in similar amounts as present in cells lysed immediately after biotinylation (hrs: 0). In cells incubated in the presence of agonist, a significant reduction in the amount of biotinylated receptor protein was observed. D and E, biotinylated receptor protein was quantitated by densitometric scanning of streptavidin overlays from multiple experiments (n ≥ 3). Results are plotted as the mean recovery of biotinylated receptor protein (relative to that isolated at t = 0). Error bars represent S.D. of three experiments. D represents relative amount of biotinylated B2AR and DOR recovered after incubation of cells for 3 h in the absence (solid bar) or presence (stippled bar) of the agonist. E, displays a time course of the amounts of biotinylated receptor recovered from agonist-treated cells.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 2

Dual localization of B2AR and DOR by confocal microscopy. Stably transfected cells coexpressing FLAG-tagged B2AR and HA-tagged DOR were treated with 10 μm isoproterenol and 10 μm etorphine for 60 min, fixed, and processed for dual localization of receptor immunoreactivity by confocal fluorescence microscopy as described under “Experimental Procedures.” DOR immunoreactivity is displayed in A. B2AR immunoreactivity is displayed in B. Colocalization of DOR (green) and B2AR (red) is indicated in the merged image (C) by yellow staining. The inset represents a 2-fold magnification of the boxed region. Solid arrows indicate examples of vesicles containing comparable amounts of B2AR and DOR immunoreactivity. Open arrows indicate examples of vesicles relatively enriched in DOR immunoreactivity. Bar, 10 μm.

B2AR and DOR Differ in Their Rate of Degradation after Agonist-induced Endocytosis

Our studies thus far address the effects of agonists on the total complement of B2AR and DOR detected in cells, including receptors present in the plasma membrane as well as various intracellular membranes. To examine specifically the trafficking of receptors from the plasma membrane, we used a biochemical method to label selectively receptors present on the cell surface. Intact cells were reacted with a membrane-impermeant biotinylation reagent (sulfo-NHS-biotin), which labels proteins exposed on the cell surface but not proteins present in intracellular membranes. After various manipulations, surface-labeled receptors were detected in receptor immunoprecipitates (prepared from whole-cell extracts) using streptavidin overlay. Whereas no detectable biotinylated receptor signal was observed in control immunoprecipitates prepared from untransfected cells, surface-biotinylated B2AR and DOR were readily detected in transfected cells (Fig.3 A). Both receptors resolved as a heterogeneous protein band by SDS-PAGE, consistent with the predominant receptor species detected by immunoblotting of whole-cell extracts. This heterogeneity resulted from complex N-linked glycosylation, as indicated by digestion to single band with theN-linked endoglycosidase PNGase F (Fig. 3 B).

The fate of B2AR and DOR present initially in the plasma membrane was examined by determining the amount of biotinylated receptor protein recovered from surface-biotinylated cells after incubation under various conditions. Immunoprecipitations were conducted under conditions of antibody excess to ensure that the amount of biotinylated receptor protein isolated in immunoprecipitates provided a reliable measure of the relative amount of surface-labeled receptor protein present in the cell extracts. In the absence of agonist, surface-biotinylated B2AR exhibited little or no degradation for prolonged periods of time, as indicated by the uniformly high recovery of biotinylated B2AR at all time points examined. Moreover, B2AR was recovered efficiently from cells incubated in the prolonged presence of saturating concentrations of isoproterenol (Fig. 3 B, upper panels). Two additional pieces of evidence support the remarkable biochemical stability of the B2AR in the presence of agonist. First, enzymatic cleavage of N-linked glycans using PNGase F (which is expected to provide a more sensitive assay for small changes in the electrophoretic mobility of receptors that could result from partial proteolysis) failed to reveal any evidence for B2AR proteolysis, even after 3 h in the continuous presence of isoproterenol (Fig.3 B, lower panels). Second, immunoprecipitation of B2AR using an antibody recognizing the distal carboxyl terminus (29, 38) (rather than the proximal amino-terminal epitope tag sequence) also revealed no evidence for agonist-induced proteolysis of the B2AR (not shown).

The same experiments conducted on the DOR yielded markedly different results. Surface-biotinylated DOR, like B2AR, was highly stable in the absence of agonist. However, in the presence of the opiate agonist etorphine, the amount of biotinylated DOR isolated from cells was rapidly and dramatically reduced. Proteolytic degradation of labeled receptors was nearly complete after 3 h and readily detectable even without enzymatic deglycosylation of receptors (Fig.3 C). Quantitation of these results by scanning densitometry of streptavidin overlays confirmed that surface-labeled B2AR and DOR differ significantly in their biochemical stability (Fig. 3,D and E). These observations were also confirmed in cells coexpressing both B2AR and DOR (tagged selectively with HA and FLAG epitopes, respectively, not shown), further confirming that these differences reflect receptor-specific differences in proteolytic degradation following endocytosis. In addition, similar results for the DOR were obtained when cells were incubated with the the peptide agonist DADLE for 3 h (not shown).

