INTRODUCTION

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The coordinated cellular functions evoked by endogenous and exogenous ligands depend on the availability of appropriate receptors at the particular surface domains where the ligand has access. In polarized cells, the non-random localization is essential for vectorial functions of the cells; in its absence, disease ensues (1, 2). We are interested in elucidating mechanisms and structural regions within G protein-coupled receptors (GPCR)¹ responsible for their polarized expression in renal epithelia.

We previously demonstrated that the _2A-adrenergic receptor subtype (_2AAR) is predominantly localized (>85%) to the basolateral surface of Madin-Darby canine kidney (MDCKII) cells, a polarized model system for renal epithelia that accurately reflects _2AAR localization in vivo (3). Mutagenesis studies demonstrated that direct basolateral targeting was unperturbed by elimination of glycosylation, deletion of the third cytoplasmic loop, truncation of the carboxyl terminus, uncoupling from G proteins by pertussis toxin, or modification of tyrosine-based endocytosis motifs (3, 4). Subsequently, similar observations were made for the thyrotropin-releasing hormone receptor, again demonstrating that regions in GPCR involved in targeting are distinct from those involved in coupling to G proteins or in ligand-induced endocytosis (5). Removal of the third cytoplasmic loop of the _2AAR did, however, interfere with the retention of the receptor once at the basolateral surface, shortening the half-life on that surface from 10-12 to 4-6 h (4). These data collectively suggest that the transmembrane regions of the _2AAR are predominantly involved in targeting, whereas the third cytoplasmic loop of the _2AAR is involved in retention at the basolateral surface.

The goal of the present studies was to delineate further the membrane-embedded sequences involved in the basolateral targeting of _2AAR. Previous studies have demonstrated that truncation and especially chimeric receptor strategies can reveal the roles of membrane-embedded or proximal sequences in GPCR while stabilizing the overall tertiary structure of these heptahelical molecules. For example, chimeras between the _2AAR and ₂AR have revealed that the seventh transmembrane (TM) domain is necessary for antagonist binding specificity (6). Chimeras also have revealed regions that confer G protein selectivity of varying receptor families (7-9), differing agonist binding properties (10), and sites for receptor-dependent sequestration and down-regulation (11, 12). Previous studies with receptor truncations and chimeras also have led to the interpretation that TM 1-5 and TM 6-7 of GPCR can operate as independent domains and that both truncations and chimeras involving these regions can be synthesized, folded, and properly delivered to the membrane surface (6, 13).

We therefore addressed the role of the basolateral targeting information of the _2AAR using deletion, truncation, and chimera strategies. We selected the A₁ adenosine receptor (A₁AdoR) as the chimeric partner for the _2AAR, as we had previously demonstrated that the A₁AdoR is apically enriched (~70%) in both Madin Darby canine kidney II (MDCKII) and porcine renal LLC-PKI polarized epithelial cells and achieves this apical enrichment by direct targeting to that surface (14). Our study of the delivery and steady-state localization of deletions and truncations in the _2AAR and chimeras with the A₁AdoR has revealed the following: 1) there is basolateral targeting information for the _2AAR in TM 1-5 of the receptor, 2) there is also basolateral targeting information in TM 6-7 of the _2AAR, and 3) targeting information for other GPCR, such as the apically directed A₁AdoR, is similarly distributed throughout the receptor molecule.

	EXPERIMENTAL PROCEDURES

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Materials-- ³⁵S-Express protein labeling mixture (1200 Ci/mmol), [³H]methoxyinulin (125.6 mCi/g), and -³⁵S-dATP (1389 Ci/mmol) were purchased from NEN Life Science Products. Biotin hydrazide and streptavidin-agarose were purchased from Pierce. The protein A-purified 12CA5 monoclonal antibody was purchased from Babco. Cy-3-conjugated donkey anti-mouse IgG was purchased from Jackson ImmunoResearch.

