INTRODUCTION

The coordinated and vectorial functioning of polarized cells mediated by endogenous and exogenous ligands depends on the availability of appropriate receptors at the particular surface domains to which the ligand has access. We are interested in elucidating the mechanisms by which G-protein-coupled receptors (GPCR)¹ attain their localization in renal epithelial cells, using polarized MDCK II cells as a model system. We have demonstrated that the G-protein-coupled _2AAR is delivered directly to and retained on the basolateral subdomain of renal epithelial cells (1), whereas the A₁AdoR predominantly is apically targeted (65-83%) in renal epithelial cells (2). The trafficking itineraries of the _2BAR and _2CAR subtypes differ from that of the _2AAR: the _2BAR is randomly delivered to both the apical and basolateral domains but selectively retained basolaterally; the _2CAR is directly delivered basolaterally, but a substantial fraction of the receptor population remains in a cytoplasmic compartment at steady state (3). These different targeting itineraries are summarized in Fig. 1.

Like receptors, including the GPCR described above, the cytoskeleton in a polarized cell also is nonuniformly distributed (Fig. 1A). It is thought that this disparate distribution of cytoskeletal proteins contributes to the polarized compartmentalization of proteins, since the development of a functionally and structurally polarized phenotype is paralleled by cytoskeletal polarization (4). In MDCK II cells grown in polarized culture, Ca²⁺-dependent induction of cell-cell contacts via E-cadherin leads to a gradual reduction of actin microfilaments on the basal surface and their apical enrichment, in parallel with an increased stability (and polarization) of microtubules, and an alignment of ankyrin and fodrin under the lateral subdomain (4).

Since apical versus basolateral targeting of membrane proteins appears to involve discrete post-trans-Golgi network vesicular populations (5), it is not surprising that different cytoskeletal elements also have been implicated in this differential transport of apically versus basolaterally targeted vesicles (e.g. see Refs. 11, 12, and 18). To date, however, the role of the cytoskeleton in the trafficking of GPCR has not been explored.

The present studies examine the impact of cytoskeletal disrupting agents on the localization and delivery of GPCR targeted to different surfaces in polarized cells. We examined the effect of agents that disrupt the cytoskeleton (colchicine, nocodazole, cytochalasin D) or vesicular transport (monensin, brefeldin A) on the steady-state localization and delivery of basolaterally targeted _2AAR and _2CAR, the randomly delivered but basolaterally retained _2BAR, and the apically targeted A₁AdoR. We postulated that the use of cytoskeletal agents in combination with the study of GPCR possessing varying trafficking itineraries (2, 3) would allow us to ascertain criteria for basolateral versus apical targeting as well as direct versus random delivery. The present data provide evidence that apical delivery of the A₁AdoR and _2BAR occurs by microtubule-dependent and -independent mechanisms, respectively, and that cytoskeletal disrupting agents may be valuable tools for elucidating distinct direct apical delivery pathways as well as the molecular mechanisms that govern them.

EXPERIMENTAL PROCEDURES

Materials

8-Cyclopentyl-1,3-di-[2,3-³H]propylxanthine ([³H]DPCPX) (109 Ci/mmol), ³⁵S-Express protein labeling mixture (1200 Ci/mmol), [³H]methoxyinulin (125.6 mCi/g), and [-³⁵S]dATP (1389 Ci/mmol) were from NEN Life Science Products. The A₁AdoR antagonist, 1,3-dipropyl-8-(4-sulfophenyl)xanthine (6), was kindly donated by Dr. Jack N. Wells (Vanderbilt University). Biotin hydrazide and streptavidin-agarose were from Pierce; protein A-purified 12CA5 monoclonal antibody was from Babco; gp135 (7) and EGF receptor (8) monoclonal antibodies were generously donated by Dr. Peter Dempsey (Vanderbilt University); the E-cadherin antibody was from the Developmental Studies Hybridoma Bank, NICHD, National Institutes of Health (Iowa City, IA); Cy-3 conjugated donkey anti-mouse IgG was from Jackson Immunochemicals; monoclonal anti--tubulin was from Amersham Corp.; and rhodamine-conjugated phalloidin was from Molecular Probes. Nocodazole, monensin, colchicine, and lumicolchicine were from Sigma; cytochalasin D and brefeldin A were from Calbiochem.

Polarized Culture of MDCK II Cells and Functional Confirmation of Intact Monolayers

MDCK II cells were maintained as described previously (2, 3). For polarity experiments, MDCK II cells were seeded at a density of 1 × 10⁶ cells/24.5-mm polycarbonate membrane filter (Transwell chambers, 0.4-µm pore size, Costar, Cambridge, MA), and cultured for 5-8 days with medium changes every day. The A₁AdoR-expressing cell lines were grown in the presence of the receptor antagonists, 60 µM theophylline and 100 µM 1,3-dipropyl-8-(4-sulfophenyl)xanthine, since previous studies had demonstrated that, if grown in their absence, A₁AdoR-expressing cells grew as multicellular layers in Transwell culture (2). Prior to each experiment, the integrity of the monolayer was assessed by monitoring [³H]methoxyinulin leak (1).

