From the Institut de Pharmacologie et de Toxicologie, Faculté de Médecine, 1005 Lausanne, Switzerland
Received for publication, February 28, 2003
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ABSTRACT |
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INTRODUCTION |
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The AP2 complex directly links the clathrin coat with cargo transmembrane proteins that are sorted into coated pits and vesicles (8) and is composed of two large subunits, and
2, of about 100 kDa and two smaller subunits, µ2 and
2, of 50 and 17 kDa, respectively (9). The AP2 adaptor can initiate endocytosis of membrane receptors by either associating directly with their cytoplasmic tail or by interacting with additional molecules, such as
-arrestins, as described for the
2-AR (5, 7). Direct interactions between AP2 and transmembrane proteins have been demonstrated, for example, for the transferrin receptor (10), the epidermal growth factor receptor (11, 12), and the cystic fibrosis transmembrane conductance regulator (13) but not for GPCRs. They are principally mediated by the µ2 subunit, which specifically associates with endocytosis signals including YXX
(where
represents a bulky hydrophobic residue) (14) and dileucine motifs (15) on the cytoplasmic portion of the transmembrane proteins (8). Interestingly, recent evidence suggests that the
2 subunit also participates in the recognition of dileucine motifs on the proteins (16).
Previous reports have shown that the 1b-AR undergoes rapid endocytosis upon exposure to the agonist. This was shown both in DDT1-FM2 smooth muscle cells expressing endogenous receptors as well as in cells expressing the recombinant receptor (17, 18). The molecular determinants involved in agonist-induced endocytosis of the
1b-AR reside within the C-tail of the receptor as demonstrated by the fact that truncation of this region almost completely abolished receptor desensitization and internalization (19). A previous report suggested that the
1b-AR can internalize in clathrin-coated vesicles as shown by that fact that
1b-AR endocytosis can be blocked by hypertonic sucrose and that internalized receptors colocalize with transferrin receptors (18). Moreover, we previously reported that agonist-induced internalization of the
1b-AR is, at least in part, mediated by
-arrestins (20). Despite this experimental evidence, our knowledge on the biochemical mechanisms and the molecular mediators controlling the clathrin-mediated endocytosis of the
1b-AR is still at an early stage.
To identify new proteins interacting with the 1b-AR that could potentially be involved in regulating receptor function, we have used the yeast two-hybrid system and identified the µ2 subunit of the AP2 complex. In this study, we demonstrate that the
1b-AR and µ2 subunit can directly interact through a polyarginine motif located on the C-tail of the receptor and that this interaction plays a role in agonist-induced internalization of the receptor. Our findings highlight a previously unappreciated mechanism that might also be involved in the endocytosis of other GPCRs.
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EXPERIMENTAL PROCEDURES |
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The full-length cDNA encoding the human µ2 subunit was PCR-amplified from EST 3538047 (accession number BE264960 [GenBank] ) and subcloned at HindIII-SalI in pEGFPN3 and pGEX4T1 to generate fusion proteins with GFP at the C terminus and GST at the N terminus of µ2, respectively. The full-length cDNAs encoding human µ1, µ3, and µ4 subunits identified in other AP complexes were PCR-amplified from EST 4954703 (accession number BG920410 [GenBank] ), EST 720361 (accession number AA2611409), and EST 748897 (accession number AI644755 [GenBank] ), respectively, and subcloned at EcoRI/SalI in pEGFPN3 to construct fusion proteins with GFP at the C terminus. For generating fusion proteins with GFP at the C terminus, DNA fragments encoding amino acids 1163, 164282, and 283435 of µ2 were PCR-amplified and subcloned at HindIII/SalI into pEGFPN3.
The full-length cDNA encoding the hamster 1b-AR (21) was PCR-amplified and inserted into the pEGFPN1 using EcoRI/AgeI to fuse GFP at the C terminus of the receptor (
1b-GFP). The T368-GFP and
371378-GFP receptor mutants were constructed by PCR-directed mutagenesis of the
1b-GFP using the Pwo DNA polymerase (Roche Applied Science). To construct HA-tagged receptor forms, the N-terminal fragment of the wild type
1b-AR was amplified using primers encoding the HA (YPYDVPDYA) epitope at the 5' end and subcloned into the pRK5 vector encoding the wild type receptor or its mutants.
