Disruption of Microtubules Reveals Two Independent Apical Targeting Mechanisms for G-protein-coupled Receptors in Polarized Renal Epithelial Cells
1997; Elsevier BV; Volume: 272; Issue: 30 Linguagem: Inglês
10.1074/jbc.272.30.19035
ISSN1083-351X
AutoresChristine Saunders, Lee E. Limbird,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoG-protein-coupled receptors demonstrate differing trafficking itineraries in polarized Madin-Darby canine kidney (MDCK II) cells. The α2A adrenergic receptor (α2AAR) is directly delivered to the basolateral subdomain; the A1 adenosine receptor (A1AdoR) is apically enriched in its targeting; and the α2BAR subtype is randomly delivered to both domains but selectively retained basolaterally (Keefer, J. R., and Limbird, L. E. (1993) J. Biol. Chem. 268, 11340–11347; Saunders, C., Keefer, J. R., Kennedy, A. P., Wells, J. N., and Limbird, L. E. (1996) J. Biol. Chem. 271, 995–1002; Wozniak, M., and Limbird, L. E. (1996) J. Biol. Chem. 271, 5017–5024). The present studies explore the role of the polarized cytoskeleton in localization of G-protein-coupled receptors in MDCK II cells. Nocodazole or colchicine, which disrupt microtubules, did not perturb lateral localization of α2AR subtypes but led to a relocalization the A1AdoR to the basolateral surface, revealed by immunocytochemical and metabolic labeling strategies. Conversely, the apical component of the random delivery of α2BAR was not affected by these agents, suggesting microtubule-dependent and -independent apical targeting mechanisms for G-protein-coupled receptors in polarized cells. Apparent rerouting of the apically targeted A1AdoR was selective for microtubule-disrupting agents, since cytochalasin D, which disrupts actin polymerization, did not alter A1AdoR or α2BAR localization or targeting. These data suggest that multiple apical targeting mechanisms exist for G-protein-coupled receptors and that microtubule-disrupting agents serve as tools to probe their different trafficking mechanisms. G-protein-coupled receptors demonstrate differing trafficking itineraries in polarized Madin-Darby canine kidney (MDCK II) cells. The α2A adrenergic receptor (α2AAR) is directly delivered to the basolateral subdomain; the A1 adenosine receptor (A1AdoR) is apically enriched in its targeting; and the α2BAR subtype is randomly delivered to both domains but selectively retained basolaterally (Keefer, J. R., and Limbird, L. E. (1993) J. Biol. Chem. 268, 11340–11347; Saunders, C., Keefer, J. R., Kennedy, A. P., Wells, J. N., and Limbird, L. E. (1996) J. Biol. Chem. 271, 995–1002; Wozniak, M., and Limbird, L. E. (1996) J. Biol. Chem. 271, 5017–5024). The present studies explore the role of the polarized cytoskeleton in localization of G-protein-coupled receptors in MDCK II cells. Nocodazole or colchicine, which disrupt microtubules, did not perturb lateral localization of α2AR subtypes but led to a relocalization the A1AdoR to the basolateral surface, revealed by immunocytochemical and metabolic labeling strategies. Conversely, the apical component of the random delivery of α2BAR was not affected by these agents, suggesting microtubule-dependent and -independent apical targeting mechanisms for G-protein-coupled receptors in polarized cells. Apparent rerouting of the apically targeted A1AdoR was selective for microtubule-disrupting agents, since cytochalasin D, which disrupts actin polymerization, did not alter A1AdoR or α2BAR localization or targeting. These data suggest that multiple apical targeting mechanisms exist for G-protein-coupled receptors and that microtubule-disrupting agents serve as tools to probe their different trafficking mechanisms. 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) 1The abbreviations used are: GPCR, G-protein-coupled receptor(s); α2AR, α2adrenergic receptor(s); A1AdoR, A1 adenosine receptor(s); MDCK, Madin-Darby canine kidney; WT, wild-type; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; BFA, brefeldin A; [3H]DPCPX, 8-cyclopentyl-1,3-di-[2,3-3H]propylxanthine; BFA, brefeldin A; EGF, epidermal growth factor. 