Development of Permanent Transformants of MDCK II Cells

Permanent clonal cell lines of MDCK II cells were developed as described previously (1-3). In each case, the first 9 amino acids after the initiating methionine encode a hemagglutinin epitope (9) recognized by the commercially available monoclonal antibody 12CA5 (Babco). The clonal cell lines evaluated in the present study include TAG-_2AAR (25 and 7 pmol/mg of protein), TAG-_2BAR (10 and 3 pmol/mg of protein), TAG-_2CAR (5 and 3 pmol/mg of protein) and TAG-A₁AdoR (37 pmol/mg of protein). The _2AAR subtype was encoded by a porcine cDNA, the _2BAR and _2CAR subtypes were encoded by a rat cDNA, and the A₁AdoR was encoded by a canine cDNA. The species origin does not alter the trafficking patterns of these receptors. For example, the porcine _2AAR is targeted directly to the basolateral surface in both canine MDCK II and porcine LLC-PK1 renal epithelial cells (1), and the canine A₁AdoR is targeted apically in both MDCK II and LLC-PKI cells (2).

Receptor Binding Assays

MDCK II particulate preparations were prepared essentially as described (1). A₁AdoR were identified using [³H]DPCPX (2), and ₂AR were identified with [³H]rauwolscine (1, 3) as described previously.

Treatment of Polarized MDCK II Cells with Agents to Disrupt Cytoskeleton or Vesicular Traffic

Colchicine is a microtubule-disrupting drug that binds slowly to soluble tubulin heterodimers, reducing them to large aggregates and rendering them incapable of polymerization for microtubule growth (10). Most incubations with colchicine were performed for 15 h, since the half-life of all four GPCR studied on their enriched membrane surfaces is 10-12 h. Thus, a 15-h incubation with colchicine permitted us to evaluate the localization of receptors already delivered to the cell surface and those that were under synthesis and delivery. Previous reports have established that increased time of incubation with colchicine beyond 4-6 h does not lead to nonspecific mechanisms for this agent (11, 12). We conducted a time course of the steady-state localization of the A₁AdoR-expressing cell line in the presence of 10 µM colchicine; the appearance of the A₁AdoR on the lateral surface was evident after only 4 h of treatment and was maximal at 18 h (data not shown). Even at 18 h, however, some trace apical staining of A₁AdoR was detectable (see "Results"), which could be due to incomplete microtubule disruption, incomplete turnover of the already delivered receptor population, or a microtubule-insensitive fraction of apically delivered receptor. Immunocytochemical analysis of treated cells using an anti -tubulin antibody confirmed that colchicine treatment of cells had indeed disrupted the microtubule network. Since a change in MDCK II cell morphology was noted after 24-48 h of colchicine treatment, these longer incubations were not used in the present studies.

Nocodazole disrupts microtubules via a molecular mechanism different from that of colchicine (10). Nocodazole binds rapidly to microtubule subunits and prevents heterodimers from repolymerizing, at either 37 or 4 °C. Incubation at 4 °C accelerates nocodazole-effected depolymerization, at least in MDCK (7) and Caco-2 (12) cells. MDCK II cells in Transwell culture were treated for 15 h, as described above for colchicine. Just as for colchicine, the ability of nocodazole treatment to disrupt microtubules was confirmed by immunocytochemical analysis with the anti--tubulin monoclonal antibody (Amersham).

The effects of nocodazole (33 µM, equivalent to 10 µg/ml) on GPCR delivery to the cell surface were performed as follows, based on earlier reports for nocodazole treatment in MDCK cells (18): on the day of the experiment, medium was replaced with 4 °C medium and incubated for 30 min, followed by 4 °C Cys/Met-free medium with or without nocodazole for 1 h, followed by 37 °C Cys/Met-free medium with or without nocodazole for 1-1.5 h, followed by [³⁵S]Cys/Met metabolic labeling at 37 °C for the desired "pulse" time with or without nocodazole.

The actin polymerization inhibitor, cytochalasin D (2 µM), was incubated with polarized MDCK II cells for 15 h prior to fixation and immunocytochemical analysis. Disruption of the actin cytoskeleton was confirmed by staining control and treated cells with rhodamine phalloidin, which reveals the actin network.

BFA is a fungal metabolite that has been shown to fuse the endoplasmic reticulum with the cis, medial, and trans cisternae of the Golgi apparatus but not with the trans-Golgi network (13). BFA has been demonstrated to both enhance transcytosis (14, 15) and inhibit transcytosis (16) or targeting (17) and was tested in these studies to explore the possible contribution of the transcytotic pathway to the apical delivery of A₁AdoR and/or the apical component of the random delivery of _2BAR. Brefeldin A was added to a final concentration of 3.5 µM, as described previously for MDCK II cells (18, 19).

Monensin is a cationic ionophore that exchanges Na⁺ for H⁺, thereby dissipating transmembrane pH gradients. Although multiple intracellular consequences of monensin treatment might be expected, most widely reported are the effects of monensin to block cell surface receptor recycling of internalized molecules to the cell surface (20-23). Monensin was evaluated at a final concentration of 1.4 µM, as described previously for MDCK II cells (18, 24).