Construction of Mutants-- To create the structures utilized in this study, polymerase chain reaction or subcloning was used, according to standard recombinant DNA strategies. The wild-type and mutant receptors were all amino-terminally epitope-tagged as described previously (3, 4, 14); the first 9 amino acids after the initiating methionine encode a hemagglutinin (HA) epitope recognized by the commercially available monoclonal antibody 12CA5 (Babco). The structures developed and amino acids that corresponded to the junctions within chimeras are noted in the legend to Fig. 1A. Positive mutations were verified using dideoxy-DNA sequencing (Sequenase 2.0, Sequenase Kit, U.S. Biochemical Corp.) utilizing T7 DNA polymerase with -³⁵S-dATP. Before making permanent cell lines in MDCKII cells, all of the resulting pCMV4 truncation and chimeric constructs were transiently transfected into COSM6 cells, and membranes from these transfectants were assayed for binding and feasibility for immunodetection with 12CA5 antibody.

Development of Permanent Transformants of MDCKII Cells-- The pCMV4 plasmids containing the epitope-tagged receptors of interest were transfected as described previously (3). G418-resistant colonies were screened for chimeric receptor expression by immunodetection. Untransfected MDCKII cells displayed no detectable staining pattern. Table I lists the camera exposure time (minutes) for all of the epitope-tagged clones evaluated in this study. Camera exposure time under the microscope (using auto-exposure) was used as an indirect measure of the level of expression of the non-binding mutant and chimeric receptors relative to the "parent" wild-type receptors, for which the receptor density can be measured by radioligand binding. The stronger the intensity of the image, the shorter the exposure time and, presumably, the greater the protein expression; alternatively, the longer the exposure time, the lower the protein expression. For comparison, the receptor density of the _2AAR clone 3 is 25 pmol/mg protein, and the A₁AdoR clone 17 is 37 pmol/mg protein. (At very high expression levels such as for the _2AAR clone 3 and the A₁AdoR clone 17, the camera exposure time as a function of fluorescence intensity may have reached a plateau.)

Cell Culture and Functional Confirmation of Intact Monolayers-- Madin-Darby canine kidney (MDCKII) cells were obtained from Enrique Rodriquez-Boulan (Cornell University, New York) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma), 100 units/ml penicillin G, and 100 µg/ml streptomycin (complete Dulbecco's modified Eagle's medium) at 37 °C, 5% CO₂.

Metabolic Labeling/Biotinylation Strategy for Determining Surface Delivery of the Chimeric Receptors-- The amount of newly synthesized chimeric receptor delivered to the apical versus basolateral surface was quantified by biotinylating one surface or the other of metabolically labeled MDCKII cells in Transwell culture and subsequently isolating radiolabeled receptor on the biotinylated surface through sequential immunoprecipitation and streptavidin-agarose chromatography. These procedures were performed essentially as described (3). Unfortunately, the _2AARTM1-5+i3 truncation, as well as the A₁AdoRTM1-5/_2AARTM6-7 and the A₁AdoRTM1-5/_2AARTM6-7+i3 chimeras were not amenable to immunoprecipitation for reasons that we have not been able to clarify. Hence, we have no insights concerning the delivery of these structures.

Immunolocalization of the Deletion, Truncation, and Chimeric Receptors-- Primary and secondary immunostaining protocols were optimized using cells plated on coverslips before evaluating cells polarized by culture in Transwells. For immunostaining of MDCKII cells on coverslips, cells were plated at confluence on glass coverslips and cultured for 2-3 days prior to staining. Cells were fixed and stained with a 1:50 dilution of 12CA5 primary antibody as described (14). Upon rinsing the cells after the incubation of the primary antibody, a 1:200 dilution of the secondary Cy3-conjugated donkey anti-mouse IgG was added to the cells in phosphate-buffered saline containing 2% bovine serum albumin and incubated for 1 h at room temperature in the dark. The cells then were mounted on glass slides with Aqua-Poly/Mount (Polysciences Inc., Warrington, PA).

Assessment of Receptor Binding of Wild-type _2AAR and Mutant Structures-- Various cDNAs encoding _2AAR and receptor truncations or chimeras were transfected into COSM6 cells as described previously (16) and evaluated for [³H]yohimbine binding 60 h after transfection (4). For each cDNA construction, 10 µg was transfected; in cells co-transfected with 2 cDNAs, a total of 20 µg of cDNA was added to the cells. A positive control (wild-type _2AAR) and negative control (either vector alone or no DNA) were included in every experiment. Specific [³H]yohimbine binding was that competed for by 10 µM phentolamine.

