Introduction

Summary Introduction Procedures Results Discussion References

The ₂-AR family consists of three highly homologous subtypes: _2a, _2b, and _2c (1, 2). They belong to the superfamily of G protein-coupled receptors and mediate the physiological actions of the endogenous catecholamines, epinephrine and norepinephrine. These receptors are targets for therapeutic agents for hypertension, anesthesia, and analgesia. Although the three ₂-ARs are highly homologous (50-60% identity), several differences in their coupling to G proteins  subunits and their desensitization have been reported. In reconstitution experiments. Kurose et al. (3) have shown that the human _2a- and _2c-ARs couple to the same G proteins, with only slight differences in efficiency. In contrast, using transfected cell lines, others (4) have shown that the _2c subtype (the rat RG10 receptor) preferentially couples to G_o, whereas _2a- and _2b-ARs couple to G_i (5). Initial studies indicated that ₂-ARs transduce their signal through a pertussis toxin-sensitive G protein (G_i or G_o); however, several more recent studies have demonstrated that all three ₂-AR subtypes can stimulate adenylyl cyclase in pertussis toxin-treated cells (6). The EC₅₀ value of agonists for stimulation of adenylyl cyclase by ₂-ARs, presumably through G_s, is higher than the EC₅₀ value for inhibition of adenylyl cyclase through G_i (6). The three ₂-AR subtypes seem to differ in their efficiency of coupling to G_s (6). Moreover, the capacity and efficacy of G_s coupling by the three ₂-ARs are dependent on the agonist (7) and the cell line used to express the ₂-AR subtype (6, 8). Similarly, others have shown that depending on the cell line studied, ₂-ARs can inhibit or increase cellular cAMP levels, suggesting that these differences are due to coupling to different G proteins or isoforms of adenylate cyclase (9). Cotecchia et al. (10) demonstrated that _2a- and _2c-ARs can stimulate phospholipase C activity. The _2a-AR has also been shown to inhibit voltage-dependent calcium currents and increase inwardly rectifying potassium currents (11); however, the ability of _2b and _2c to couple to these ion channels has not been reported. Finally, differences in receptor regulation have been described for the three ₂-ARs. Agonist-promoted desensitization has been observed for all ₂-AR subtypes; however, the extent of desensitization is greater for the _2a subtype (12, 13). It has also been shown that phosphorylation of the _2a-AR is required for desensitization (14, 15).

In addition to these functional differences in signal transduction and regulation, recent studies have identified differences in the intracellular trafficking of this receptor family. The _2a- and _2b-ARs have been observed to reside primarily in the plasma membrane, whereas a large portion of the _2c-AR is found in an intracellular compartment (16, 17). We previously reported that there is no detectable agonist-induced internalization of the _2a-AR in cells in which rapid agonist-induced internalization of the ₂-AR is seen (16). Previously, agonist-induced internalization of the _2b and _2c-AR subtypes have not been thoroughly characterized using immunocytochemical techniques; however, these receptors are structurally and functionally more similar to the _2a-AR than to the ₂-AR and might be predicted to have similar trafficking properties. In this study, we examined the steady state distribution and agonist-induced subcellular sorting of each of the three ₂-AR subtypes in the same cell line. We observed that each subtype has distinctive trafficking properties. The _2a- and _2b-ARs are located on the plasma membrane at steady state, whereas most of the _2c-AR is found in the endoplasmic reticulum. The _2b- and _2c-AR subtypes are internalized into endosomes after exposure to agonists, whereas the _2a subtype is not.

	Experimental Procedures

Summary Introduction Procedures Results Discussion References

Materials. [³H]Atipamezole (50 Ci/mmol) and dexmedetomidine were kindly provided by Orion-Farmos (Turku, Finland). All other ligands and chemicals were purchased from Sigma Chemical (St. Louis, MO) unless indicated.

