ARTICLE |
Correspondence to: Katja Holappa, Dept. of Biochemistry, University of Oulu, PO Box 3000, FIN-90014 Oulu, Finland. E-mail: kholappa@cc.oulu.fi
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Na+-independent Cl-/HCO3- exchangers (AE1, AE2, AE3) are generally known as ubiquitous, multispanning plasma membrane proteins that regulate intracellular pH and transepithelial acidbase balance in animal tissues. However, previous immunological evidence has suggested that anion exchanger (AE) proteins may also be present in intracellular membranes, including membranes of the Golgi complex and mitochondria. Here we provide several lines of evidence to show that an AE protein is indeed a resident of the Golgi membranes and that this protein corresponds to the full-length AE2a isoform in fibroblasts. First, both the N- and C-terminal antibodies to AE2 (but not to AE1) detected an AE protein in the Golgi membranes. Golgi localization of this AE2 antigen was evident also in cycloheximide-treated cells, indicating that it is a true Golgi-resident protein. Second, our Northern blotting and RT-PCR analyses demonstrated the presence of only the full-length AE2a mRNA in cells that show prominent Golgi staining with antibodies to AE2. Third, antisense oligonucleotides directed against the translational initiation site of the AE2a mRNA markedly inhibited the expression of the endogenous AE2 protein in the Golgi. Finally, transient expression of the GFP-tagged full-length AE2a protein resulted in predominant accumulation of the fusion protein in the Golgi membranes in COS-7 and CHO-K1 cells. Golgi localization of the AE2a probably involves its oligomerization and/or association with the recently identified Golgi membrane skeleton, because a substantial portion of both the endogenous AE2a and the GFP-tagged fusion protein resisted detergent extraction in cold. (J Histochem Cytochem 49:259269, 2001)
Key Words: Golgi, anion exchanger, fibroblast, expression, protein sorting, cytoskeleton, immunocytochemistry
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The functional family of bicarbonate transporters constitutes a versatile class of plasma membrane proteins that include Na+-dependent and -independent Cl-/HCO3- exchangers, Na+/HCO3- co-transporters, organic anion/HCO3- transporters, and a K+/HCO3- co-transporter. The Na+-independent Cl-/HCO3- exchanger (AE) gene family is the best characterized of these and consists of three well-defined members, termed AE1, AE2, and AE3 (
AE proteins are encoded by multiple transcripts that appear to be expressed in a tissue- or cell type-specific fashion (
|
AE polypeptides also show remarkable variability in their subcellular localization in cells and tissues. For example, in kidney tubules, the N-terminally truncated variants of AE1 and of AE2 are localized to the basolateral plasma membrane of polarized epithelial cells (
The biological rationale for the existence of multiple AE size variants is not known. One possibility is that the N-terminal sequences that are variable among the transcripts contain regulatory elements (e.g., phosphorylation sites) that can be used either to inhibit or to stimulate their anion transport properties in specified target cells (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antibodies and Reagents
The antibody against the AE2 C-terminal peptide (amino acids 12291241 of AE2) was prepared as described (
An N-terminal peptide antibody against AE2 (amino acids 485497 of AE2) was a kind gift from William Horne (Yale University; New Haven, CT). C-terminal and N-terminal AE1-specific polyclonal and monoclonal antibodies were generous gifts from Drs. Ron Kopito (Stanford University; San Francisco, CA), Marcin Czerwinski (BIII.136; Ludwik Hirszfeld Institute of Immunology, Wroclaw, Poland), and David Anstee (Bric 130, Bric 169; International Blood Group Laboratory, Bristol, UK).
A monoclonal anti-GFP antibody and the EGFP-C1-vector were obtained from Clontech. Tetramethylrhodamine isothiocyanate (TRITC)-conjugated swine anti-rabbit and fluorescein isothiocyanate (FITC)-conjuaged goat anti-mouse secondary antibodies were obtained from Dakopatts (Copenhagen, Denmark). Peroxidase-conjugated sheep anti-rabbit and goat anti-mouse antibodies were from BioSys (Compiegne, France). Antibodies against ß-COP (Affinity Bioreagents; Denver, CO) and giantin (a kind gift from Dr. Hans-Peter Hauri; University of Basel, Switzerland) were used as Golgi markers.
Stock solutions of cycloheximide, nocodazole (Sigma, St Louis, MO; both 10 mg/ml) and brefeldin A (BoehringerMannheim, Mannheim, Germany; 5 mg/ml) were dissolved in ethanol, stored at -20C, and used at 1:1000 dilution.
Cell Cultures
Human embryonic skin fibroblasts were grown in MEM (Gibco; Grand Island, NY) with glutamax supplemented with 10% newborn calf serum, penicillinstreptomycin (Gibco), and 50 µg/ml ascorbic acid. Treatment of cells with cycloheximide, brefeldin A, and nocodazole was for 15 hr, 30 min, and 4 hr, respectively. The COS-7, NRK, and CHO-K1 cells were obtained from ATCC (Rockville, MD) and were cultured in media recommended by ATCC.