Steady-state Localization of GPCR and of Cell Surface Proteins by Immunolocalization

Immunostaining of cells grown in Transwell culture was performed as described previously (2, 3) with the following concentration of primary antibody: a 1:50 dilution of 12CA5 primary antibody, purified as described previously (3), for the localization of hemagglutinin epitope-tagged GPCR (1); 15 µg/ml mouse monoclonal EGF receptor antibody (25); a 1:10 dilution of mouse monoclonal gp135 antibody (7); or a 1:8 dilution of mouse monoclonal E-cadherin antibody (RR1, Developmental Studies Hybridoma Bank). Except when indicated otherwise, the antibody-containing and antibody wash buffers contained 0.1% Triton X-100, to permit detection of epitope either on the cell surface or in the cell interior. Treatment with the secondary Cy3 conjugated donkey anti-mouse IgG (1:200) was performed as described (2, 3). Samples were visualized by confocal microscopy on a Zeiss Axiovert 135 Micro Systems LSM (Germany). The samples were first visualized in the xy plane and then in the xz plane. In the images shown, the bottom 3/4 represent the xy plane, the conventional view of the cells as one looks down upon them. The white line that is shown in the xy plane confocal images indicates where the laser took a cross-section of the cells to generate the z scan. The top 1/4 of the images represents the xz plane (or z scan), the cortical section perpendicular to the plane of the cell layer. Images were analyzed using Showcase software on a Silicon Graphics Iris Indigo workstation.

Metabolic Labeling/Biotinylation Strategy for Determining Surface Delivery of GPCR

The amount of newly synthesized receptor delivered to the apical versus basolateral surface of polarized, metabolically labeled MDCK II cells grown in Transwell culture was quantified by surface biotinylation of either the apical or basolateral surface with NHS-biotin followed by isolation of radiolabeled receptor on the biotinylated surface by sequential Protein A- and streptavidin-agarose chromatography. These procedures were performed essentially as described (1), except that wherever drugs were used, they were added in the Cys/Met-free medium for the duration that preceded and included pulse labeling. Briefly, the cells were pretreated with Cys/Met-free medium for 2 h, followed by [³⁵S]Cys/Met metabolic labeling for the durations given in the figure legends. If the experiment was designed to examine the retention time of the receptor on the cell surface, then chase times were included after the pulse with or without drug.

RESULTS

To examine the effect of colchicine (10 µM), nocodazole (33 µM) and cytochalasin D (2 µM) on the steady-state localization of the GPCR, _2AAR, _2BAR, _2CAR, and A₁AdoR in renal epithelial cells (Fig. 2), cell lines permanently expressing epitope-tagged versions of these receptor populations were incubated for 15 h with the drugs of interest as described under "Experimental Procedures." Since the receptor half-life of the ₂AR subtypes on the basolateral surface (3) and that of the A₁AdoR on the apical surface (2) is approximately 10-12 h, this 15-h incubation with cytoskeletal disrupting agents provided optimal opportunity to explore the impact of these agents on nascent as well as pre-existing receptor populations. Previous studies have demonstrated that overnight incubations like those we utilized in the present studies do not lead to nonspecific effects of these drugs; e.g. although the maximal effects of colchicine often are noted after only 4 h of incubation with 10 µM colchicine (26), others have shown that treatment with nocodazole requires at least 6 h for complete microtubule depolymerization (27). Each experiment in the present study was preceded with an assessment of transepithelial leak to confirm the integrity of the polarized MDCK II cell monolayer under control and treated conditions (see "Experimental Procedures"). Similarly, the impact of these agents on endogenous polarized "marker" proteins in MDCK II cells also was examined (Fig. 3).

As shown in Fig. 2, the most dramatic effect of the cytoskeletal disrupting agents is seen for the steady-state localization of the A₁AdoR, where treatment with both of the microtubule-disrupting agents, colchicine and nocodazole, altered the apical staining pattern characteristic of the wild-type receptor under control conditions to a basolateral staining profile. This change in A₁AdoR polarization is evident in both the xy plane confocal images and the xz plane images, which effectively provide a laser "slice" through the polarized MDCK II cells (Fig. 2). In contrast to the dramatic changes in A₁AdoR polarization, microtubule disruption by either colchicine or nocodazole did not alter the steady state basolateral polarization of any of the three ₂AR subtypes.

Although the microtubule-directed agents, colchicine and nocodazole, did not redirect the steady-state localization of the ₂AR subtypes, they nonetheless altered the relative surface:internal compartment localization of the _2BAR (Fig. 2). The intracellular localization of the _2BAR is interpreted to result from ligand-induced internalization (28) or from catecholamines in serum-containing medium.² The fluorescence intensity of the lateral staining pattern for the _2BAR in the presence of colchicine was increased dramatically concomitant with a decrease in intracellular staining of this receptor. These findings suggest that in the presence of colchicine, and to a lesser extent in the presence of nocodazole (Fig. 2), the fraction of the _2BAR population that existed in the cell interior was now enriched on the lateral surface. In contrast, no change in relative lateral membrane staining of the _2AAR or _2CAR was observed following colchicine or nocodazole treatment. The intracellular enrichment of the _2CAR subtype at steady state has been reported previously in HEK293 cells (47) and, in later studies, in polarized MDCK II cells (3). The _2CAR internal compartment can be distinguished functionally and morphologically from endocytosing cell surface GPCR (47). The relative surface:internal distribution of the _2CAR subtype was resistant to treatment with microtubule disrupting agents. The existence of internal _2BAR and _2CAR in independent compartments may explain why colchicine or nocodazole influences _2BAR redistribution without impact on the intracellular _2CAR pool.