Confirmation That Internalized B2ARs Recycle Efficiently to the Plasma Membrane in the Continuous Presence of Agonist

The dramatically different rates of agonist-induced degradation of surface-labeled B2AR and DOR, together with the effects of inhibitors of lysosomal proteolysis, suggest that receptors differ significantly in trafficking between recycling and lysosomal pathways after endocytosis. To examine this hypothesis in greater detail, we devised several assays to measure specifically the recycling of receptors to the plasma membrane after agonist-induced internalization to steady-state levels (i.e. 30 min agonist treatment (23,29)).

Recycling of internalized B2AR was first estimated using fluorescence flow cytometry to measure the recovery of surface receptors in the plasma membrane after removal of agonist from the culture medium. Consistent with previous immunocytochemical and pharmacological studies (42-45), this “surface recovery” assay indicated that internalized B2AR undergoes rapid recycling to the plasma membrane following removal of agonist (Fig. 4 A). After a brief lag, approximately 50% of internalized B2AR recycled within 15 min, and nearly complete recycling was observed within 30 min. Rapid recovery of surface B2AR was observed even in cells incubated in the presence of the protein synthesis inhibitor cycloheximide (200 μm), indicating that this recovery resulted from receptor recycling rather than from the biosynthesis of new receptor protein.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 4

Internalized B2AR recycles rapidly and efficiently to the plasma membrane. Two fluorescence flow cytometric assays were used to estimate recycling of internalized B2AR under various conditions. A, represents the surface recovery assay. Cells were incubated with 10 μm isoproterenol for 30 min (indicated by dotted line), washed, and then incubated with 10 μm alprenolol for the indicated times to stop further endocytosis. Receptors present in the plasma membrane at each time point were labeled with fluorescein-conjugated M1 antibody and quantitated by flow cytometry, as described under “Experimental Procedures.” Data (open circles) indicate the mean surface receptor fluorescence of cells (relative to surface fluorescence of cells not exposed to agonist). Error bars represent S.E. of mean fluorescence data collected from multiple experiments (n ≥ 4). Some experiments were performed in the presence of 200 μm cycloheximide to exclude the possible contribution of new receptor synthesis (closed triangle).B, represents the “loss of internal receptor” assay. Internalized B2AR was specifically labeled with monoclonal antibody and the loss of cell-associated antibody (which indicates recycling of the receptor protein) was measured by flow cytometry, as described under “Experimental Procedures.” Closed triangles indicate efflux of internalized receptors observed from cells incubated in the absence of agonist, and open circles represent receptor efflux in cells incubated in the presence of 10 μmisoproterenol.

The biochemical stability of B2AR in the continuous presence of saturating concentrations of agonist (Figs. 1 and 3) as well as previous studies by others (42-44) predict that the B2AR recycles efficiently to the plasma under these conditions. To confirm this, we assayed the efflux of antibody-labeled receptors from endocytic vesicles in cells incubated in calcium-depleted medium. Antibody attached to receptors accessible at the cell surface is efficiently dissociated within seconds in this medium, allowing recycling of receptors pre-labeled with the fluorochrome-conjugated antibody to be monitored directly using fluorescence flow cytometry. In the absence of agonist, recycling of antibody-labeled B2AR was readily observed (Fig.4 B, open circles and solid line) and occurred with similarly rapid kinetics as recycling of receptors estimated by the surface recovery assay (Fig. 4 A). Moreover, antibody-labeled receptors returned to the plasma membrane at a similarly rapid rate in the continuous presence of isoproterenol (Fig.4 B, closed triangle and dashed line), further validating the flow cytometric assay and directly confirming the ability of internalized B2AR to recycle rapidly even in the continuous presence of agonist.

Internalized DOR Is Selectively Retained at an Early Stage in the Endocytic Pathway

In contrast to the rapid rate and complete extent of recycling of the B2AR (Fig.5 A, closed circles anddotted line), the surface recovery assay indicated that internalized DOR returned to the plasma membrane to a significantly smaller extent (<50%), even after incubation of cells in the presence of antagonist for 45 min (Fig. 5 A, open circles andsolid line). We typically conducted these experiments by adding the antagonist naloxone to the culture medium after agonist washout. This was done to block possible effects of residual agonist that may remain associated with cells after agonist washout (46). We do not believe that the failure of DOR to recycle is due to the presence of antagonist binding as naloxone induces neither detectable endocytosis (not shown) nor proteolytic degradation (see below and Fig.6 B, lane e) of DOR.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 5