	RESULTS

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Immunolocalization of the Deletion and Truncation Mutant _2AAR in MDCKII Cells-- Because introduction of the HA epitope into the amino terminus does not appear to alter the localization of _2AAR or the A₁AdoR in MDCKII cells (3, 4, 14, 17), we evaluated clones of MDCKII cells permanently expressing the hemagglutinin epitope-tagged versions of receptors mutated by deletion and truncation. As seen in Fig. 2A, confocal microscopy images confirm that the steady-state localization of the wild-type _2AAR is lateral. Even though previous surface biotinylation strategies confirm that this lateral localization is at the surface (3, 4, 14), the signal is enhanced in the presence of Triton X-100, suggesting that the epitope is more accessible to antibody recognition under these conditions. Quantification of the fluorescence signal for the wild-type _2AAR in the presence of Triton X-100 reveals a surface:intracellular value of 3.17 ± 0.09 (Fig. 1B). As shown in Fig. 2B, the deletion of the large third intracellular loop of the _2AAR (_2AARi3) does not perturb its lateral localization; this structure also manifests a surface:intracellular staining ratio similar to wild-type _2AAR, 3.16 ± 0.4 (Fig. 1B). In contrast, truncation of the _2AAR to a structure including only TM 1-5 and the third intracellular loop, _2AARTM1-5+i3, results in a staining profile that is largely basolateral (Fig. 2C) but also has enriched intracellular staining, consistent with reduced surface:intracellular staining ratio for the _2AARTM1-5+i3 structure of 1.5 ± 0.03 (Fig. 1B). When we examined the localization of a truncated _2AAR that includes only TM 1-5 and lacks the third intracellular loop (_2AARTM1-5), we detected only marginal basolateral localization at steady state and a large population of detectable truncated receptor inside the cell, consistent with the calculated surface:intracellular staining ratio of 1.07 ± 0.06 (Fig. 1B). The greater intracellular accumulation of _2AARTM1-5 versus _2AARTM1-5+i3 confirms previous findings that the third cytoplasmic loop (i3) contributes to stabilization on the basolateral surface (4). These data suggest that the TM 1-5 domain has some information that permits lateral localization (Fig. 2, C and D); however, TM 6-7 must also impart some of the lateral localization information, since in the absence of this domain, exclusive surface basolateral localization characteristic of the wild-type _2AAR and _2AARTMi3 mutant structure is lost.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the mutant receptors evaluated in these studies and their surface:intracellular distribution in permanent transformants of MDCKII cells. A, amino-terminally epitope-tagged structures of the porcine wild-type 2AAR (thin black line) and the canine wild-type A1AdoR (thick gray line) were used as the parent molecules from which mutant and chimeric molecules were made as described under "Experimental Procedures." Shown below each schematic receptor are the amino acids that encode the wild-type 2AAR and the A1AdoR, and the respective truncations, deletions and chimeras. B, quantitation of surface to intracellular receptor distribution at steady state for wild-type and mutant 2AAR. The pixel intensity of the immunofluorescence for the stained cells was quantified by means of the program NIH Image as described under "Experimental Procedures." Immunocytochemical experiments were performed at least three times for one clone in a mutant type, and 2-3 clones were examined for each mutant receptor (data not shown). A nonparametric analysis of variance followed by a Dunn's multiple comparisons test was performed to determine which surface to intracellular ratios were significantly different from each other (* = p < 0.05). The reference point was the wild-type 2AAR, which demonstrated a surface:intracellular value of 3.17 ± 0.08 (n = 3). The loop deletion mutant, 2AARi3, had a value similar to wild-type: 3.2 ± 0.46 (n = 3). The truncation mutant, 2AARTM1-5, had the lowest surface:intracellular value, 1.07 ± 0.06, which was significantly lower than the wild-type and loop deletion mutant (n = 3; p < 0.05). The other truncation mutant, 2AARTM1-5+i3, had a surface:intracellular value of 1.5 ± 0.03 (n = 4). The three chimeras had values as follows: A1AdoRTM1-5/2AARTM6-7+i3 had a ratio of 1.83 ± 0.28 (n = 3); A1AdoRTM1-5/2AARTM6-7 was 1.21 ± 0.09 (n = 3); and 2AARTM1-5/A1AdoRTM6-7 was 1.4 ± 0.05 (n = 3).