The apparent increase in _2BAR density at the lateral surface detected morphologically was paralleled by an increase in functional _2BAR density detected using radioligand binding assays (Fig. 4). Saturation binding analyses revealed that the increase in functional _2BAR binding was due to an increase in maximal receptor density (B_max = 1 ± 0.06 pmol/mg of protein for control versus B_max = 2.96 ± 0.67 pmol/mg of protein for colchicine-treated cell lines; n = 3; p < 0.05), with no change in _2BAR receptor affinity for the radiolabeled antagonist [³H]rauwolscine (K_d = 1.86 ± 0.18 nM for cells treated under control conditions versus 2.23 ± 0.16 nM in colchicine-treated cells). This effect depended on the microtubule-disrupting properties of colchicine, since -lumicolchicine, a chemical analog of colchicine that does not disrupt microtubules, had no effect on radioligand binding density (data not shown). This marked increase in binding density was uniquely observed for this ₂AR subtype; although effects of colchicine on _2CAR binding were observed, they were significantly smaller (approximately a 20% increase in binding; Fig. 4). The absence of any loss of binding for any of the GPCR following treatment with these microtubule-disrupting agents provides additional evidence that these agents are not having nonspecific deleterious effects on the MDCK II cells under the conditions of our experiments.

The functional density of the _2BAR subtype is uniquely increased by colchicine.

We also evaluated the effect of microtubule-disrupting agents on the localization of endogenous MDCK II proteins that serve as "markers" of the polarized state: EGF receptor (localized laterally) (25), gp135 (localized apically) (7), and E-cadherin (a quintessential marker of cell polarity, localized laterally and involved in the development of polarity in MDCK cells) (29). Neither colchicine nor nocodazole had a significant effect on the localization of the basolateral markers (Fig. 3). Interestingly, the apical marker gp135 also remained enriched apically, but its distribution on the apical surface was modified in about 50% of the cells, creating an apparently "black" cellular interior. Earlier reports have noted a similar clustering of gp135 occurs in MDCK cells treated with cytochalasin D (7). It is unclear why, however, nocodazole but not colchicine caused this "black hole" phenomenon; perhaps it reflects differing kinetic effects of those agents.

As shown in Fig. 2, disruption of microtubules with colchicine or nocodazole led to relocalization of A₁AdoR from a predominantly apical localization to a predominantly lateral expression pattern. To determine whether this A₁AdoR relocalization results from altered delivery to or retention on the basolateral versus the apical surface, we examined the effects of colchicine and nocodazole on delivery and turnover of this receptor in metabolically labeled MDCK II cells (Fig. 5). Nocodazole treatment reversed the predominant apical delivery (70:30, apical:basolateral) of the A₁AdoR such that a major portion of the apical receptor population is delivered basolaterally (40:60, apical:basolateral) after treatment (Fig. 5A). This reversal of the targeting pattern for the A₁AdoR seen with nocodazole treatment also is seen with colchicine, where the apical:basolateral ratio of the A₁AdoR delivery went from 63:37 under control conditions to 45:55 following colchicine treatment (Fig. 5B). In the autoradiogram shown, the apparent reversal appeared more dramatic for nocodazole than colchicine. As mentioned earlier, these subtle differences in quantitative effects may reflect kinetic differences in colchicine versus nocodazole action (10). The studies shown in Fig. 5A follow a 90-min pulse labeling with [³⁵S]Cys/Met-containing medium, but indistinguishable findings were observed following a 60-min pulse. The colchicine or nocodazole-induced basolateral enrichment of the A₁AdoR was retained over time, as evidenced following pulse-chase studies (data not shown). These data suggest that microtubule disruption alters the delivery of the direct apically targeted A₁AdoR and that the morphological manifestation of the steady-state redistribution to the lateral subdomain is due, at least in part, to an enhanced delivery to the basolateral surface.