Inefficient recycling and selective endocytic retention of internalized DOR. A, the surface recovery assay was used to estimate recycling of receptors following agonist-induced internalization. Open circles (solid line) represent recovery of immunoreactive DOR in the plasma membrane. Error bars represent S.E. calculated from the mean fluorescence of cells in multiple experiments (n = 3).Closed circles (dashed line) represent surface recovery of B2AR measured by the same assay (see Fig. 4 A).B–D, receptor recycling was measured biochemically by the loss of internalized receptor protein specifically labeled with disulfide-linked (cleavable) biotin, as described under “Experimental Procedures.” Control experiments (B) display surface-biotinylated B2AR or DOR detected by streptavidin overlay (lanes 1 and 3). Surface-biotinylated receptors were completely cleaved by glutathione (lanes 2 and4), confirming that any glutathione-resistant signal represents internalized receptor protein that is inaccessible to added glutathione. C, a strong signal of internalized B2AR was observed in cells incubated with 10 μm isoproterenol for 30 min (iso = 30). Additional incubation of agonist-treated cells for 30 min in the presence of 10 μmalprenolol (iso → alp) caused the complete disappearance of internalized B2AR. D, the identical experiment performed on cells expressing DOR (left lanes) indicated that a significant fraction of biotinylated DOR remained internalized after incubation for 30 min with 10 μmnaloxone. Significant retention of internalized DOR was observed even in cells incubated with etorphine for only 10 min and subsequently incubated in the presence of naloxone for 30 min (right lanes). The results shown are representative of three independently conducted experiments.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 6

Degradation of the retained pool of DOR does not require the continuous presence of agonist. Degradation of biotinylated receptors was examined under various conditions to determine the ligand dependence of this process. A outlines the experimental protocol. B displays biotinylated DOR detected by streptavidin overlay under each of the following conditions: a, untreated; b, treated with 10 μm etorphine (et) for 30 min; c,treated with etorphine for 1 h; d, treated with 10 μm etorphine for 2 h; e, treated with 10 μm naloxone (nal) for 2 h; f,treated with 10 μm etorphine for 30 min, washed, and then treated with 10 μm naloxone for 30 min; g,treated with 10 μm etorphine for 30 min, washed, and then treated with 10 μm naloxone for 1.5 h.

To determine whether the failure of internalized DOR to recycle reflects a bona fide sorting event or is simply a consequence of proteolytic degradation of the receptor, we devised a biochemical assay to analyze DOR retained in cells after agonist-induced endocytosis. Cells expressing B2AR or DOR were surface-biotinylated at 4 °C using “cleavable” sulfo-NHS-S-S-biotin, and endocytosis of receptors was induced by incubating cells with the appropriate agonist at 37 °C for 30 min. Cells were then washed at 4 °C in the absence of agonist and subsequently incubated at 37 °C in the presence of the appropriate antagonist for 30 min, in order to block additional endocytosis of surface-biotinylated receptors and to allow sufficient time for “maximal” recycling of receptors to occur (Fig. 5 A). Following this incubation, cells were incubated at 4 °C in the presence of a membrane-impermeant reducing agent that cleaves biotinylated receptors present in the plasma membrane (Fig. 5 B, lanes 1–4). Under these conditions, only those receptors that were internalized from the cell surface and failed to recycle in the presence of antagonist remained biotinylated. Essentially no residual biotinylated B2AR was detected in the endocytic pathway under these conditions (Fig. 5 C) whereas, in marked contrast, a substantial amount of biotinylated DOR failed to recycle (Fig.5 D, 1st and 2nd lanes). Furthermore, residual biotinylated DOR detected under these conditions resolved with an electrophoretic mobility indistinguishable from that of the full-length receptor protein, suggesting that internalized DOR is retained in endocytic pathway without any detectable proteolytic degradation. Moreover, although it was difficult to quantitate precisely the fraction of retained DOR using this complex assay, a significant amount of internalized DOR was reproducibly retained within the endocytic pathway, even after preincubation of cells for as little as 10 min with agonist (Fig. 5 D, 3rd and 4th lanes). These observations, which were confirmed in three separate experiments, strongly suggest that intracellular retention of rapidly internalized DOR can be distinguished from, and significantly precedes, the proteolytic degradation of receptors observed after more prolonged incubation of cells with agonist.

Later Stages of Membrane Trafficking Leading to Proteolytic Degradation of Retained DOR Do Not Require the Continuous Presence of Agonist

We next examined the fate of internalized DOR after removal of agonist from the culture medium. Based on recent studies of the V2 vasopressin receptor (47, 48), one might expect internalized DOR to remain in the endocytic pathway for a prolonged period of time without proteolytic degradation. However, since the DOR is rapidly proteolyzed in the continuous presence of agonist, it is possible that internalized DOR is delivered to lysosomes and degraded, even after removal of agonist. To distinguish between these possibilities, we used a pulse-chase protocol (Fig. 6 A) in which surface-biotinylated cells were incubated for 30 min with agonist (pulse) to induce substantial internalization of biotinylated DOR without causing detectable proteolysis (Fig. 3) and then incubated for an additional 90 min under various conditions (chase). Degradation of DOR was estimated by the recovery of biotinylated receptor protein in immunoprecipitates (Fig. 6 B).