Plasmid construction and receptor expression in tissue culture cells. All cells were grown in DMEM (University of California, San Francisco Cell Culture Facility) supplemented with 10% fetal bovine serum (Gemini Bio-Products, Calabasas, CA) and 25 µg/ml gentamicin (Boehringer-Mannheim Biochemicals, Indianapolis, IN). The three mouse ₂-ARs were cloned into pBC 12BI, pREP4, or pcDNA3 expression vectors (InVitrogen, San Diego, CA) and epitope-tagged as previously described (16). Briefly, the 12CA5 epitope (sequence MGYPYDVPDYA) was inserted at the amino terminus of the three mouse ₂-ARs, using oligonucleotide linker-adapters into the NcoI site located at the 5-end of the receptor coding sequence. The sequence of the recombinant ₂-ARs was confirmed by dideoxy sequencing. To monitor the trafficking pathway of ₂-ARs between intracellular organelles and the cell surface, a double epitope tag with a thrombin cleavage site between the two epitopes was added to the amino termini of the _2a- and _2c-ARs (see Fig. 6). For this purpose, part of the extracellular amino-terminal sequence of a flag epitope-tagged thrombin receptor (18, 19) was combined with the coding sequences of the murine _2a- and _2c-ARs. The thrombin epitope tag consists of the following domains: prolactin signal sequence, flag epitope (italic), amino acids 53-64 of the human thrombin receptor containing a thrombin cleavage site (dash) and the hirudin domain epitope (bold) (20) (amino acid sequence: MDSKGSSLLCQGVVSDYKDDDDVDATLDPR-SFLLRNPNDKY EPFWEDEEKNESGLTEYRLVSINDS). The sequence of the recombinant ₂-ARs was confirmed by dideoxy sequencing and subcloned into the pREP4 plasmid for expression in eukaryotic cells. The thrombin epitope tag can be detected by immunostaining using the monoclonal M1 antibody (Kodak IBI, Rochester, NY) recognizing the flag epitope and a polyclonal antiserum that was raised against a peptide containing the hirudin epitope domain of the human thrombin receptor (19). Epitope-tagged receptors were transfected by either DEAE-Dextran, calcium-phosphate precipitation, or electroporation methods. Stable cell lines were selected under 250-500 µg/ml G418 (GIBCO, Grand Island, NY) or 200 IU/ml hygromycin (Boehringer-Mannheim) in the media.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Amino-terminal thrombin/M1 Flag epitope-tagged 2c-AR. A recombinant 2c-AR was constructed with an additional amino-terminal sequence containing the M1 Flag epitope (first box), preceding a thrombin cleavage site (arrow) and the hirudin epitope (second box). When this epitope-tagged receptor is present in the plasma membrane of intact cells, thrombin treatment cleaves off the flag epitope, resulting in a loss of staining for flag, but retains staining for hirudin. Therefore, this construct allowed the differentiation between intracellular 2c-AR, which had not yet been delivered to the plasma membrane (stains positive for both epitopes), and receptor presented to the plasma membrane, which had its M1 flag epitope irreversibly cleaved by thrombin (stains positive for hirudin only). This thrombin epitope-tagged receptor binds the antagonist atipamezole and agonists epinephrine and norepinephrine similar to wild-type 2c-AR (Table 1) and shows subcellular distribution typical of the wild-type 2c-AR (see Fig. 7, E and F).

Radioligand binding assay. For binding studies, COS-7 cells were transiently transfected using the DEAE-Dextran method, and cell membranes were harvested 3 days after transfection as previously described (21). Binding experiments were performed in 500-µl volumes of binding buffer (75 mM Tris, 12.5 mM MgCl₂, and 1 mM EDTA, pH 7.4) for 90 min at room temperature. The bound radioactivity was separated from free by filtration through GF/C filters and washed three times with 5 ml of ice-cold binding buffer using a Brandel cell harvester. Saturation isotherms were performed by incubating the membranes with varying concentrations of [³H]atipamezole. Nonspecific binding was determined by the addition of 100 µM yohimbine. Competition binding experiments were carried out by incubating membranes with varying concentrations of competing ligand and 2 nM [³H]atipamezole. Nonspecific binding was determined by the addition of 100 µM yohimbine. Equilibrium dissociation constants were determined from saturation isotherms and competition curves using GraphPAD software (San Diego, CA). All binding experiments were done in duplicate and repeated at least three times.