Nocodazole and colchicine reverse the polarity of apically delivered A₁AdoR. A, MDCK II cells expressing epitope-tagged A₁AdoR grown in Transwell culture in the presence of A₁AdoR antagonists (see "Experimental Procedures") were metabolically labeled with 1 µCi/µl of [³⁵S]Met/Cys protein labeling mix (150 µl) for 1 h in the absence or presence of nocodazole (33 µM) and then harvested and processed using sequential immunoprecipitation and streptavidin-agarose chromatography as described under "Experimental Procedures." The autoradiogram shown is for an SDS-polyacrylamide gel exposed for 3 weeks to Kodak X-Omat film between two Quanta III screens at 70 °C. Gel slices corresponding to the position of the A₁AdoR signal on autoradiograms were excised and counted in 10 ml of NEN-963 scintillation fluid in a -scintillation counter. In this representative autoradiogram, under control conditions, the apical:basolateral ratio for A₁AdoR was 70:30; in the presence of nocodazole, it was 40:60. The findings from multiple individual experiments are summarized as follows. The mean apical:basolateral ratio under control conditions was 68:32, and for nocodazole treatment it was 44:56 (n = 5). B, MDCK II cells expressing epitope-tagged A₁AdoR grown in Transwell culture were metabolically labeled in the absence or presence of colchicine (10 µM) with 1 µCi/µl of [³⁵S]Met/Cys protein labeling mix (150 µl) for 1 h and then processed as in Fig. 5A. In this representative autoradiogram, the apical:basolateral ratio A₁AdoR under control conditions was 63:37; in the presence of colchicine, it was 45:55. The mean apical:basolateral ratio under control conditions was 62:38, and for colchicine treatment it was 42:58 (n = 2). C, the retention time of the A₁AdoR on the apical surface in the presence of microtubule-disrupting agents was not significantly different from control. The loss of the receptor from the apical surface in the presence or absence of colchicine or nocodazole was assessed by means of "pulse-chase" experiments. MDCK II cells expressing epitope-tagged A₁AdoR grown in Transwell culture were metabolically labeled in the absence or presence of colchicine (10 µM) or nocodazole (33 µM) with 1 µCi/µl [³⁵S]Met/Cys protein labeling mix for 1 h, chased for 8 (t₀) and then 20 h (t₁), and then processed as in Fig. 5A. This was done twice for each drug at these time points. The ranges of the percentage remaining on the apical surface at 20 h were as follows: 50-67% (average, 57.3%; n = 3) for untreated (control) conditions, 51-79% (average, 65%; n = 2) in the presence of colchicine, and 57-58% (average, 57.5%; n = 2) in the presence of nocodazole.

Since steady-state localization reflects a balance of the delivery or targeting to a surface domain and the retention on that surface domain, we examined the effects of colchicine or nocodazole on the retention of the A₁AdoR on the apical surface. Neither colchicine nor nocodazole altered the half-life of this receptor on the apical surface (Fig. 5C). These findings suggest that the relative basolateral enrichment of the A₁AdoR following treatment with microtubule-disrupting agents is not due to accelerated apical turnover of the A₁AdoR. However, pulse-chase studies undertaken to evaluate whether the altered apical delivery profile was detected at multiple time points also revealed that the A₁AdoR was retained on the basolateral surface for longer periods of time (t1/2 > 30 h) when compared with the apical retention in the presence of nocodazole (t1/2 = 13 h); the t1/2 of the A₁AdoR in the presence of nocodazole is indistinguishable for the t1/2 in control, untreated cells (2). Previous studies of aminopeptidase N and sucrose-isomaltase in polarized Caco-2 intestinal epithelial cells also have noted a preferential retention of proteins on the basolateral versus apical surface in the presence of microtubule-disrupting agents (26).

The Apical Component of the Random Delivery of Nascent _2BAR Is Not Perturbed by Microtubule-disrupting Agents

Our observation that the A₁AdoR was apparently rerouted from the apical to the basolateral surface by microtubule-disrupting agents raised the question of whether the apical component of the random delivery of the _2BAR also might be modified following similar treatments. As shown in Fig. 6A, the equivalent delivery of the _2BAR to both the apical and basolateral surfaces is maintained in the presence of nocodazole: 47:53 apical to basolateral under control conditions versus 42:58 in the presence of nocodazole. Unexpectedly, the [³⁵S]Cys/Met signal for both the apical and basolateral _2BAR is increased 1.5-2 times that of the signal under control conditions in the presence of nocodazole (Fig. 6A). Similarly, a 2-3-fold increase in metabolically labeled _2BAR at both the apical and basolateral surfaces was observed in the presence of colchicine (Fig. 6B). This unexpected and unexplained increase in receptor delivery of the _2BAR to the cell surface in the presence of microtubule-disrupting agents may be due to a generalized increase in protein synthesis. Consistent with this hypothesis is our finding that a 15-h (but not 3-h) treatment with cycloheximide (data not shown) inhibited the colchicine-induced increase in radioligand binding, directly correlating with the time-dependent effects of colchicine to increase _2BAR radioligand binding, as described earlier in Fig. 4. The direct targeting of the _2AAR and the _2CAR to the basolateral surface was not altered by the microtubule-disrupting drugs, nocodazole and colchicine (data not shown).

Neither nocodazole nor colchicine disturbs _2BAR random delivery to both apical and basolateral surfaces.

We have shown previously that the _2AAR subtype is delivered directly to the basolateral domain (1). This apparent direct basolateral delivery, however, might instead result from active exclusion of this receptor subtype from the apical surface due to, for example, a cytoskeletal "screen" (30). Consequently, we examined the effect of cytochalasin D, which disrupts the actin-based cytoskeleton, on the localization of the _2AAR and other GPCR. Cytochalasin D did not alter the lateral localization of the _2AAR, suggesting that the direct basolateral delivery of this subtype is not due to exclusion from the actin-rich sub-apical surface. Interestingly, however, the _2BAR acquired a random localization at steady state following exposure to cytochalasin D, revealed by morphological detection of the _2BAR subtype on both the apical and basolateral surfaces (Fig. 2). Morphological analysis demonstrated that cytochalasin D also caused a redistribution of the basolaterally expressed endogenous EGF receptor and E-cadherin. Following cytochalasin D treatment, the EGF receptor appeared to be distributed on all surfaces as well as in the cell interior, and E-cadherin was apically as well as laterally localized (cf. x-z scans, Fig. 3). Thus, it would appear that the _2BAR, albeit not the _2AAR, mirrors the cytochalasin D-effected redistribution pattern of endogenous MDCK II proteins that typically are enriched on the lateral subdomain. Cytochalasin D also caused the apical marker protein gp135 to localize in a punctate, aggregated form on the apical surface (Fig. 3), as reported previously (7), without significantly altering the distribution of the apically targeted and localized A₁AdoR.