In cells incubated in the continuous presence of etorphine (pulse and chase), pronounced degradation of DOR occurred over the 2-h time course (Fig. 6 B, lanes a–d), fully consistent with the agonist-induced degradation of receptors observed previously (Fig. 3). In contrast, no detectable degradation of DOR was observed after incubation of cells for this time in the continuous presence of the opiate antagonist naloxone (Fig. 6 B, lane e). However, in cells pulsed with etorphine for 30 min followed by agonist washout and chase incubation in the presence of naloxone (which completely blocks receptor-mediated inhibition of adenylyl cyclase in intact cells, not shown), significant proteolytic degradation of biotinylated DOR was observed after an initial lag period of approximately 30 min (Fig.6 B, lanes f and g). These results further confirm that internalized DOR fail to recycle to the plasma membrane following agonist removal, and they indicate that, in marked contrast to the stability of internalized V2 receptors shown previously (47, 48), internalized DOR undergo substantial proteolytic degradation even after removal of agonist from the culture medium.

Visualization of the Endocytic Trafficking of Surface-labeled Receptors by Fluorescence Microscopy

Internalized DOR could fail to recycle because they remain physically retained in the same early endocytic compartment through which other membrane proteins rapidly recycle, as has been suggested to occur for internalized epidermal growth factor receptors (49). Alternatively, it is possible that internalized DOR are rapidly segregated out of the clathrin-mediated early endocytic pathway and delivered to a distinct population of endocytic vesicles that do not recycle. To begin to examine these hypotheses, we used an immunocytochemical pulse-chase assay to estimate the degree to which internalized B2AR or DOR remains associated with endocytic vesicles that can be labeled with Texas Red-labeled transferrin, a well established marker of early and recycling endosomes that mediate rapid recycling (50, 51).

FLAG-tagged B2AR or DOR present in the plasma membrane was specifically labeled by incubating intact cells with monoclonal antibody, and then agonist was added to cells to initiate a 30-min pulse of endocytosis. Cells were chilled to 4 °C, and antibodies bound to receptors remaining in the plasma membrane were eluted, in order to label selectively the newly internalized receptors. Washed cells were then warmed to 37 °C in the absence of agonist and chased for an additional 15 min in the presence of labeled transferrin, conditions which label both early and recycling endosomes (50). The extent of colocalization between the pulse of internalized B2AR or DOR and the endocytic tracer was examined using dual-label confocal microscopy.

Many vesicles containing internalized B2AR colocalized with endocytosed transferrin, (Fig. 7 A, colocalization is indicated by the numerous yellowstructures observed in the merge panel), consistent with the rapid recycling of internalized B2AR via early and recycling endosomes (29). However, the same experiment conducted with internalized DOR yielded significantly different results. In this case we observed a large number of endocytic vesicles containing DOR but no detectable transferrin (Fig. 7 B, arrows indicate examples of such vesicles, which appear green in the merge panel). This difference in the endocytic trafficking of surface-labeled DOR was confirmed by quantitation of vesicular colocalization observed in multiple cells examined in coded specimens (Fig. 7 C). These observations support the idea that the failure of internalized DOR to recycle rapidly is mediated, at least in part, by sorting of internalized receptors to a population of endocytic vesicles distinct from those that constitute the conserved recycling pathway marked by transferrin.

View larger version:

• In this window
• In a new window

• Download as PowerPoint Slide

Figure 7

Pulse-chase analysis of internalized B2AR and DOR relative to endocytosed transferrin. Confocal fluorescence microscopy of double-labeled cells stably expressing B2AR (A) or DOR (B) was used to examine colocalization between a 30-min pulse of antibody-labeled receptor (green) followed by a 15-min chase with transferrin (red) in the absence of agonist, as described under “Experimental Procedures.” A representative region of labeled cells is indicated in each panel. Extensive overlap between an internalized pulse of B2AR (A) with the transferrin chase is indicated by the numerousyellow vesicular structures visualized in the merged color image (A), whereas vesicles containing internalized B2AR without detectable labeled transferrin were observed relatively rarely (e.g. arrow). In contrast, in the same experiment conducted using DOR-expressing cells (B), we observed numerous DOR-enriched vesicles that contained no detectable labeled transferrin (arrows). These observations are quantitated inC, which indicates the mean fraction of receptor-containing endocytic vesicles that also contained labeled transferrin. Error bars represent the S.D. of these data collected from multiple cells (n = 6) examined in coded specimens.Bar, 5 μm.