1The abbreviations used are: GPCR, G-protein-coupled receptor(s); α2AR, α2adrenergic receptor(s); A1AdoR, A1 adenosine receptor(s); MDCK, Madin-Darby canine kidney; WT, wild-type; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; BFA, brefeldin A; [3H]DPCPX, 8-cyclopentyl-1,3-di-[2,3-3H]propylxanthine; BFA, brefeldin A; EGF, epidermal growth factor. attain their localization in renal epithelial cells, using polarized MDCK II cells as a model system. We have demonstrated that the G-protein-coupled α2AAR is delivered directly to and retained on the basolateral subdomain of renal epithelial cells (1Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar), whereas the A1AdoR predominantly is apically targeted (65–83%) in renal epithelial cells (2Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The trafficking itineraries of the α2BAR and α2CAR subtypes differ from that of the α2AAR: the α2BAR is randomly delivered to both the apical and basolateral domains but selectively retained basolaterally; the α2CAR is directly delivered basolaterally, but a substantial fraction of the receptor population remains in a cytoplasmic compartment at steady state (3Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). These different targeting itineraries are summarized in Fig. 1.Figure 2The steady-state localization of heterologously expressed GPCR in the absence or presence of cytoskeletal disrupting drugs. MDCK II cells expressing GPCR were grown in Transwell culture for one week to ensure a polarized phenotype. For experiments, the cells were treated overnight with or without colchicine (10 μm), nocodazole (33 μm), or cytochalasin D (2 μm). Before beginning the immunocytochemistry, the integrity of the monolayer was assessed by assaying for the transepithelial leak of [3H]methoxyinulin from the apical to the basolateral compartment after 1 h of incubation at 37 °C. A leak greater than 3% was excluded from the study (except for cytochalasin D, where leaks often were 5–8%). The cells were then stained on their Transwell filter supports, as described under "Experimental Procedures," and analyzed by confocal microscopy.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Nocodazole 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 β-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. 5 A. 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 (t 0) and then 20 h (t 1), and then processed as in Fig.5 A. 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Polarized localization of cytoskeletal elements (A) and GPCR (B) in MDCK II cells. A, a schematic diagram showing the structurally and functionally distinct apical and basolateral surface membrane domains and their relationship to the polarized cytoskeleton. The tight actin-rich network of microfilaments is close to the microvilli of the apical domain of the cell. Randomly organized microtubules also underlie the apical surface domain; the apical surface is denoted by the wavy line at the top of the cell shown. Polarized microtubules run vertically along the lateral surface domain of the cell, with the minus ends of the microtubule facing the apical surface and the plus ends facing the basal region of the cell. Polarized microtubules "grow" from the minus end toward the plus end. γ-Tubulin binds α/β-tubulin heterodimers, the former thus serving as a nucleation site onto which tubulin dimers assemble (10Hyams J.S. Lloyd C.W. Microtubules. Wiley-Liss, New York, NY1994: 33-45Google Scholar). The cortical cytoskeleton underlying the lateral subdomain is composed of fodrin (non-erythrocyte spectrin) and ankyrin, which often serves to tether transmembrane proteins to this lateral scaffold. B, polarization of GPCR in MDCK II cells. Previous studies reveal that the Gi/Go-coupled subtypes achieve basolateral localization by differing trafficking itineraries (1Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar, 3Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). A substantial fraction of the α2CAR is localized intracellularly at steady state (3Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 47von Zastrow M. Link R. Daunt D. Barsh G. Kobilka B. J. Biol. Chem. 1993; 268: 763-766Abstract Full Text PDF PubMed Google Scholar). In contrast, the Gi/Go-coupled A1AdoR is preferentially targeted to and sustained on the apical surface by a direct targeting mechanism (2Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). These findings indicate that these four GPCR serve as unique reagents to explore the role of the polarized cytoskeleton on GPCR trafficking and polarization.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Like receptors, including the GPCR described above, the cytoskeleton in a polarized cell also is nonuniformly distributed (Fig.1 A). 