Since cytochalasin D appeared to alter the steady-state localization of a variety of endogenous MDCK proteins, we did not pursue this agent further as a tool to reveal distinct mechanisms for delivery and retention of GPCR in polarized renal epithelial cells. However, it should be noted that we evaluated the effects of cytochalasin D on GPCR radioligand binding and found that binding was not altered; the percentage of control binding for the GPCR following treatment with cytochalasin was 94 ± 7% for _2AAR, 115 ± 29% for _2BAR, 82 ± 25% for _2CAR, and 115 ± 19% for A₁AdoR (n = 3 for all GPCR). Thus, despite the impact of cytochalasin D on the morphological localization of both heterologously expressed GPCR and endogenous proteins that serve as markers for the polarized state, this agent did not have deleterious effects on the functional binding properties of the ₂AR subtypes or of the A₁AdoR.

Table I summarizes the morphological consequences of treatment with the microtubule-disrupting agents, colchicine and nocodazole, and of the actin cytoskeleton-disrupting agent, cytochalasin D, on the localization of GPCR and of polarized surface marker proteins in MDCK II cells.

Table I. Summary of effects of agents that disrupt the cytoskeleton or vesicular transport on the steady-state localization of proteins in MDCK II cells Drug Heterologous expression Endogenous proteins  2AAR A1AdoR  2BAR  2CAR EGFR gp135 E-cadherin None (control) Ba Ab > B B >> Ic B  I B A B Microtubule-disrupting agents   Colchicine B >> A, I B > A B B  I B A B   Nocodazole B >> A B > A B B  I B >> A A >> B B Actin-disrupting agents   Cytochalasin Dd B >> A, I B, I > A A, I, B B  A, I B, A, I A > I B >> A Vesicular transport-disrupting agents   Monensin B A >> B B > I, A B  I > Ae B A B   Brefeldin A B >> A A B > A B  I B >> A A B a B, basolateral. b A, apical. c I, intracellular compartment. d Note that cytochalasin D alters the polarization of endogenous proteins used as control "markers," such that changes in distribution of heterologously expressed receptors cannot be interpreted with confidence. e Pattern of drug-induced apical staining is punctate.

Monensin is a Na⁺/H⁺ electroneutral ionophore that dissipates transmembrane pH gradients. This agent has been demonstrated to inhibit receptor-mediated endocytosis (31) or the recycling (20-23) of cell surface receptors and has been used as a tool to prevent resensitization of GPCR (23). We evaluated the effects of monensin (1.4 µM) on the steady-state localization of GPCR to determine whether receptor endocytosis/recycling played a demonstrable role in their ultimate polarization (4-fold higher concentrations of this agent had similar effects on receptor localization; data not shown). Monensin altered the relative surface:internal compartment distribution of the _2BAR (Fig. 7A) without any appreciable effect on the localization of the other three GPCR examined (data not shown). Strikingly, there is an increased lateral staining intensity for the _2BAR in the presence of monensin compared with the control. As we observed with colchicine, these effects of monensin on lateral surface enrichment of the _2BAR are specific for this receptor subtype and are not seen for the other two ₂AR subtypes. The localization of the _2BAR in the absence of 0.1% Triton X-100 (surface receptors only) demonstrates that the _2BAR is enriched solely on the lateral surface at steady state, both in the absence and presence of monensin. However, monensin dramatically increased the fraction of _2BAR detected intracellularly, as seen in confocal images of cells incubated with anti-epitope antibody in the presence of 0.1% Triton X-100 (Fig. 7A), a manipulation that permeabilizes cells and allows the entire receptor population to be evaluated (surface and intracellular). Thus, there is receptor "trapped" inside the cells following treatment with this drug. These findings are analogous to the effects of monensin on the VSV G-protein trafficking in MDCK cells reported previously (24). Monensin did not interfere with the polarization of the endogenous proteins that serve as surface markers for MDCK II cells: the EGF receptor, gp135, and E-cadherin (Fig. 7B). Also, a 4-fold higher concentration of monensin (5.6 µM) similarly did not alter the polarization of these marker proteins (data not shown).

It is possible that the intracellular accumulation of the _2BAR subtype following monensin treatment results from a blockade of recycling (as a potential first step in apical to basolateral transcytosis and thus rerouting) of the randomly delivered _2BAR following its rapid removal (t1/2 = 15 min; Ref. 3) from the apical surface. As shown in Fig. 8, monensin did not alter the random delivery of the _2BAR, although it did increase the amount of nascent receptor delivered to both cell surfaces, again similar to findings with colchicine (and to some extent, nocodazole). Longer pulse time points were also evaluated; however, there was no change in the random targeting of _2BAR other than increased signal intensity delivered to both surfaces of polarized MDCK cells (data not shown). Monensin also did not alter the targeting of the A₁AdoR, as assessed in metabolic labeling and nascent receptor delivery studies (data not shown). We were not successful in monitoring the surface delivery of nascent _2AAR and _2CAR in the presence of monensin, perhaps because the drug interfered with the glycosylation of these two receptor subtypes (13), thereby rendering it incapable of reaching the cell surface (the _2BAR subtype is not glycosylated (32)). Monensin had no significant effect on radioligand binding for the GPCR: percentage of control binding for the GPCR following treatment with monensin was 86 ± 14% for _2AAR, 126 ± 15% for _2BAR, 156 ± 22% for _2CAR, and 110 ± 20% for A₁AdoR (n = 3-4 for all GPCR).