Yeast Two-hybrid ScreeningThe yeast strain L40 was transformed with the C-tail-pLexA plasmid encoding the 1b-AR C-tail fused to LexA, and clones were selected and subsequently transformed with 250 µg of a human brain Matchmaker cDNA library in the pACT2 vector (Clontech). Of 13 million double transformants, 40 exhibited moderate to strong growth on histidine-deficient plates. The library plasmids isolated from positive clones were used to cotransform the L40 strain with either the C tail-pLexA or the empty pLexA plasmid, and the specificity of the interactions was confirmed by growth on histidine-deficient plates as well as by
-galactosidase activity (Yeast Protocols Handbook; Clontech).
Expression and Purification of Recombinant Proteins in Bacteria GST-tagged fusion proteins of µ2 and 1b-AR C-tail were expressed using the bacterial expression vector pGEX4T1 in the BL21DE3 strain of Escherichia coli and purified. Bacterial extracts containing GST fusion proteins were prepared by centrifugation of bacterial cultures, followed by lysis of the pelleted bacteria in buffer A (20 mM Tris, pH 7.4, 50 mM NaCl, 5 mM MgCl2, 0.5% (w/v) Triton X-100, 1 mM benzamidine, 2 µg/ml leupeptin, 2 µg/ml pepstatin), sonication, and centrifugation at 38,000 x g for 30 min at 4 °C. After incubating the supernatants with glutathione-Sepharose beads (Amersham Biosciences) for 2 h at 4 °C, the resin was washed with 10 bed volumes of buffer A and stored at 4 °C. GST fusion proteins were eluted from the resin with 5 mM reduced glutathione for 15 min at room temperature, dialyzed, and stored at -20 °C.
His6-tagged fusion protein of the 1b-AR C-tail was expressed using the bacterial expression vector pET30 in BL21DE3 bacteria and purified. Extracts containing His6-tagged fusion proteins were prepared by centrifugation of bacterial cultures and lysis of pelleted bacteria in buffer B (20 mM Hepes, pH 7.8, 500 mM NaCl, 10 mM imidazole, 1 mM benzamidine, 2 µg/ml leupeptin, 2 µg/ml pepstatin). After a 1-min sonication, the lysates were centrifuged at 38,000 x g for 30 min at 4 °C. The His6-tagged fusion proteins were purified by incubating the supernatant with nickel-nitrilotriacetic acid chelating resin (Amersham Biosciences) for 1 h at 4 °C. The resin was then washed five times with 10 bed volumes of buffer B and stored at 4 °C. His6-tagged fusion proteins were eluted from the resin with 20 mM Hepes, pH 7.8, 500 mM NaCl, 300 mM imidazole, 1 mM benzamidine, 2 µg/ml leupeptin, 2 µg/ml pepstatin for 1 h at room temperature, dialyzed, and stored at -20 °C. The protein content of the eluates was assessed by Coomassie staining of SDS-PAGE gels.
Cell Culture and TransfectionsHEK-293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum and gentamycin (100 µg/ml) and transfected at 5080% confluence in 35- or 100-mm dishes using the calcium-phosphate method. After transfection, cells were grown for 48 h in DMEM supplemented with 10% fetal calf serum before harvesting. The total amount of transfected DNA was of about 0.51 µg/35-mm dish and 10 µg/100-mm dish.
GST Pull-down and Immunoprecipitation ExperimentsFor GST pull-down, HEK-293 cells expressing the various constructs grown in 100-mm dishes were lysed in 1 ml of buffer C (20 mM Tris, pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% (w/v) Triton X-100, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and centrifugation at 100,000 x g for 30 min at 4 °C. Glutathione-Sepharose beads coupled to the different GST fusion proteins were incubated with 1.5 mg of proteins derived from the cell lysates in a total volume of 1 ml overnight at 4 °C. The beads were then washed five times with buffer C and resuspended in SDS-PAGE sample buffer. Eluted proteins were analyzed by SDS-PAGE and Western blotting.
For immunoprecipitation experiments, HEK-293 cells expressing the various HA-tagged constructs grown in 100-mm dishes were lysed in 1 ml of buffer D (20 mM Tris, pH 7.4, 100 mM NaCl, 5 mM EDTA, 1% digitonin, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Cell lysates were incubated overnight at 4 °C on a rotating wheel. The solubilized material was centrifuged at 100,000 x g for 30 min at 4 °C, and the supernatant was incubated for 4 h at 4 °C with 5 µg of a rabbit anti-HA polyclonal antibody (Santa Cruz) or with control nonimmune IgG. After the addition of 40 µl of protein A-Sepharose, the incubation was continued for 2 h at 4 °C, followed by a brief centrifugation on a bench top centrifuge. The pellet was washed five times with buffer D and twice with PBS and then dissolved in SDS-PAGE sample buffer for 1 h at 37 °C. Imunoprecipitated proteins were analyzed by SDS-PAGE and Western blotting.