View larger version (114K): [in this window] [in a new window]   Fig. 2.   Partial structures of the 2AAR are localized to the basolateral domain of MDCKII cells. The amino-terminally epitope-tagged wild-type 2AAR (A) was stained in the presence of Triton X-100 to permeabilize the cells and gain access to any epitope associated with an intracellular receptor population in the absence of Triton X-100, which should stain only the cell-surface receptor pool. Comparison of immunofluorescence in the absence and presence of Triton X-100 was also performed for the mutant receptor with the third loop deletion, 2AARi3 (B), a truncation mutant without the transmembrane domains 6-7, 2AARTM1-5+i3 (C), and a truncation mutant encoding TM 1-5, 2AARTM1-5 (D). The confocal gallery of the z sections in each panel gives a complete representation of the localization of these structures at steady state when stained in the presence of Triton X-100. Each square of the nine-member composite gallery represents a 1-µm z section through the cell. Section 1 represents the upper (apical) portion of the cells and section 9 represents the lower (basal) portion of the cells.

Transmembrane Domains 6 and 7 of the _2AAR Also Contain Basolateral Targeting Information-- To evaluate further the role of TM 6-7 of the _2AAR in conferring surface localization information, two independent approaches were used. First, we determined if TM 6-7 of the _2AAR could redirect apical A₁AdoR localization. Thus, the front half of the A₁AdoR (TM 1-5) was joined to the back half of the _2AAR (TM 6-7) in the presence or absence of the third intracellular loop of the _2AAR. As seen in Fig. 3B, A₁AdoRTM1-5/_2AARTM6-7+i3 chimera has a steady-state localization which is largely lateral: the gallery of the z sections shows that the lateral staining is present in all 9-µm sections. The apparent intracellular fluorescence is not visible in the cells stained in the absence of permeabilization and is mostly seen in the middle sections of the gallery, suggesting that this immunocytochemical signal is indeed intracellular rather than emerging from the apical surface (which would primarily be detectable in the top sections). As seen in Fig. 3C, the A₁AdoRTM1-5/_2AARTM6-7 chimera looks similar in its steady-state localization to the A₁AdoRTM1-5/_2AARTM6-7+i3 chimera, but with greater intracellular fluorescence, again consistent with previous observations that lack of the third intracellular loop of the _2AAR reduces basolateral retention (4).

View larger version (105K): [in this window] [in a new window]   Fig. 3.   Chimeras containing structures from both the A1AdoR and the 2AAR lose the preferential apical localization characteristic of wild-type A1AdoR. A, the steady-state localizations of the wild-type 2AAR and A1AdoR in the presence of Triton X-100. The steady-state localizations of A1AdoRTM1-5/2AARTM6-7+i3 (B), A1AdoRTM1-5/2AARTM6-7 (C), and 2AARTM1-5/A1AdoRTM6-7 (D) are shown in the presence and absence of permeabilization for the immunocytochemical procedure; the surface:intracellular values were 1.83 ± 0.28, 1.21 ± 0.1, and 1.4 ± 0.057, respectively. The galleries of z sections are from experiments in the presence of Triton X-100.