Production of antisera. The antigenic epitopes selected for the polyclonal antibodies were the carboxyl termini of each ₂-AR. The antigenic peptides were synthesized and MBS-coupled to thyroglobulin, purified, and injected into New Zealand White rabbits. Polyclonal sera directed against the _2b and _2c epitopes were affinity-purified over peptide coupled to epoxy-activated Sepharose columns and eluted with either 3 M KSCN, pH 7.4, or 20 mM glycine, pH 3.0. The three polyclonal antisera are specific for their respective ₂-AR subtype and do not cross-react with other ₂-AR subtypes at the dilutions used (_2a, 1:500; _2b and _2c affinity-purified antisera, 1:100; data not shown). The previously described ₂ antibody was kindly provided by Dr. M. Von Zastrow (University of California, San Francisco).

Immunocytochemical studies and techniques. Receptor trafficking and subcellular distribution were examined using indirect immunocytochemical staining. Nonspecific binding was blocked with blotto (5% nonfat dry milk in 50 mM Tris, pH 7.6) for 45 min. For staining in permeabilized cells, nonionic detergent (Nonidet P-40, Sigma) was added to blotto incubations to a final concentration of 0.2%. Cells were stained using 12CA5 hemagglutinin antibody (Berkeley Antibody Co., Berkeley, CA) monoclonal antibody (at 1:500 in blotto). For nonpermeabilized studies, receptors present on the plasma membrane were stained by incubation of live cells grown on coverslips at 37° with 6% CO₂ for 60 min with 12CA5 monoclonal antibody at 1:500 in DMEM supplemented with 10% fetal calf serum. After a 60-min incubation, cells were washed with cold PBS and fixed with 4% formaldehyde for 30 min and then rinsed three times in room-temperature PBS. Nonpermeabilized immunocytochemistry was also performed by growing cells on coverslips for 2 or 3 days and then fixing them for 30 min in 4% formaldehyde and deleting detergent from the blotto. Secondary antibodies were either fluorescein-conjugated (fluorescein isothiocyanate) or Texas red-conjugated directed against the species of primary antibody used (Jackson ImmunoResearch, West Grove, PA).

ELISA. For quantification of receptor internalization, ELISA assays were performed. Briefly, nonclonal stable HEK 293 cell lines were plated onto 24-well tissue culture dishes. To improve adhesion of this cell line to plastic, the wells were treated for 30 min with 0.0005% poly-L-lysine in PBS immediately before plating out ~10 × 10⁴ cells/well. At ~48 hr later, when cells were ~75% confluent, they were incubated for 30 min at 37° with 6% CO₂ in serum-free DMEM or 10 µM norepinephrine in serum-free DMEM. After incubations, the medium was aspirated, and cells were fixed with 4% paraformaldehyde for 5 min at room temperature. Wells were washed three times with room-temperature PBS. Nonspecific binding was blocked with 1% BSA for 45 min with occasional gentle shaking. The 12CA5 antibody was diluted to 1:1000 in 1% BSA for 60 min, followed by three gentle PBS washes. Cells were briefly reblocked with 1% BSA for 15 min and then incubated with anti-mouse conjugated alkaline phosphatase (BioRad, Richmond, CA) diluted 1:1000 in 1% BSA for 60 min with occasional gentle shaking. Wells were then washed three times with PBS, and a colorimetric alkaline phosphate substrate was added. Plates were continuously but gently shaken (40 rotations/min) until an adequate color change had occurred, at which time a 100-µl sample was taken for colorimetric readings. Nontransfected cells were studied concurrently to determine background. All experiments were done in triplicate.

	Results

Summary Introduction Procedures Results Discussion References

Immunocytochemical techniques were used to examine the distribution of ₂-AR subtypes expressed in COS 7, HEK 293, MDCK, NRK and Rat1 fibroblast cell lines. Subtype-specific rabbit polyclonal antibodies were prepared against a carboxyl-terminal peptide as described in Experimental Procedures. The specificity of these antibodies was verified by staining untransfected COS 7 cells or COS 7 cells transfected with each of the ₂-AR subtypes (data not shown). In addition, we used receptors modified with an amino-terminal 12CA5 epitope, which is recognized by a commercially available monoclonal antibody. The amino-terminal tag permits examination of cell surface receptor density in nonpermeabilized cells. We previously observed that the use of this epitope does not alter the trafficking of the ₂-AR (16). Moreover, the distribution and trafficking of epitope tagged ₂-ARs (examined with either 12CA5 monoclonal antibody or polyclonal subtype-specific antibody) are indistinguishable from those of nontagged receptors (examined with polyclonal antibody; data not shown). Binding affinity for the antagonist atipamezole, the endogenous catecholamines epinephrine and norepinephrine, and the specific ₂ agonist dexmedetomidine are also comparable between wild-type and epitope-tagged ₂-ARs (Table 1).