SDS-PAGE and Western BlottingSamples were denatured in SDS-PAGE sample buffer (65 mM Tris, 2% SDS, 5% glycerol, 5% -mercaptoethanol, pH 6.8) for 1 h at room temperature, separated on 10% acrylamide gels and electroblotted onto nitrocellulose membranes. The blots were incubated in TBS-Tween (100 mM Tris, pH 7.4, 140 mM NaCl, 0.05% Tween 20) containing 5% (w/v) nonfat dry milk overnight at room temperature, washed three times with TBS-Tween, and then incubated with the specific primary antibody diluted in TBS-Tween for 2 h at room temperature. After three washes with TBS-Tween, the membranes were probed with horseradish peroxidase-conjugated secondary anti-mouse antibodies (Amersham Biosciences) for 1 h, washed three times with TBS-Tween, and developed using the enhanced chemiluminescence detection system (Amersham Biosciences).
The following affinity-purified primary antibodies were used for immunoblotting: mouse monoclonal anti-HA (200 µg/ml, 1:250 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse monoclonal anti-GFP (400 µg/ml, 1:500 dilution; Roche Applied Science), mouse monoclonal anti-µ2 (1:250 dilution; Transduction Laboratories), mouse monoclonal anti--adaptin (1:250 dilution; Transduction Laboratories), and mouse monoclonal anti-
-adaptin (1:250 dilution; Transduction Laboratories).
Solid Phase Overlay AssayAfter SDS-PAGE and electroblotting of the samples, the nitrocellulose filters were incubated with TBS-Tween containing 5% (w/v) nonfat dry milk and 1% bovine serum albumin for 1 h at room temperature and with 100,000 cpm/µl of32P-labeled His6-tagged 1b-AR C-tail in TBS-Tween containing 5% nonfat dry milk and 0.1% bovine serum albumin for 16 h at room temperature. After extensive washes in TBS-Tween, the blots were visualized by autoradiography.
Purified His6-tagged C-tail (4 µg) was radiolabeled following its incubation with the catalytic subunit of protein kinase A (0.1 µg) and [-32P]ATP (50 µCi) in 50 mM MOPS, pH 6.8, 50 mM NaCl, 2 mM MgCl2, 1 mM dithiothreitol, 0.1 mg/ml bovine serum albumin for 1 h at 30 °C. The radiolabeled protein was separated from free [
-32P]ATP on an Excellulose GF-5 desalting column (Pierce) equilibrated in TBS-Tween.
Confocal MicroscopyHEK-293 cells grown on glass coverslips were transfected with the cDNAs encoding different GFP-tagged receptors. 48 h after transfection, cells were incubated in serum-free DMEM for 1 h and treated for various times with 10-4 M epinephrine (Sigma) at 37 °C. After the treatment, cells were placed on ice, washed twice with ice-cold PBS, fixed for 10 min in PBS plus 3.7% formaldehyde, and mounted using Prolong (Molecular Probes, Inc., Eugene, OR). In the experiments measuring the effect of K44A dynamin mutant on receptor endocytosis, HEK-293 cells were cotransfected with the cDNAs encoding the GFP-tagged 1b-AR and the HA-tagged dynamin K44A mutant. 48 h after transfection, cells were incubated in serum-free DMEM for 1 h and treated for 1 h with 10-4 M epinephrine at 37 °C. After two washes with ice-cold PBS, cells were fixed for 10 min in PBS plus 3.7% formaldehyde and permeabilized for 5 min with 0.2% (w/v) Triton X-100 in PBS. Cells were incubated in PBS plus 1% bovine serum albumin for 1 h and with 1:250 dilution of anti-HA polyclonal antibody (Santa Cruz Biotechnology) for 1 h, followed by another incubation with Texas Red-conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch) for 1 h. The cells were then mounted using Prolong (Molecular Probes). GFP fluorescence or immunofluorescent staining were visualized on a laser-scanning confocal microscope (Zeiss).