Assessment of the Functional Integrity of Truncations and Chimeras by Binding Rescue Studies-- The greater intracellular localization of the _2AAR truncations and chimeras compared with the wild-type _2AAR may reflect a perturbed structure that is inefficiently delivered to the surface. Since the ultimate goal of these studies was to reveal structural regions within the receptor that directly target the _2AAR to the basolateral surface, we wanted to ascertain whether the truncation and chimeric structures that reached the surface membrane were capable of supporting receptor functions, implying that the structure of the domains expressed in _2AAR truncations or chimeras retained the structure characteristic of those domains in the native receptor. To this end, we co-expressed the truncation mutants, _2AARTM1-5 and _2AARTM1-5+i3, with a point mutation of the _2AAR, D113N_2AAR, that lacks ligand binding capabilities (18). Similar strategies have been valuable in assessing the structural integrity of the _2CAR (19), M3 muscarinic receptor (19, 20), and the V2 vasopressin receptor (21). As shown in Fig. 4A, neither the _2AARTM1-5 or _2AARTM1-5+i3 truncations nor the D113N_2AAR mutant bind the ₂AR antagonist [³H]yohimbine when expressed alone. However, when expressed in combination, the co-expressed structures reveal readily detectable binding in multiple experiments. Binding to the "rescued" receptors is indistinguishable in affinity for the radioligand, assessed using saturation analysis; the K_d for [³H]yohimbine was 3 nM for both the wild-type _2AAR as well as the co-expressed _2AARTM1-5 and D113N_2AAR. The rescued binding achieved upon co-expression represented 3-19% of the wild-type _2AAR binding, which is in the range of rescue reported previously for other GPCR (19, 20, 22).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   Co-expression of 2AAR truncations, chimeras, and binding defective mutants in COSM6 cells. Binding of [3H]yohimbine was evaluated in particulate preparations of COSM6 cells 60 h following transfection with the cDNAs encoding receptor truncations (A) and chimeras (B). A, specific [3H]yohimbine binding (that competed for by 10 µM phentolamine) was only 200-450 cpm/incubation following expression of the individual D113N2AAR, 2AARTM1-5, or 2AARTM1-5+i3 structures. However, [3H]yohimbine binding detected upon co-expression of the D113N2AAR with the 2AARTM1-5 ranged from 2200 to 6540 cpm in three separate experiments, corresponding to 3-19% of binding for the wild-type 2AAR. For co-expression of the D113N2AAR with the 2AARTM1-5+i3, the binding ranged from 3950 to 4630 cpm for three separate experiments, corresponding to 4-6% of binding for the wild-type 2AAR. B, binding for the individual constructs, 2AAR/A1AdoRTM6-7, A1AdoRTM1-5/2AARTM1-5, or A1AdoRTM1-5/2AARTM1-5+i3 ranged from 250 to 440. Binding of [3H]yohimbine following co-expression of A1AdoRTM1-5/2AARTM1-5 with 2AARTM1-5+i3 ranged from 0.5 to 2% of wild-type 2AAR binding (n = 3). Co-expression of the 2AAR/A1AdoRTM6-7 with the A1AdoRTM1-5/2AARTM1-5+i3 ranged from 0.5 to 2% of wild-type binding (n = 3). The [3H]yohimbine binding detected upon co-expression of the D113N2AAR with the 2AAR/A1AdoRTM6-7 ranged from 1545 to 2500 cpm (n = 2), corresponding to 2-3.5% of binding for the wild-type 2AAR. Co-expression of A1AdoRTM1-5/2AARTM1-5+i3 with 2AARTM1-5 resulted in 3560-8800 cpm/incubation (n = 3), representing 8.5-10.5% of wild-type 2AAR binding.