View this table: [in this window] [in a new window]   TABLE 1 Equilibrium dissociation constants of various adrenergic ligands for wild-type and amino-terminal 12CA5 or thrombin epitope-tagged mouse 2-adrenergic receptors expressed in COS-7 cells Binding assays were performed as described in Experimental Procedures. Values (in nM) represent an average of at least three independent binding assays.

Fig. 1 shows the distribution of the three ₂-AR subtypes in HEK 293 cells at steady state and after agonist treatment. It can be seen that _2a and _2b subtypes are predominantly localized in the plasma membrane at steady state, whereas a large portion of _2c-AR is found in an intracellular compartment. After agonist treatment (10 µM norepinephrine) for 30 min at 37°, the _2b-AR is internalized, but the _2a subtype remains in the plasma membrane. This analysis does not allow us to determine whether the _2c-AR is internalized because of the large background of ₂-AR already in an intracellular membrane compartment. This intracellular pool of _2c-AR is be further characterized below.

View larger version (78K): [in this window] [in a new window]   Fig. 1.   Distribution of the three 2-AR subtypes in permeabilized HEK 293 cells at steady state and after agonist treatment. Agonist treatment, cell fixation, permeabilization, and staining are described in Experimental Procedures. A, At steady state, the 2a-AR was primarily localized to the plasma membrane. B, After a 30-min incubation with 10 µM norepinephrine, the 2a-AR did not redistribute. C, Similar to the 2a, at steady state the 2b-AR was primarily localized in the plasma membrane. D, However, 10 µM norepinephrine treatment caused the 2b-AR to internalize into intracellular vesicles. E, In contrast to 2a- and 2b-AR sorting at steady state, the 2c-AR was primarily localized in an intracellular compartment; the 2c-AR appears as punctate intracellular staining. In nonpermeabilized cells at steady state, there is a relatively small amount of the 2c-AR present in the plasma membrane (data not shown). F, A 30-min incubation with 10 µM norepinephrine did not cause notable redistribution of the 2c-AR.

To further investigate differences in agonist-mediated internalization, 12CA5 epitope-tagged _2b-AR was coexpressed with a nontagged _2a-AR, allowing us to examine differences in trafficking between these two subtypes in the same cell. The _2a distribution was monitored with the polyclonal _2a subtype-specific antibody, and the epitope-tagged _2b-AR distribution was monitored with a monoclonal antibody to the 12CA5 epitope. At steady state, both receptors are found in the plasma membrane of the same cell (Fig. 2, A and B). After exposure to 10 µM norepinephrine for 30 min, only the _2b-AR is internalized (Fig. 2, C and D). Similar treatment with 10 nM of the nonselective ₂ agonist dexmedetomidine also selectively internalized the _2b-AR in cotransfected cells (Fig. 2, E and F). Additional agonist treatment paradigms with the _2a-AR (30-min incubations with 10 µM epinephrine or 100 µM norepinephrine or 4-hr incubation with 10 µM norepinephrine) resulted in similar findings (data not shown).

View larger version (80K): [in this window] [in a new window]   Fig. 2.   Receptor distribution after agonist treatment in HEK 293 cells coexpressing 2a- and 2b-ARs. The 12CA5 epitope-tagged 2b-AR was transiently transfected into a nonclonal HEK 293 cell line stably expressing the 2a-AR. After treatment, cells were fixed and permeabilized as described in Experimental Procedures. At steady state, receptor was localized to the plasma membrane in cells expressing both (A) 2a- and (B) 2b-AR subtypes. C, After a 30-min incubation with 10 µM norepinephrine, the 2a-AR did not redistribute. D, In contrast, agonist treatment caused the 2b-AR coexpressed in the same cell to internalize into endosomes. Similar treatment with a 10 nM concentration of the nonselective 2 agonist dexmedetomidine also selectively internalized the 2b-AR (F), whereas no 2a-AR internalization could be detected in the same cells (E).