Cell Surface Biotinylation ExperimentsHEK-293 cells grown in 100-mm dishes were transfected with the cDNAs encoding the HA-tagged 1b-AR or its T368 and
371378 mutants. 48 h after transfection, cells were incubated in serum-free DMEM for 1 h and treated for various times with 10-4 M epinephrine (Sigma) at 37 °C. After the incubation, cells were placed on ice and washed twice with ice-cold PBS. Surface proteins were biotinylated by incubating cells with 500 µg/ml of the membrane-impermeable biotin analogue sulfo-NHS-S-S-biotin (Pierce) in PBS for 30 min at 4 °C. Unreacted biotin was quenched and removed by three washes with ice-cold TBS at 4 °C. Biotinylated cells were then lysed with hypo-osmotic buffer (10 mM Tris, pH 7.4, 5 mM EDTA, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride), and the cellular homogenate was centrifuged at 30,000 x g for 15 min at 4 °C. The pellet was resuspended in buffer D (20 mM Tris, 100 mM NaCl, 5 mM EDTA, 1% digitonin, 5 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride), sonicated for 30 s, and stirred for 6 h at 4 °C. The solubilized material was centrifuged at 100,000 x g for 30 min at 4 °C, and the supernatant was incubated overnight with 40 µl of streptavidin-Sepharose beads (Amersham Biosciences) at 4 °C. The beads were pelleted by brief centrifugation and washed five times with buffer D and twice with PBS. Biotinylated proteins were eluted by incubating the beads with 200 mM dithiothreitol for 2 h at 37 °C and separated on SDS-PAGE followed by Western blotting. The biotinylated receptors were revealed by immunoblotting using anti-HA monoclonal antibodies (Santa Cruz Biotechnology) as described above. The intensity of the band was quantified by densitometry of films exposed in the linear range, imaged using the Molecular Imager FX (Bio-Rad), and analyzed using NIH Image software (National Institutes of Health). In control experiments, we determined that the biotinylation reagent did not cross the plasma membrane, as shown by the fact that the catalytic subunit of protein kinase A, which is cytoplasmic, could not be detected in streptavidin-Sepharose precipitates (results not shown).
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RESULTS |
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To provide biochemical evidence that the 1b-AR can associate with the µ2 subunit of the AP2 complex, pull-down experiments were performed by incubating the GST-tagged C-tail of the receptor with cell extracts of HEK-293 cells. We found that the µ2 endogenously expressed in HEK-293 cells specifically bound to GST-C-tail but not to GST alone (Fig. 1B).
To assess whether the association between the 1b-AR and the µ2 subunit of the AP2 complex occurs through a direct interaction or is mediated through another protein, we monitored the ability of purified GST-µ2 to associate with the purified His6-tagged C-tail using a solid phase overlay assay. The autoradiography shown in Fig. 1C (left panel) indicates that the C-tail of the32P-labeled His6-tagged C-tail of the
1b-AR could specifically interact with GST-µ2, but not with GST alone. Control experiments showed that the GST-µ2 did not interact with 100 nM of an unrelated radiolabeled protein (regulatory subunit of protein kinase A) (results not shown). Altogether, these results strongly suggest that the µ2 subunit of the AP2 complex can directly interact with the C-tail of the
1b-AR.
Identification of the Binding Site for µ2 on the 1b-ARThe µ2 subunit of the AP2 complex has been shown to recognize endocytosis signals including tyrosine-based motifs (YXX
) and dileucine motifs on the cytoplasmic portion of membrane receptors (9). Analysis of the primary sequence of the C-tail of the
1b-AR revealed the presence of two potential tyrosine-based motifs at positions 386 and 442 and of two potential dileucine motifs at positions 450 and 473 (Fig. 2). Moreover, two additional YXX
sequences can be found at positions 144 and 153 in the second intracellular loop of the receptor (Fig. 2). To assess whether these motifs can mediate the interaction between the
1b-AR and µ2, we generated GST-C-tail fusion proteins in which tyrosines 386 and 442 as well as the leucine doublets 450451 and 473474 were individually substituted by alanines and assessed their ability to interact with µ2 endogenously expressed in HEK-293 cells. Surprisingly, none of these mutations was able to disrupt the interaction between the C-tail of the receptor and µ2 (Fig. 3A). Similar results were obtained using the yeast two-hybrid system as an interaction assay (Fig. 3B). To investigate whether the two YXX
sequences located at positions 144 and 153 of the
1b-AR were involved in the interaction with µ2, we also constructed a GST fusion protein, including the second intracellular loop of the receptor. This fusion protein was not able to interact with the µ2 from HEK-293 cell extracts (results not shown). Altogether, these results suggest that the structural determinants of the
1b-AR mediating its interaction with µ2 are different from the canonical tyrosine-based or dileucine motifs.