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 (4Nelson W.J. Cell Biol. 1991; 2: 375-385Google Scholar). 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 (4Nelson W.J. Cell Biol. 1991; 2: 375-385Google Scholar). Since apical versus basolateral targeting of membrane proteins appears to involve discrete post-trans-Golgi network vesicular populations (5Griffiths G. Simons K. Science. 1986; 234: 438-443Crossref PubMed Scopus (756) Google Scholar), 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. 11Lafont F. Burkhardt J.K. Simons K. Nature. 1994; 372: 801-803Crossref PubMed Scopus (169) Google Scholar, 12Matter K. Bucher K. Hauri H.-P. EMBO J. 1990; 9: 3163-3170Crossref PubMed Scopus (76) Google Scholar, and 18Arreaza G. Brown D.A. J. Biol. Chem. 1995; 270: 23641-23647Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). 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 α2AAR and α2CAR, the randomly delivered but basolaterally retained α2BAR, and the apically targeted A1AdoR. We postulated that the use of cytoskeletal agents in combination with the study of GPCR possessing varying trafficking itineraries (2Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 3Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) would allow us to ascertain criteria for basolateralversus apical targeting as well as direct versusrandom delivery. The present data provide evidence that apical delivery of the A1AdoR and α2BAR 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. 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 [α-35S]dATP (1389 Ci/mmol) were from NEN Life Science Products. The A1AdoR antagonist, 1,3-dipropyl-8-(4-sulfophenyl)xanthine (6Daly J.W. Shamin M.T. Butts-Lamb P. Waters J. J. Med. Chem. 1985; 28: 487-492Crossref PubMed Scopus (235) Google Scholar), 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 (7Ojakian G.K. Schwimmer R. J. Cell Biol. 1988; 107: 2377-2387Crossref PubMed Scopus (153) Google Scholar) and EGF receptor (8Gill G.N. Kawamoto T. Cochet C. Le A. Sato J.D. Masui H. McLeod C. Mendelsohn J. J. Biol. Chem. 1984; 259: 7755-7760Abstract Full Text PDF PubMed Google Scholar) 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-β-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. MDCK II cells were maintained as described previously (2Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 3Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). 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 (2Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Prior to each experiment, the integrity of the monolayer was assessed by monitoring [3H]methoxyinulin leak (1Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar). Permanent clonal cell lines of MDCK II cells were developed as described previously (1Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar, 2Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 3Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In each case, the first 9 amino acids after the initiating methionine encode a hemagglutinin epitope (9Wilson I.A. Niman H.L. Houghten R.A. Cherenson A.R. Connolly M.L. Lerner R.A. Cell. 1984; 37: 767-778Abstract Full Text PDF PubMed Scopus (651) Google Scholar) recognized by the commercially available monoclonal antibody 12CA5 (Babco). The clonal cell lines evaluated in the present study include TAG-α2AAR (25 and 7 pmol/mg of protein), TAG-α2BAR (10 and 3 pmol/mg of protein), TAG-α2CAR (5 and 3 pmol/mg of protein) and TAG-A1AdoR (37 pmol/mg of protein). The α2AAR subtype was encoded by a porcine cDNA, the α2BAR and α2CAR 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 α2AAR is targeted directly to the basolateral surface in both canine MDCK II and porcine LLC-PK1 renal epithelial cells (1Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar), and the canine A1AdoR is targeted apically in both MDCK II and LLC-PKI cells (2Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). MDCK II particulate preparations were prepared essentially as described (1Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar). A1AdoR were identified using [3H]DPCPX (2Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and α2AR were identified with [3H]rauwolscine (1Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar, 3Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) as described previously. 