The _2b- and ₂-ARs internalize by a similar mechanism. The mechanism of agonist-mediated internalization of the ₂-AR has been well characterized. We therefore coexpressed the epitope-tagged _2b-AR along with the ₂-AR and examined the distribution of the two receptors after agonist treatment. At steady state, both receptors are on the plasma membrane (Fig. 3, A and B). Only the ₂-AR is internalized in cells exposed to the -AR agonist isoproterenol (Fig. 3, C and D), and only the _2b-AR is internalized in cells exposed to the ₂-AR agonist dexmedetomidine (Fig. 3, E and F). Using confocal microscopy, we demonstrated that both receptors are internalized by exposure to the common agonist norepinephrine and seem to reside in the same endocytic vesicles (Fig. 3, G and H).

View larger version (78K): [in this window] [in a new window]   Fig. 3.   Effect of subtype-selective and -nonselective agonist treatment of HEK 293 cells coexpressing 2b- and 2-ARs. The 12CA5 epitope-tagged mouse 2b-AR was transiently cotransfected with the human 2-AR in HEK 293 cells. At ~24 hr after transient transfection, cells were plated onto glass coverslips and grown for 2 days. For treatments, coverslips were incubated for 30 min in serum-free DMEM (control) or with the appropriate agonist in serum-free DMEM at 37° and 6% CO2. After fixation, cells were permeabilized before staining. A and B, At steady state, both receptors are localized in the plasma membrane. In cells coexpressing both ARs, 10 µM isoproterenol, a -selective agonist, induced internalization of 2-ARs (D), whereas 2b-ARs did not redistribute (C). Similarly, 10 nM dexmedetomidine, an 2-selective agonist, promoted internalization of 2b-ARs (E), whereas 2-ARs did not redistribute (F). When cells coexpressing both 2b- and 2-ARs were treated with 10 µM norepinephrine, a common agonist for 2- and 2-ARs, both receptors internalized into the same endocytic vesicles as observed by confocal microscopy (G and H, respectively).

The _2c-AR is found in the endoplasmic reticulum in Rat1 fibroblast cells. Although there is some plasma staining, the _2c-AR seems to reside primarily in an intracellular compartment in HEK 293 cells (Fig. 1C). To further study this, we expressed the _2a- and _2c-ARs in a variety of cell lines. Similar to studies reported by Wozniak et al., (17) when expressed in a MDCK II cell line, the abundance of _2c-AR was localized in an intracellular compartment, whereas the _2a-AR was targeted to the basolateral plasma membrane (Fig. 4, A and B). When the 12CA5 epitope-tagged _2c-AR was transiently cotransfected in an HEK-293 cell line stably expressing wild-type _2a-AR, the _2c subtype was also localized to an intracellular compartment (Fig. 4, C and D). Coexpression of the _2c did not alter the trafficking of the _2a-AR subtype. We attempted to quantify the amount of 12CA5 epitope-tagged _2c-AR in the plasma membrane using the ELISA method discussed above. In contrast to the _2a- and _2b-AR subtypes, the signal generated from _2c-AR transfected cells was not significantly different from that of untransfected cells. Thus, although we were able to detect a small amount of 12CA5 epitope-tagged _2c-AR in the plasma membrane of nonpermeabilized cells by the sensitive technique of immunofluorescence microscopy, the amount was too small to quantify by ELISA.

View larger version (61K): [in this window] [in a new window]   Fig. 4.   Distinct subcellular localization of 2a- and 2c-ARs at steady state. Immunolocalization of 12CA5 epitope-tagged (A) 2a- and (B) 2c-ARs in permeabilized MDCK cells is shown. A, At steady state in stably transfected MDCK cells, the 2a-AR was localized to the basolateral plasma membrane. B, The 2c-AR subtype was predominantly localized in an intracellular compartment when stably transfected in MDCK cells. Consistent with our findings of subcellular distribution in other cell lines, a relatively small amount of this receptor is localized in the basolateral plasma membrane (arrows). The 12CA5 epitope-tagged 2c-AR was also transiently cotransfected in HEK 293 cells stably expressing the wild-type 2a-AR. C, Field of permeabilized cells viewed by confocal microscopy at 40× magnification. D, Confocal image at higher magnification (63×). At steady state, the 2a-AR is predominantly sorted to the plasma membrane (stained red). Although some 2c-AR is localized in the plasma membrane (seen as yellow from overlap of red and green), the abundance of this receptor is localized in an intracellular compartment (stained green).