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To further investigate the binding site for µ2 on the C-tail of the 1b-AR, we fused to GST a series of fragments of the C-tail carrying progressive truncations (Fig. 2) and assessed their ability to interact with µ2 endogenously expressed in HEK-293 cells. As shown in Fig. 3C, whereas different fusion constructs truncated up to residue 380 could interact with µ2, the GST-T368 fusion construct did not, thus suggesting that the region included between residues 380 and 368 is crucial for this interaction. Interestingly, the deletion of eight arginines at positions 371378 completely abolished the binding of the C-tail to µ2, suggesting that the
1b-AR interacts with the µ2 subunit of the AP2 complex through a novel arginine-based binding domain (Fig. 2).
Identification of the Binding Site for the 1b-AR on µ2 Recently, the crystal structure of the µ2 subunit of the AP2 complex has been solved (12, 24). The first 157 residues of the protein are organized in a predominantly
-helical structure, whereas the C-terminal fragment of µ2 is largely composed of
-sheet structures that are folded into two subdomains (Fig. 4A) (12). The first subdomain, comprising residues 158282, contains the binding site for tyrosine-based endocytic motifs, whereas the second subdomain, comprising residues 283435, contains an interaction site for synaptotagmin, a neuronal AP2-binding protein involved in synaptic vesicles exocytosis (25).
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To identify the region within µ2 that interacts with the C-tail of the 1b-AR, we tagged with GFP the three fragments of the µ2, 1157, 158282, and 283435, and expressed them in HEK-293 cells. Pull-down experiments were performed by incubating the GST-C-tail construct with cell extracts overexpressing different GFP-tagged µ2 fragments (Fig. 4A). We found that the 1157 and 282435 fragments retained the ability to bind the C-tail of the
1b-AR, whereas the 158257 fragment did not (Fig. 4B). These findings led us to conclude that the molecular determinants of µ2 involved in the recognition of the polyarginine motif on the C-tail of the
1b-AR are located on two domains at the N and C terminus, respectively, of the µ2 molecule. Interestingly, these domains are distinct from the µ2 region that binds tyrosine-based internalization signals, which is located between residues 157 and 282 (12).
The 1b-AR/µ2 Interaction Occurs in the Cells and Is Regulated by Agonist-induced Receptor ActivationTo demonstrate that the
1b-AR and µ2 can form a complex inside the cells, we performed coimmunoprecipitation experiments from HEK-293 cells transiently expressing the HA-tagged
1b-AR. After immunoprecipitating the receptor using polyclonal anti-HA antibodies, monoclonal anti-HA as well as anti-µ2 antibodies were used to immunoblot the immunoprecipitated samples. The Western blots revealed that the µ2 endogenously expressed in HEK-293 cells could specifically co-immunoprecipitate with the
1b-AR, whereas no bands were immunoprecipitated by IgG (Fig. 5A, top panel, lanes 2 and 3). These findings demonstrate that beyond their ability to interact in vitro, the
1b-AR and µ2 can associate inside the cells.
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To confirm that the 1b-AR/µ2 interaction in intact cells was mediated by the polyarginine motif identified above as the binding site for µ2, coimmunoprecipitation experiments were performed from HEK-293 cells overexpressing the HA-tagged receptor mutants truncated at residue 368 (T368) or carrying a deletion of the polyarginine motif (
371378). The T368 and
371378 receptor mutants displayed pharmacological properties similar those of the wild type
1b-AR (results not shown). In addition, as previously shown (26), tagging the wild type or mutated receptors with HA or GFP at their N and C terminus, respectively, did not affect the pharmacological properties of the receptor (results not shown). As shown in Fig. 5A, the endogenous µ2 did not coimmunoprecipitate either with the T368 or with the
371378 receptor mutants, thus suggesting that the polyarginine stretch between residues 371 and 378 represents the only binding site of the
1b-AR for µ2.