Assessment of the Delivery of Mutant _2AAR to the Apical Versus the Basolateral Cell Surface-- To examine how these mutant receptors achieved their steady-state localization in polarized MDCKII cells, we assessed appearance of metabolically labeled receptors to either the apical or basolateral surface. Both the wild-type _2AAR (3) and the _2AARi3 deletion mutant (4) achieve their steady-state localization on the basolateral surface by means of direct delivery to that membrane domain, as described previously and confirmed here for the _2AARi3 at pulse times of 90 and 120 min (Fig. 5A). As seen in Fig. 5B, delivery studies revealed that the truncation of the _2AAR to _2AARTM1-5 yielded a structure that was first delivered preferentially, but not exclusively, basolaterally; however, within a very short window of time, i.e. about 15-30 min, metabolically labeled receptor could also be detected on the apical surface. The lack of exclusive surface targeting of the _2AARTM1-5 truncation is in marked contrast to the wild-type _2AAR (3), suggesting, as do the steady-state localization data in Fig. 2D, that basolateral delivery cannot rely solely on this single domain. The key observation is that exclusive basolateral targeting characteristic of the wild-type _2AAR and _2AARi3 structures is not afforded by a structure encoding only TM 1-5 of the _2AAR. The changing relative distribution of this truncation mutant on the apical versus the basolateral surfaces as a function of metabolic labeling times could be due to rapid turnover, recycling, and/or re-routing. For example, pulse-chase experiments revealed that the half-life of this structure on the apical surface was ~40 min and that on the basolateral surface ~20 min (data not shown), in contrast to the basolateral half-lives of 10-12 h for the wild-type _2AAR or 4-6 h for the _2AARi3 structure (3, 4).

View larger version (59K): [in this window] [in a new window]   Fig. 5.   Delivery of deletion or truncated 2AAR and chimeric 2AAR/ A1AdoR reveals regions critical for basolateral targeting of 2AAR in MDCKII cells. Metabolic labeling coupled with surface biotinylation as described under "Experimental Procedures" was used to track the delivery of the 2AARi3 (A), 2AARTM1-5 (B), and 2AARTM1-5/A1AdoRTM6-7 (C) to either the apical or basolateral surface in MDCKII cells in Transwell culture. These are representative autoradiograms of three to four separate experiments for each structure examined in one clonal cell line; similar results were obtained in another clonal cell line expressing each structure.

	DISCUSSION

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A number of previous studies have led to the identification of basolateral targeting motifs for single transmembrane spanning receptors in polarized renal epithelial cells. For example, Thomas and Roth (23) have shown that the basolateral sorting signal for vesicular stomatitis virus G lies within the cytoplasmic domain and is encoded by the aliphatic sequence YXX. By making chimeras with transferrin receptor, Odorizzi and Trowbridge (24) demonstrated that a dihydrophobic motif in the cytoplasmic tail of the major histocompatibility complex class II invariant chain targets it basolaterally in MDCK cells. The molecular basis for basolateral targeting of these and other single transmembrane proteins has been reviewed previously (23, 25-27).

The basolateral targeting motifs for multi-spanning proteins have been more difficult to deduce, as deletion or insertion mutants, useful in delineating motifs in single membrane-spanning proteins, can create confounding results in polytopic membrane proteins. Consequently, chimeras between proteins of similar structural families but with distinct trafficking itineraries have yielded the most informative insights. For example, chimeras between the 10 transmembrane-spanning ATP-powered Ca²⁺ pump in the plasma membrane and the closely related pump in the sarco(endo)plasmic reticulum revealed that the first transmembrane domain of sarco(endo)plasmic reticulum was sufficient to target chimeras between these two proteins to the endoplasmic reticulum (28). In contrast to the role of transmembrane-spanning domains in the Ca²⁺-ATPase targeting, chimeras between the apically localized GLUT 5 and the basolaterally localized GLUT 1 isoform of glucose transporters revealed that the intracellular loops of these 12 transmembrane-spanning transporters confer apical versus basolateral localization in polarized Caco-2 intestinal cells (29). In a related molecular family, Perego et al. (30) demonstrated that basolateral sorting information lies in the cytoplasmic tail of the betaine transporter, whereas apical sorting information for the closely related -aminobutyric acid transporter (GAT-1) does not and may be embedded in the bilayer or in endofacial domains, or both. These examples emphasize that there are no demonstrated consensus motifs or unifying mechanisms that confer basolateral targeting of polytopic membrane proteins in polarized epithelial cells.