View larger version (91K): [in this window] [in a new window]   Fig. 5.   Immunocytochemical identification of the subcellular compartment containing 2c-ARs. Colocalization studies using antibodies that specifically stain various subcellular compartments and antibodies specific for the wild-type (polyclonal) or 12CA5 (monoclonal) epitope-tagged 2c-AR have allowed identification of the distribution of this receptor in permeabilized Rat1 fibroblast cells. A, Cell is stained for the 12CA5 epitope-tagged 2c-AR. B, Same cell is costained for BiP, which specifically labels the endoplasmic reticulum. There is considerable overlap between these two antibodies, indicating that a significant proportion of the 2c-AR is localized to this organelle. Similarly, the wild-type 2c-AR (C) colocalizes with mannosidase II (Mann. II), a marker for cis/medial Golgi (D). Conversely, the 12CA5 epitope-tagged 2c-AR (E) does not overlap with M6PR, which labels the trans-Golgi network and endosomes (F). There also is no significant colocalization between the wild-type 2c-AR (G) and lgp120, a lysosomal marker (H).

View larger version (74K): [in this window] [in a new window]   Fig. 7.   Thrombin cleavage of the amino-terminal flag epitope distinguishes intracellular 2c-AR from that delivered to the plasma membrane. For these experiments, NRK cells were studied because they are large and have a large ratio of cytoplasm to nucleus, which enabled better visualization of intracellular receptor compared with similar studies in HEK-293 cells. Intact NRK cells transfected with the thrombin/flag epitope-tagged 2c-AR were incubated in DMEM (Control) or 5 nM thrombin in DMEM for 30 min. After fixation, immunocytochemical staining was performed using both nonpermeabilizing conditions (A-D) or after cellular permeabilization with 0.2% nonionic detergent (E-H). In nonpermeabilized stained control cells, (A) flag epitope and (B) hirudin epitope colocalized to the plasma membrane. Thrombin treatment caused a complete loss of flag staining in the plasma membrane (C), whereas hirudin staining was retained (D). In control cells that were permeabilized for immunocytochemical staining after fixation, (E) flag epitope and (F) hirudin epitope were colocalized. Thrombin treatment cleaved off the flag epitope of receptor present in the plasma membrane (G, arrow), whereas the intracellular flag epitope remained intact (G). Costaining with hirudin in thrombin-treated cells identifies flag-cleaved receptor in the plasma membrane (H, arrow).

View larger version (95K): [in this window] [in a new window]   Fig. 8.   Agonist treatment does not induce redistribution of the intracellular pool of thrombin/flag epitope-tagged 2c-ARs. To investigate whether agonist treatment could induce a shift in the subcellular localization of 2c-ARs, intact NRK cells transfected with amino terminal thrombin/flag epitope-tagged 2c-ARs were studied. Incubation with 10 µM cycloheximide did not result in an observable loss of 2c-AR from the plasma membrane or shift in the intracellular receptor pool to the plasma membrane over a 4-hr treatment (A and B). Simultaneous 4-hr incubations with 10 µM cycloheximide and 5 nM thrombin showed a loss of receptor flag staining in the plasma membrane (C, arrow), whereas hirudin staining remained intact (D, arrow). There was no reduction of the intracellular pool of 2c (staining with both antibodies). Finally, the effect of agonist on the subcellular trafficking of thrombin/flag epitope-tagged 2c-AR was studied. Concurrent 4-hr incubations with 10 µM cycloheximide, 5 nM thrombin, and 10 µM epinephrine did not produce any observable shift in the intracellular pool of 2c-AR (E and F), indicating that the intracellular pool of 2c-AR is relatively stable. Moreover, the cleavable thrombin/flag epitope on the amino terminus of the 2c-AR allowed us to identify that some receptor is internalized with agonist treatment. In the presence of thrombin, agonist-promoted receptor internalization from the plasma membrane resulted in a relatively small population of intracellular vesicles that stained only for the hirudin epitope (F, arrow), not the flag epitope (E, arrow).