To investigate whether the whole heterotetrameric AP2 complex could interact with the 1b-AR, we determined whether additional subunits of the AP2 complex could be coimmunoprecipitated with the receptor. As shown in Fig. 5B, the
and
2 subunits endogenously expressed in HEK-293 cells were coimmunoprecipitated with the wild type receptor but not with the
371378 receptor mutant, thus suggesting that the whole AP2 complex can associate with the
1b-AR through the interaction mediated by its µ2 subunit.
To assess whether the 1b-AR/µ2 interaction could be modulated by the agonist-induced activation of the receptor, HEK-293 cells expressing the HA-tagged
1b-AR were incubated for 15 min in the absence or presence of 10-4 M epinephrine prior to immunoprecipitation of the receptor. As shown in Fig. 6, treatment with epinephrine induced a 2-fold increase in the amount of endogenous µ2 coimmunoprecipitated with receptor when compared with untreated cells (Fig. 6, A (lanes 6 and 8) and B). This strongly suggests that the
1b-AR/µ2 interaction is dynamically regulated by the agonist-induced activation of the
1b-AR, which might increase the amount of µ2 associated with the receptor.
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Specificity of the 1b-AR/µ2 InteractionSo far, four different adaptor protein complexes (APs) involved in sorting of membrane proteins have been identified and characterized (9). AP1 is involved in the formation of clathrin-coated vesicles from the trans-Golgi network and the trafficking of proteins from the trans-Golgi network to the plasma membrane (8). AP2 plays a role in the clathrin-mediated endocytosis of plasma membrane receptors (8). AP3 has been shown to mediate the sorting of proteins form early endosomes to lysosomes (27). Finally, AP4 has been shown to participate in the polarized transport of proteins to the basolateral membrane in Madin-Darby canine kidney cells (28). To assess whether the
1b-AR preferentially associates with the AP2 as compared with other AP complexes, we performed coimmunoprecipitation experiments from HEK-293 expressing the HA-tagged
1b-AR and GFP-tagged µ1, µ2, µ3, and µ4 subunits. Interestingly, Western blotting using anti-GFP antibodies indicated that, whereas µ2 could be coimmunoprecipitated with the HA-tagged
1b-AR, µ1, µ3, and µ4 could not (results not shown). This suggests that, inside the cells, the
1b-AR specifically associates with the µ2 subunit of the AP2 complex rather than with other AP complexes.
The 1b-AR/µ2 Interaction Is Involved in Clathrin-mediated Endocytosis of the ReceptorTo confirm that the
1b-AR can internalize in clathrin-coated vesicles, we assessed whether hypertonic sucrose and the overexpression of a dominant negative mutant of dynamin (K44A) could inhibit the agonist-induced internalization of the GFP-tagged
1b-AR transiently expressed in HEK-293 cells. Incubation of cells with 0.45 M sucrose for 15 min prior to stimulation with epinephrine completely inhibited agonist-induced internalization (Fig. 7, C and D). Similarly, overexpression of the K44A dynamin mutant impaired receptor endocytosis when compared with control cells (Fig. 7, E and F). These results support the notion that in HEK-293 cells the
1b-AR undergoes internalization through clathrin-mediated endocytosis.
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It is well established that the AP2 complex controls the early steps of membrane receptor endocytosis, allowing clathrin to be recruited to the receptor. Since the 1b-AR represents the first GPCR to be shown to directly interact with AP2, and since this interaction seems to occur through a noncanonical µ2 binding motif on the receptor, we sought to establish whether the
1b-AR/µ2 interaction might play a role in the regulation of receptor endocytosis.
To test this hypothesis, we determined whether deleting the µ2 binding site on the C-tail of the 1b-AR could affect agonist-induced receptor internalization. The GFP-tagged forms of the wild type
1b-AR (WT-GFP) and of its mutants T368 (T368-GFP) and
371378 (
371378-GFP) were transiently expressed in HEK-293 cells and tested for their ability to undergo agonist-induced internalization. Cells expressing the different GFP-tagged receptors were treated with 10-4 M epinephrine for 15, 30, and 60 min, and agonist-induced internalization was assessed by monitoring receptor redistribution by confocal microscopy. As shown in Fig. 8 (upper panels), incubation of cells expressing the WT-GFP receptor with epinephrine caused a rapid redistribution of the receptor from the cell surface to intracellular compartments. Internalization was already detectable 15 min after agonist exposure, whereas a more pronounced redistribution was observed after 60 min. The T368-GFP receptor was completely impaired in its ability to undergo internalization (Fig. 8, middle panels) in agreement with our previous findings indicating that the integrity of the C-tail is required for receptor endocytosis and desensitization (19). Interestingly, epinephrine-induced endocytosis of the
371378-GFP receptor mutant was delayed as compared with that of the WT-GFP receptor (Fig. 8, lower panels). In fact, internalization was barely detectable after 30 min of exposure to the agonist, and at 60 min a significant portion of the receptor was still present at the cell surface. Altogether, these results strongly suggest that the
1b-AR/µ2 interaction is involved in receptor internalization, since receptor mutants lacking the binding site for µ2 are clearly impaired in receptor endocytosis.