Monensin did not alter the targeting of the _2BAR in MDCK II cells.

Brefeldin A inhibits vesicular transport and secretion by selective disruption of the Golgi apparatus. It often has been used to explore trafficking itineraries in MDCK cells, and the results have been conflicting. BFA perturbs basolateral to apical transcytosis (14, 16-18, 33) but also enhances apical endocytosis (14) and transcytosis (14, 15). We were interested in determining whether or not the apical trafficking of the A₁AdoR might result from transcytosis, thus accounting for the different consequences of microtubule disruption on apical delivery of the A₁AdoR versus the _2BAR. A concentration of 3.5 µM BFA did not change the steady-state localization of any of the GPCR or any of the "marker" proteins (data not shown). As MDCK cells have been reported to be relatively resistant to BFA, we also tried a 10-fold higher concentration. At 35 µM BFA, the findings for the A₁AdoR, _2AAR, and _2BAR were similar to those in the presence of 3.5 µM BFA. Only the _2CAR localization was slightly altered following exposure to 35 µM BFA, such that the intracellular compartment described previously (3, 47) was no longer as visible (data not shown). Although we do not know the significance of the ability of BFA to eliminate detection of the intracellular compartment of the _2CAR, the data suggest that the intracellular pool of the _2CAR either is in a different vesicular compartment than internal pools of _2BAR (see "Discussion") or is delivered to or maintained in the cell interior via a different, BFA-sensitive mechanism. BFA had no significant effect on radioligand binding for the GPCR.

Table I summarizes the morphological consequences of treatment with the agents that interfere with vesicular traffic, monensin and brefeldin A.

DISCUSSION

It is important to understand the molecular mechanisms that govern polarized expression of epithelial cell proteins, since this polarity is an intrinsic part of the vectorial functioning of these cells. The importance of receptor localization in signal transduction is inferred from the number of pathophysiologic states that result from mislocalized proteins. In autosomal polycystic kidney disease and renal ischemia, the Na⁺,K⁺-ATPase and epidermal growth factor receptors of the basolateral plasma membrane are redistributed from the lateral surface to the apical membrane (34). In familial hypercholesterolemia, the low density lipoprotein receptor is unable to internalize and subsequently cannot transport cholesterol for storage as cholesterol esters (35). In cystic fibrosis, the most common F508 mutation restricts the CFTR chloride ion transporter to the endoplasmic reticulum and prevents its delivery to the cell surface (34). Mislocalized GPCR also contribute to disease. For example, one form of retinitis pigmentosa results from intracellular trapping of the G-protein-coupled receptor for light, rhodopsin (51). In addition, diabetes insipidus can result from a variety of mutations in the V₂ vasopressin receptor, many of which lead to receptor misfolding and lack of surface delivery (36). Understanding the mechanisms that govern the trafficking of receptors and signal transducing proteins should provide insights into the operation of receptor-mediated signal transduction events in polarized cells under physiologic conditions and reveal potential molecular culprits in pathophysiologic states.

We are interested in elucidating the mechanisms conferring the basolateral or apical polarization of GPCR. Our previous studies have shown that the basolaterally localized ₂AR subtypes have differing trafficking itineraries that lead to their ultimate shared polarity at steady state (1, 3); in contrast, the A₁AdoR is apically targeted and resides principally on the apical surface at steady state (2). In this study, we used experimental manipulations that previously have been demonstrated to disrupt the cytoskeleton or to interfere with vesicular transport of proteins as tools to explore possible differences in apical versus basolateral polarization mechanisms in more detail.

The present studies have revealed that the apical delivery of the A₁AdoR and _2BAR is differentially affected by microtubule-disrupting agents. Thus, apical delivery of the A₁AdoR, but not of the _2BAR, is inhibited by microtubule disrupting agents, leading to enriched delivery of the A₁AdoR to the basolateral surface (Fig. 5, A and B) and localization there at steady state (Fig. 2). Whether or not this relocalization is due to the direct rerouting of A₁AdoR delivery from the apical to the basolateral surface or, alternatively, to blockade of basolateral to apical transcytosis of the A1AdoR is not known. However, it should be noted that previous studies examining the delivery of metabolically labeled A₁AdoR to the apical surface are consistent with the interpretation that this is directly delivered to the apical surface (2). The ability of colchicine and nocodazole to disrupt apical delivery of the A₁AdoR is consistent with previous reports describing the requirement of an intact microtubule matrix for apical delivery of a number of proteins (12, 37-39) and lipids (40) in epithelial cells of the intestine, another absorptive epithelium. In polarized MDCK cells, apical but not basolateral membrane proteins (18, 41, 42) and apically but not basolaterally secreted proteins (43) were reported to be missorted to the basolateral surface following exposure to microtubule-disrupting drugs.