The present studies were undertaken to establish targeting motifs that confer basolateral targeting of seven transmembrane-spanning GPCR, using the directly targeted _2AAR as a model molecule. Previous studies in our laboratory have provided evidence that basolateral targeting of the _2AAR relies on membrane-embedded or proximal sequences (4). The present studies exploit receptor truncations and chimeras to explore the membrane-spanning regions involved in basolateral targeting of the _2AAR. The ability of _2AAR truncations and chimeras to restore binding of the _2AAR antagonist, [³H]yohimbine, to a binding-defective D113N_2AAR mutant suggests that these independently expressed domains possess a structure that, at least functionally, resembles these domains within the native receptor. Furthermore, the observation that multiple independent clonal cell lines for each structure examined revealed indistinguishable findings for receptor delivery and steady-state localization adds further confidence to our interpretations and argues that the truncations and chimeras evaluated are providing informative insights into subdomains of the _2AAR critical for direct basolateral delivery.

The findings presented here, and summarized in Fig. 6, indicate that there is basolateral targeting information in TM1-5 of the _2AAR that is stabilized by juxtaposition of the third intracellular loop (Fig. 2C). However, TM 1-5 of the _2AAR does not contain all of the necessary basolateral targeting information. This conclusion is based on the observation that the truncated _2AARTM1-5 is not exclusively delivered to the basolateral surface like the wild-type _2AAR. The basolateral signals missing in _2AARTM1-5 must lie within TM 6-7, since the mutant lacking the third cytoplasmic loop (_2AARi3) does not show any signs of apical delivery. Chimeric structures of the _2AAR with the A₁AdoR also support the importance of the TM 6-7 domain of the _2AAR in conferring basolateral localization information; the absence of TM 6-7 in the _2AARTM1-5/A₁AdoRTM6-7 chimera leads to initial apical delivery (Fig. 5C), and introduction of TM 6-7 of the _2AARTM onto TM 1-5 of the A₁AdoR to create the A₁AdoRTM1-5/_2AARTM6-7+i3 and the A₁AdoRTM1-5/_2AARTM6-7 chimeras results in structures with demonstrably lateral staining patterns (Fig. 3, B and C). Our interpretation of these collective findings is that information conferring basolateral localization of the _2AAR exists in both the TM 1-5 and TM 6-7 domains.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Steady-state localization of wild-type and mutant GPCR in MDCKII cells affirms the existence of multiple, non-contiguous localization motifs. This table summarizes what has been learned from evaluating deletion, truncation, and chimeric receptor structures of the 2AAR and A1AdoR. The greater the amount of the 2AAR encoded by the structure expressed, the greater the basolateral expression of that structure in the MDCKII cells. Consistent with the data obtained in metabolic labeling and in confocal images of immunocytochemical studies, we conclude that targeting of the 2AAR to the basolateral surface involves multiple, non-contiguous targeting signals embedded in or proximal to the bilayer of this seven transmembrane-spanning protein.

An enigmatic observation in our studies is that the _2AARTM1-5 truncation is localized basolaterally and intracellularly at steady state, although the kinetic profile reveals targeting to both surfaces, with an apical t1/2 of ~40 min and a basolateral t1/2 of ~15-30 min (Fig. 5B). Predictions from these kinetic data would lead to an expectation of greater apical than basolateral localization at steady state, if the steady-state localization solely reflected direct delivery. There are two possible explanations for these unexpected findings. The first possibility is that the truncated receptor is quickly removed from both surfaces but that removal from the apical surface is followed by degradation or intracellular retention, whereas removal from the basolateral surface is followed by recycling back to the basolateral surface. We know from our own studies with the _2BAR, for example, that its short half-life on the apical surface (t1/2 15-30 min) is not paralleled by detectable apical localization at steady state via confocal microscopy, suggesting that removal from the apical surface is followed by degradation (17). Thus, it may be a common trafficking itinerary for GPCR that apical delivery is followed by removal and degradation, and not recycling, in contrast to findings for molecules on the basolateral surface. A second possible explanation of our findings that no apical localization of the _2AARTM1-5 is seen at steady-state in confocal images is that, after removal from the apical surface, the receptor transcytoses to the basolateral surface, where it then "stays" by virtue of recycling back and forth to the basolateral domain. The independent recycling properties of proteins internalized from polarized surfaces via a distinct subset of exocytotic vesicles in MDCK cells has already been demonstrated for single transmembrane-spanning proteins (31). Thus, either of these two scenarios is possible. Extant tools for study of the _2AAR do not permit us to distinguish between these two possible explanations.