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To quantify receptor endocytosis, we used a biotinylation assay to selectively label the receptors expressed at the cell surface. HEK-293 cells expressing HA-tagged forms of the wild type 1b-AR (HA-WT) and of its mutants T368 (HA-T368) and
371378 (HA-
371378) were exposed to 10-4 M epinephrine for various times and subsequently incubated with a membrane-impermeant biotinylation reagent (sulfo-NHS-biotin) (see "Experimental Procedures"). Biotinylated cell surface receptors were precipitated using streptavidin-Sepharose beads and detected by Western blotting using anti-HA antibodies. The results of this biochemical assay indicated that the HA-WT, HA-T368, and HA-
371378 receptors were expressed at similar levels at the cell surface (Fig. 9, lanes 1, 5, and 9). HA-tagged receptors could not be detected in streptavidin-Sepharose precipitates when cells were not incubated with the biotinylation reagent, thus confirming that, in our experimental conditions, only biotinylated receptors could be isolated (results not shown).
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Treatment of cells expressing the HA-WT receptor with 10-4M epinephrine induced a rapid receptor endocytosis, which was 23% after 15 min, 61% after 30 min, and 80% after 60 min (Fig. 9, lanes 14). As expected, the HA-T368 receptor mutant was resistant to agonist-induced internalization as demonstrated by the fact that 94% of the receptors were still present at the cell surface after 60 min of exposure to epinephrine (Fig. 9, lanes 58). In agreement with the results of confocal imaging (Fig. 8), epinephrine-induced internalization of the HA-371378 receptor mutant was significantly decreased compared with that of the wild type receptor with 60% of the receptor remaining at the cell surface after a 60-min exposure to the agonist (Fig. 9, lanes 912). Altogether, these findings support the hypothesis that agonist-induced internalization of the
1b-AR is controlled, at least in part, by the interaction of µ2 with the C-tail of the receptor.
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DISCUSSION |
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One unexpected finding of our study was the observation that µ2 recognizes a noncanonical binding motif on the 1b-AR, which consists of eight consecutive arginines included between residues 371 and 378 of the C-tail of the receptor (Fig. 3). Deletion of this motif completely abolished the
1b-AR/µ2 interaction inside the cells measured in the coimmunoprecipitation experiments (Fig. 5). Interestingly, the deletion of the µ2 binding motif also abolished the interaction of the
1b-AR with the
and
subunits of the AP2 complex, suggesting that this motif represents the only point of contact between the AP2 complex and the receptor. The polyarginine motif of the
1b-AR is reminiscent of the binding site for µ2 previously identified in synaptotagmin, a neuronal AP2 binding proteins involved in recycling of synaptic vesicles, which contains six positively charged residues (KRLKKKK) (29).
The recent publication of the crystal structure of the entire AP2 complex has provided a better understanding of how the different subunits of the complex contact each other and of how the µ2 subunit interacts with tyrosine-based endocytic motifs located on membrane receptor (24). Whereas the YXXF motifs have been shown to bind to a hydrophobic pocket located in the subdomain A (residues 157282) of µ2, recent studies indicate that the positively charged motif of synaptotagmin contacts the subdomain B (residues 283435) of µ2 (25). Similarly, we could show that the subdomain B (residues 283435) together with the N-terminal region of µ2 (residues 1157) participate in the interaction with the polyarginine motif of the 1b-AR (Fig. 4). Therefore, it appears that the N-terminal region and the subdomain B of µ2 might provide a docking surface for positively charged motifs exposed on the cytoplasmic face of membrane receptors. A systematic scanning mutagenesis of these domains will be required to determine whether the
1b-AR and synaptotagmin bind to similar structural determinants of the µ2.