View larger version (71K): [in this window] [in a new window]   Fig. 9.   Trafficking studies of the amino-terminal thrombin/flag epitope-tagged 2a-AR indicates that within 4-hr, all receptor has been processed and inserted into the plasma membrane. Intact NRK cells transfected with the thrombin/flag epitope-tagged 2a-AR were grown on coverslips and incubated in (A and B, Control) DMEM, (C and D) 5 nM thrombin in DMEM for 5 min, or (E and F) 5 nM thrombin, 10 µM cycloheximide, and 10 µM norepinephrine for 4 hr. After fixation, immunocytochemical staining was performed after cellular permeabilization as described in Experimental Procedures. Similar to wild-type or 12CA5 epitope-tagged 2a-ARs, at steady state, thrombin/flag epitope-tagged receptor is primarily localized to the plasma membrane (A and B). Thrombin treatment caused a complete loss of flag staining in the plasma membrane (D), whereas hirudin staining was retained (C). In cells that have been treated with thrombin, cycloheximide, and norepinephrine for 4 hr, there is a complete loss of flag staining (F), whereas hirudin staining in the plasma membrane is retained (E).

View larger version (59K): [in this window] [in a new window]   Fig. 10.   Immunocytochemical identification of agonist-induced redistribution of 2c-ARs into endosomes. Thrombin/flag epitope-tagged 2c-ARs were stably expressed in NRK cells and grown on coverslips as described in Experimental Procedures. Coverslips were incubated with monoclonal flag antibody at 4° for 60 min, labeling epitope-tagged 2c-ARs present on the cell surface. After gentle washing, coverslips were returned to 37° with 6% CO2 and incubated in serum-free DMEM in the presence or absence of agonist (10 µM epinephrine) for 30 min. After this 30-min incubation, cells were fixed in 4% PFA, permeabilized, and stained as described. A and C, 2c-AR distribution in control and agonist-treated cells, respectively. After fixation and permeabilization, the polyclonal antibody to M6PR was used to label the trans-Golgi network and endosomes in (B) control and (D) agonist-treated cells. C, Some 2c-AR has internalized with agonist treatment and is localized in the perinuclear region (arrow). This area overlaps with that labeled in D with the M6PR antibody (arrow).

	Discussion

Summary Introduction Procedures Results Discussion References

In this study, we report differences in steady state targeting and agonist-induced internalization of the three ₂-AR subtypes. The _2b-AR behaves more like the ₂-AR than the _2a-AR. This is particularly interesting in light of the high degree of identity shared by _2a and _2b (55%, _2a versus _2b; 21%, ₂ versus _2b). Moreover, we extend previous observations regarding the intracellular distribution of the _2c-AR subtype. The roles of intracellular targeting and trafficking in signal transduction are not well understood; however, agonist-induced internalization has been shown to play a role in receptor regulation. Studies have implicated agonist-induced internalization in the process of resensitization. Blocking internalization by selective receptor mutations or treatment of cells with concanavalin A or sucrose prevents dephosphorylation of the receptor (22). Recent studies have suggested that binding of -arrestin to the ₂-AR after G protein-coupled receptor kinase phosphorylation is necessary for internalization (23). Of interest, the _2a-AR has been shown to undergo phosphorylation by G protein-coupled receptor kinase (14, 15). The inefficient agonist-induced internalization in the _2a-AR may suggest that this receptor does not bind to -arrestin. These results may also suggest that the _2a-AR is tethered to the plasma membrane and is prevented from undergoing internalization even when phosphorylated and bound to -arrestin. This may lead to a difference in the rate of resensitization and therefore account for the more extensive desensitization observed for the _2a-AR.

We observed a small amount of agonist-induced internalization of _2a using a sensitive ELISA technique for quantitative changes in cell surface density of receptor antigen. However, there was no significant accumulation of receptor in endosomes that could be detected by immunocytochemistry. This suggests that the small amount of _2a internalization may be occurring by a different mechanism than that used for internalization of _2b- and ₂-ARs. The inefficient agonist-induced internalization of _2a relative to _2b is in contrast to previous reports (12, 24); Eason et al. found that a 30-min exposure to agonist resulted in 35% sequestration of the _2b-AR and 26% sequestration in the _2a-AR. In those studies, CHO cells were used, and internalization was assayed by ligand binding techniques that used hydrophilic agonists to distinguish between cell surface and intracellular receptor. Several possibilities might account for these different results. First, CHO cells may express other proteins that are critical for agonist-induced sequestration in the _2a-AR. Although _2b- and ₂-ARs undergo agonist-promoted receptor internalization in HEK 293 cells, it is possible that this cell line lacks a component necessary for sequestration of _2a-ARs that is present in CHO cells. Another possible explanation is that different methods were used to assess sequestration in the two studies. In the current study, we used immunocytochemical methods to examine receptor internalization. These techniques are not influenced by the state of receptor/G protein coupling. This may not be the case when ₂-AR internalization is examined by using a hydrophilic agonist to quantify cell surface receptor density. It is possible that agonist treatment could reduce agonist binding of desensitized plasma membrane _2a-AR even in the absence of receptor internalization.