Assigning the regions of the receptor that govern receptor targeting requires an appreciation that trafficking itineraries are likely controlled by both negative and positive signals. For example, interpretations of our chimeric receptor studies must be qualified by the realization that the addition of TM 6-7 of the A₁AdoR to TM 1-5 of the _2AAR could lead to changes in delivery or steady-state localization either because of the addition of a new targeting signal to the chimera or because of a loss of a targeting signal inherent in the parent molecule. Thus, if A₁AdoRTM1-5/_2AARTM6-7 and A₁AdoRTM1-5/_2AARTM6-7+i3 have lateral localization due to the presence of a basolateral targeting signal in TM6-7 of the _2AAR, then this signal is able to "override" any signal that might be expressing apical targeting in TM1-5 of A₁AdoR. In this case, the interpretation implies hierarchical targeting signals. Alternatively, it may be that the absence of TM6-7 of the A₁AdoR, which may encode apical targeting, results in chimeras with a more lateral phenotype because the lateral surface is encoded by "default." However, the _2AARTM1-5/A₁AdoRTM6-7 is not exclusively apical. Adding TM6-7 of the A₁AdoR to _2AARTM1-5 gives the receptor a more apical phenotype, suggesting that for another GPCR, the A₁AdoR, the apical targeting information is non-contiguous, since the A₁AdoRTM1-5/_2AARTM6-7 and A₁AdoRTM1-5/_2AARTM6-7 both express some apical localization as well.

The possibility that several regions of a polytopic protein can impart targeting information has been demonstrated previously. Verhey et al. (32) have demonstrated that signals in both the amino and carboxyl termini of GLUT 1 (localized intracellularly and at the cell membrane) and GLUT 4 (localized only intracellularly) confer targeting information to the two isoforms. Marks et al. (33) demonstrated the presence of two distinct and saturable protein targeting processes, one tyrosine-based and the other dileucine-based, in the cytoplasmic region of transmembrane proteins (e.g. the  chain of HLA-DM/H-2M) in HeLa cells. In a third example, Alanso et al. (34) showed that multiple sequences are responsible for the apical localization of the nonglycosylated type I membrane protein, CD3-, in MDCK cells. Multiple targeting signals have also been demonstrated in some single transmembrane-spanning proteins, both for basolaterally targeted (23-25, 35-37) and apically targeted proteins (38-40).

The present studies also provide insights into the stabilization of the _2AAR on the basolateral surface. Multiple lines of evidence confirm that the third intracellular loop stabilizes lateral retention of GPCR. Findings summarized in Figs. 1B and Fig. 2, C and D demonstrate that higher surface:intracellular expression is seen for truncations that include the third intracellular loop of the _2AAR. Similarily, in receptor chimeras, association of the third intracellular loop with the last two transmembrane regions of the _2AAR (A₁AdoR/_2AAR+i3) stabilizes whatever basolateral inclination those domains of the _2AAR possess (Fig. 3B versus 3C). In addition and not previously appreciated, we noted that the TM 6-7 domain of GPCR may also serve as a hydrophobic stabilizing anchor for receptors on polarized surfaces. This conclusion is based on the observation that the surface half-life of _2AARTM1-5 (20-40 min) is dramatically shorter than that of the _2AARTM1-5/A₁AdoRTM6-7 chimera, which has an estimated half-life of 1.5-2 h.

The present data reveal that basolateral targeting of the _2AAR, and likely other GPCR, is encoded by structural information within both TM 1-5 and TM 6-7 domains. These findings provide the first molecular insights for adrenergic receptors which demonstrate that targeting to the basolateral surface of polarized epithelia involves multiple, non-contiguous structural information. It is probable that simple sequence motifs for surface targeting will not be identifiable for GPCR, in contrast to tyrosine and dileucine motifs for basolateral sorting of single transmembrane-spanning proteins. Ingenious strategies to reveal "surfaces" created by non-contiguous elements that serve as recognition motifs for targeting machinery will be required to fully understand the targeting of GPCR to basolateral versus apical surfaces of polarized epithelial cells.