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 (10Hyams J.S. Lloyd C.W. Microtubules. Wiley-Liss, New York, NY1994: 33-45Google Scholar). 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 (11Lafont F. Burkhardt J.K. Simons K. Nature. 1994; 372: 801-803Crossref PubMed Scopus (169) Google Scholar, 12Matter K. Bucher K. Hauri H.-P. EMBO J. 1990; 9: 3163-3170Crossref PubMed Scopus (76) Google Scholar). 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 β-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 (10Hyams J.S. Lloyd C.W. Microtubules. Wiley-Liss, New York, NY1994: 33-45Google Scholar). 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 (7Ojakian G.K. Schwimmer R. J. Cell Biol. 1988; 107: 2377-2387Crossref PubMed Scopus (153) Google Scholar) and Caco-2 (12Matter K. Bucher K. Hauri H.-P. EMBO J. 1990; 9: 3163-3170Crossref PubMed Scopus (76) Google Scholar) 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-β-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 (18Arreaza G. Brown D.A. J. Biol. Chem. 1995; 270: 23641-23647Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar): 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 (13Rosa P. Mantovani S. Rosboch R. Huttner W.B. J. Biol. Chem. 1992; 267: 12227-12232Abstract Full Text PDF PubMed Google Scholar). BFA has been demonstrated to both enhance transcytosis (14Prydz K. Hansen S.H. Sandvig K. van Deurs B. J. Cell Biol. 1992; 119: 259-272Crossref PubMed Scopus (86) Google Scholar, 15Wan J. Taub M.E. Shah D. Shen W.-C. J. Biol. Chem. 1992; 267: 13446-13450Abstract Full Text PDF PubMed Google Scholar) and inhibit transcytosis (16Hunziker W. Male P. Mellman I. EMBO J. 1990; 9: 3515-3525Crossref PubMed Scopus (129) Google Scholar) or targeting (17Low S.H. Tang B.L. Wong S.H. Hong W. J. Cell Biol. 1992; 118: 51-62Crossref PubMed Scopus (57) Google Scholar) 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 α2BAR. Brefeldin A was added to a final concentration of 3.5 μm, as described previously for MDCK II cells (18Arreaza G. Brown D.A. J. Biol. 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Pharmacol. 1995; 47: 666-676PubMed Google Scholar). Monensin was evaluated at a final concentration of 1.4 μm, as described previously for MDCK II cells (18Arreaza G. Brown D.A. J. Biol. Chem. 1995; 270: 23641-23647Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 24Alonso F.V. Compans R.W. J. Cell Biol. 1981; 89: 700-705Crossref PubMed Scopus (39) Google Scholar). Immunostaining of cells grown in Transwell culture was performed as described previously (2Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 3Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) with the following concentration of primary antibody: a 1:50 dilution of 12CA5 primary antibody, purified as described previously (3Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), for the localization of hemagglutinin epitope-tagged GPCR (1Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar); 15 μg/ml mouse monoclonal EGF receptor antibody (25Dempsey P.J. Coffey R.J. J. Biol. Chem. 1994; 269: 16878-16889Abstract Full Text PDF PubMed Google Scholar); a 1:10 dilution of mouse monoclonal gp135 antibody (7Ojakian G.K. Schwimmer R. J. Cell Biol. 1988; 107: 2377-2387Crossref PubMed Scopus (153) Google Scholar); 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 (2Saunders C. Keefer J.R. Kennedy A.P. Wells J.N. Limbird L.E. J. Biol. Chem. 1996; 271: 995-1002Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 3Wozniak M. Limbird L.E. J. Biol. Chem. 1996; 271: 5017-5024Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Samples were visualized by confocal microscopy on a Zeiss Axiovert 135 Micro Systems LSM (Germany). The samples were first visualized in thexy plane and then in the xz plane. In the images shown, the bottom ¾ represent the xy plane, the conventional view of the cells as one looks down upon them. Thewhite 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 ¼ 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. The amount of newly synthesized receptor delivered to the apicalversus 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 (1Keefer J.R. Limbird L.E. J. Biol. Chem. 1993; 268: 11340-11347Abstract Full Text PDF PubMed Google Scholar), except that wherever drugs were used, they were added in the Cys/Met-free medium for the dur
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