The physiological or functional significance of the large intracellular pool of the _2c-AR subtype is not known at this time. Both we and others have reported this unique intracellular distribution in a variety of cell lines, including COS-7, HEK 293, MDCK II, Rat1 fibroblasts, and NRK (16, 17). Moreover, this subcellular distribution is not a species-specific idiosyncrasy to the mouse _2c-AR because the wild-type human receptor shows similar intracellular localization when transfected in COS-7 or HEK-293 cells (data not shown). Wozniak and Limbird (17) have shown that in MDCK II cells, this distribution is independent of receptor expression levels; consistent with our findings that a relatively small proportion of the _2c-AR is targeted to the plasma membrane (Fig. 4, B-D), the authors showed that this receptor is targeted directly to the basolateral membrane of MDCK II cells. It is also noteworthy that when _2c- and _2a-ARs are coexpressed in the same cell, these differences in receptor sorting are maintained (Fig. 4, C and D). The _2c-AR may require a specific chaperone protein for efficient targeting to the plasma membrane, or it may require a specific protein that anchors the receptor to the cytoskeleton. In the latter case, one would expect the receptor to traffic normally to the plasma membrane but not be retained. However, this mechanism is not consistent with the results of experiments using the thrombin/flag epitope-tagged receptor (Fig. 8) that demonstrate no detectable cycling of receptor between intracellular compartment and the plasma membrane.

We noted that plasma membrane _2c-AR was more easily visualized in NRK cells (Figs. 7, 8, and 10) than in HEK 293 cells. It may be that NRK cells are somewhat better in translocating the _2c-AR to the plasma membrane than are HEK 293 cells; however, this may also be due to the fact that the NRK cells are larger and very flat, permitting better visualization of a larger amount of plasma membrane in a single plane of focus.

We were unable to detect agonist-induced internalization of the _2c-AR using conventional immunocytochemical techniques (Fig. 1). This is in part due to the large preexisting pool of intracellular _2c-AR. However, using a cleavable thrombin/flag epitope on the amino terminus of the _2c-AR allowed us to identify that some receptor is internalized with prolonged agonist treatment (Fig. 8, E and F). This was confirmed by prelabeling the amino-terminal M1 flag epitope of plasma membrane _2c-AR with antibody before agonist treatment (Fig. 10).

The differences in intracellular trafficking observed for the ₂-AR subtypes is somewhat unexpected considering the high degree of amino acid identity and the functional similarity with respect to ligand-binding properties and G protein coupling. The functional significance of differences in receptor trafficking is unknown but may be more important in vivo in highly differentiated cells. It has been proposed that receptor subtypes may be targeted to specific plasma membrane microdomains in which receptors are found in close proximity with specific G proteins and effector enzymes (25). Thus, unique trafficking behavior of receptors may enable receptors to couple to specific effector systems.

Summary. We reported that in several cell lines, the three ₂-ARs display unique patterns of subcellular distribution and sorting. At steady state, the _2a- and _2b-AR subtypes are localized in the plasma membrane. Agonist treatment induces internalization of the _2b-AR into endosomes, presumably using the same cellular machinery as the ₂-AR, whereas no accumulation of _2a-AR in endosomes could be detected after agonist stimulation. In contrast, at steady state the _2c-AR is targeted to the plasma membrane; however, a significant proportion of this receptor is localized in an intracellular pool that has not yet been delivered to the plasma membrane. Through colocalization studies, we determined that the intracellular pool of _2c-ARs is predominantly localized in the endoplasmic reticulum and cis/medial Golgi. Agonist treatment of the _2c-AR results in some receptor internalization from the plasma membrane. The functional significance of the observed differences in subcellular sorting of the three relatively highly homologous ₂-ARs remains to be established.