It is reasonable to postulate that disruption of A₁AdoR delivery to the apical surface by microtubule-directed agents reveals a role for these cytoskeletal elements in directing A₁AdoR-containing vesicles to that surface, by analogy with previous reports for apically targeted membrane proteins in MDCK cells described above (12, 37-39). In contrast, the apical component of the random delivery of the _2BAR must utilize a distinct, microtubule-independent mechanism to achieve apical trafficking. Although a formal possibility is that the apical delivery of the _2BAR is due to transcytosis from the basolateral membrane, two lines of evidence argue against this hypothesis. First, the half-life of the _2BAR on the basolateral surface is 10-12 h, whereas its half-life on the apical surface is 10-15 min. These data are more consistent with a random delivery, followed by an internalization of the apical receptor and possible (although there is no direct evidence) rerouting to the basolateral surface (3), as has been noted for Na⁺/K⁺-ATPase in certain clones of MDCK II cells (44). Second, we observed no effect of brefeldin A on GPCR polarization, previously shown either to interfere with (45) or to enhance (14, 15) transcytosis in MDCK cells. The mechanism contributing to the apical component of the random delivery of the _2BAR remains to be elucidated. However, these findings are of particular importance in establishing that there is a greater complexity of direct vesicular traffic to a given surface of polarized cells than simply the distinction between apical and basolaterally destined vesicles emerging from the trans-Golgi network (46) or, alternatively, transcytosis (42).

Another important contribution of the present studies is that they provide an additional line of evidence (47) that the intracellular compartment of the _2BAR is distinct from that occupied by the _2CAR. As indicated under "Results," the fraction of the _2BAR detected in the cell interior (Fig. 2) is thought to result from the continuous endocytosis/recycling of this receptor subtype in response to exogenous agonist or to catecholamines in serum-containing medium²; consistent with this interpretation are the present findings that treatment with monensin, an agent that blocks receptor recycling to the surface (20-22), increased the intracellular localization of the _2BAR subtype. In a seemingly reciprocal manner, colchicine enhanced the basolateral surface labeling of the _2BAR with a concomitant reduction in the fraction of detectable intracellular receptor (Fig. 2). This surface enrichment detected morphologically was paralleled by dramatic increases in the density of functional _2BAR binding sites (Fig. 4). Neither of these colchicine-induced phenomena occurred with the _2CAR subtype. A similar discordance of _2BAR localization in comparison with that of an endocytosing GPCR, the ₂-adrenergic receptor, was observed in HEK 293 cells (47). It should be noted that nocodazole is quantitatively less effective than colchicine in eliciting the apparent redistribution of _2BAR to the lateral surface (Fig. 2) and in enhancing receptor binding density (Fig. 4), consistent with earlier reports that these agents, although both disrupting microtubules, can elicit quantitatively different effects on cells (12). The unique increase in signal intensity in the autoradiograms of metabolically labeled _2BAR-expressing cells seen in the presence of nocodazole and colchicine, as well as in the presence of monensin, are intriguing. However, at present, we have no data that reveal a molecular basis for these findings.

Although we evaluated the effects of the actin cytoskeleton-disrupting agent, cytochalasin D, on the polarization of A₁AdoR, ₂AR subtypes, and endogenous markers for polarization in MDCK cells, we chose not to pursue these studies beyond our morphological characterization, since we noted that the localization of E-cadherin, which initiates and then maintains polarization of epithelial cells (4), is perturbed by treatment with cytochalasin D (Fig. 3). Interestingly, the staining profile of the _2BAR is the one most altered by cytochalasin D, and this is the only receptor of the four studied known to undergo extensive ligand-induced endocytosis. Previous studies have shown that cytochalasin D selectively inhibits apical but not basolateral endocytosis of VSV G-protein, cationized ferritin, and Lucifer yellow in MDCK cells (48). It also is interesting that cytochalasin D-induced relocalization of the basolateral GPCR evaluated in these studies parallels that of the basolateral markers, EGF receptor and E-cadherin, whereas the morphological changes in distribution of the apically targeted and polarized A₁AdoR are paralleled by those of the gp135 apical marker. These findings with the actin-disrupting agent cytochalasin D are in distinct contrast to the consequences of disruption of the microtubule-dependent cytoskeleton by colchicine and nocodazole, which led to differential effects on the localization of the GPCR with no remarkable changes in the localization of gp135, E-cadherin, or the EGF receptor.

The present studies provide the first evidence that multiple pathways exist for the direct targeting of G-protein-coupled receptors of the G_i/G_o-coupled structural family, the A₁AdoR and _2BAR, to the apical surface of polarized renal epithelial cells. These findings extend earlier demonstrations that apical and basolateral proteins are packaged into different vesicular carriers following egress from the trans-Golgi network (49) and suggest that there must be at least one other bifurcation at the level of apically directed vesicles. The ability of microtubule-disrupting agents to discriminate between the apical delivery mechanisms for the A₁AdoR and the _2BAR suggest that these drugs will be powerful tools in delineating the molecular mechanisms underlying these diverse apical delivery pathways. Elucidation of the molecular mechanisms responsible for microtubule-dependent versus -independent apical delivery may provide novel therapeutic advantage in settings where apical delivery defects contribute to human pathology, as in cystic fibrosis (50).