The 1b-AR/µ2 interaction is dynamically regulated as demonstrated by the fact that activation of the receptor by the agonist increases the association of µ2 to the receptor by 2-fold (Fig. 6). This suggests that µ2 preferentially recognizes the agonist-occupied form of the
1b-AR. One can speculate that binding of epinephrine to the
1b-AR might trigger a conformational change that exposes the polyarginine motif, thus promoting the association of the receptor with the AP2 complex. A similar model has been proposed for the epidermal growth factor receptor-mediated recruitment of the AP2 complex in which the receptor exposes a high affinity binding site for µ2 only upon activation by epidermal growth factor (11, 12).
An important finding of our study is that the interaction of the 1b-AR with µ2 of the AP2 complex plays a role in receptor endocytosis. This was mainly demonstrated by the fact that deleting the µ2 binding motif in the C-tail of the
1b-AR markedly decreased agonist-induced receptor internalization as shown by confocal microscopy (Fig. 8) as well as by surface receptor biotinylation (Fig. 9). Hypertonic sucrose as well as the dominant negative dynamin mutant K44A almost completely inhibited epinephrine-induced endocytosis of the GFP-tagged
1b-AR, thus supporting the notion that the
1b-AR can internalize in clathrin-coated vesicles (Fig. 7). Since the AP2 complex links the clathrin coat to transmembrane proteins sorted into coated pits, the
1b-AR/µ2 interaction might represent a mechanism directly involved in targeting the
1b-AR to clathrin-coated vesicles.
In a previous study, we reported that agonist-induced internalization of the 1b-AR is, at least in part, mediated by
-arrestins. This was mainly demonstrated by two observations: (a) the stimulation of the
1b-AR with epinephrine induced a marked translocation to the cell surface of
-arrestin; (b) a dominant negative mutant of
-arrestin 1 (V53D) decreased the internalization of the
1b-AR (20). Therefore, in addition to the direct association of the receptor with the AP2 complex,
-arrestins also play an important role in the clathrin-mediated endocytosis of the
1b-AR. This is supported by the observation that the deletion of the µ2 binding motif did not completely abolish receptor internalization, suggesting that additional mechanisms regulate the endocytosis of the
1b-AR (Figs. 8 and 9). Interestingly, overexpression of a dominant negative mutant of
-arrestin 1 (V53D) (30) abolished the residual internalization observed for the
371378 mutant of the
1b-AR lacking the µ2-binding site (results not shown). Additional determinants involved in receptor endocytosis are likely to be localized on the C-tail of the
1b-AR, since the T368 receptor mutant, lacking most of the C-tail, was almost totally impaired in its ability to undergo agonist-induced internalization (Figs. 8 and 9). Altogether, these findings suggest that agonist-induced endocytosis of the
1b-AR results from multiple mechanisms involving the interaction of the receptor with both the AP2 complex and
-arrestins.
The findings of our study suggest that the molecular mechanisms underlying the internalization of the 1b-AR seem to differ from those controlling the endocytosis of other GPCRs, like the
2-AR, for which it is believed that their redistribution to clathrin-coated vesicles is mediated by
-arrestins. However, the possibility that GPCRs can directly interact with the AP2 complex has not been investigated so far, and it will be important to establish whether this interaction represents a common mechanism occurring at other GPCRs in addition to the
1b-AR. The discovery that the AP2 complex can directly interact with the
1b-AR raises several questions about the molecular mechanisms underlying receptor endocytosis. In particular, future studies will aim at elucidating the relationship between the structural determinants of the
1b-AR involved in binding the AP2 complex versus
-arrestins, the respective role of the AP2 complex and
-arrestins in targeting the receptor to clathrin-coated vesicles as well as their interplay with other yet unidentified mechanisms regulating receptor trafficking and function.
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FOOTNOTES |
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To whom correspondence should be addressed: Institut de Pharmacologie et de Toxicologie, Rue du Bugnon 27, 1005 Lausanne, Switzerland. Tel.: 41-21-692-5400; Fax: 41-21-692-5355; E-mail: Susanna.Cotecchia{at}ipharm.unil.ch.
1 The abbreviations used are: GPCR, G protein-coupled receptor; AR, 1b-adrenergic receptor; AP1, -2, -3, and -4, adaptor complex 1, 2, 3, and 4, respectively; EST, expressed sequence tag; GFP, green fluorescent protein; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; HA, hemagglutinin; NHS, N-hydroxysuccinimide.
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ACKNOWLEDGMENTS |
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REFERENCES |
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