Indirect Immunofluorescence
Cells were processed for indirect immunofluorescence as described elsewhere (
Northern Blotting Experiments
Isolation of total RNA from cells and human testicular tissue (obtained from Oulu University Hospital) was performed using the acid guanidinium thiocyanatephenolchloroform extraction method. Northern blotting experiments were performed as previously described (
RT-PCR
RT-PCR was utilized to amplify overlapping fragments of AE2 cDNA (each about 1 kb in length) from total RNA samples isolated either from human testicular tissue or from cultured human fibroblasts. For reverse transcription, 1 µg of total RNA was used, and two subsequent PCR rounds were performed with AE2-specific primer pairs. For the second round PCR, one of the primers in each primer pair was nested with respect to the first-round primer. As a control template, we used the full-length human testicular AE2a cDNA inserted in pGEM-4Z plasmid vector (
Antisense Oligonucleotide Treatments
Phosphorothioate antisense oligonucleotides (24-mer; 5'-GCGCTGCTCATGGCCGAATCTTAG-3') directed against the translational initiation site and flanking sequences of the AE2a mRNA were ordered from Amersham-Pharmacia Biotech (Poole, UK) and manufactured by Genosys Biotechnologies. A nonsense oligonucleotide (22-mer) was used as a control in the experiments. Both antisense and nonsense oligonucleotides (25 µg/ml) were added directly to 1-day-old COS-7 cell cultures and the cells were then cultivated in medium containing serum for 3 additional days before washing with PBS, fixation, and analyses by indirect immunofluorescence with the anti-AE2 antibody.
GFP Fusion Protein Constructs and Transfections
To prepare the full-length AE2GFP fusion protein, pGEM-4Z plasmid containing human AE2a cDNA (cloned from human testicular tissue) was first mutated by using the Quick Change mutagenesis kit (Stratagene; La Jolla, CA) to eliminate the stop codon just before the ATG translational initiation site and to create a new 5'-HindIII site for insertion into the GFP vector (pEGFP-C1; Clontech, Palo Alto, CA). The mutated full-length AE2 cDNA was subcloned into the EGFP-C1 vector after digestion with EcoRI and HindIII restriction enzymes. After transformation into JM 109 cells, cDNA was isolated using the plasmid purification kit of Qiagen. Transfections into COS-7 cells plated 1 day earlier were done using 12 µg of purified AE2a cDNA/30-mm plate and the FUGENE6-transfection reagent (BoehringerMannheim) according to the manufacturer's instructions. Transfected cells were examined 2024 hr after transfection.
Immunoblotting
Cells were washed and scraped directly into hot SDS sample buffer with a rubber policeman. The sample was collected, vortexed rigorously for 2 hr to eliminate viscosity, and heated for 3 min at 94C before subjection to SDS-PAGE (7.59% acrylamide gel). Size-separated samples were transferred onto a nitrocellulose filter and immunoblotted with the anti-GFP and anti-AE2 antibodies. Stained bands on the blots were visualized using the ECL detection system (Amersham) and FUJI RX X-ray film.
Trypsin Treatments and Cell Surface Biotinylation
To quantitate the level of expression of the fusion protein at the cell surface, transfected COS-7 cells were treated on plates at either 4C or 37C with 0.2% trypsin for 3060 min. Cells were then collected by centrifugation, rinsed with PBS, and solubilized directly into SDS sample buffer and analyzed by SDS-PAGE and immunoblotting with the anti-GFP antibody. As an alternative approach, transfected cells were biotinylated using 0.5 mg/ml sulfo-NHS-biotin (Pierce; Rockford, IL) in PBS on ice. Cells were dissolved in 0.5% Triton X-1000.5% deoxycholate in PBS and biotinylated proteins were absorbed by using immobilized streptavidin (BoehringerMannheim). Unbound and absorbed material was collected and analyzed by SDS-PAGE.
Detergent Extraction with Triton X-100
Fibroblasts and COS-7 cells were subjected to detergent extraction protocol (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Both N- and C-terminal Antibodies Against AE2 Stain the Golgi Membranes in Different Cell Lines
Previously, the presence of a Golgi-associated AE was detected by using a C-terminal peptide antibody against the erythrocyte AE1 (
|
Because both of these antibodies (anti-AE1 and -AE2) recognize AE1- and AE2-derived C-terminal peptides (nine of 12 amino acids are identical), it was not possible to ascertain which of the two isoforms (AE1 or AE2) is expressed in the Golgi. Therefore, we next used antisera that were specific for the N-terminal cytoplasmic domains of either AE1 or AE2. However, none of the three N-terminal anti-AE1 antibodies we obtained (see Materials and Methods) stained the Golgi membranes or the plasma membrane in fibroblasts, nor did they recognize any bands in immunoblotting experiments with fibroblast total-cell lysates (data not shown). However, they recognized the human erythrocyte AE1 polypeptide (a 100-kD band). In contrast, an AE2-specific N-terminal peptide antiserum (raised against amino acids 482497 of AE2) gave Golgi staining in fibroblasts (Fig 2B) and in COS-7 cells (data not shown). These data indicate that the AE2 isoform is the endogenous Golgi-associated AE polypeptide in fibroblasts and in COS-7 cells.
Double-staining experiments in COS-7 cells with the anti-AE2 antibody (Fig 2E) and monoclonal antibodies against giantin (Fig 2F), ß-COP, and KDEL receptor confirmed that the endogenous AE2 protein also co-localizes with these Golgi markers. Brefeldin A treatment of fibroblasts also caused a rapid redistribution of the AE2 into the ER (Fig 3A3D). These findings are fully compatible with our previous immunoelectron microscopic data, which have shown that the AE-related antigen was concentrated in the cisternal membranes of the Golgi stack (
|
Finally, we assessed whether the Golgi AE2 is merely in transit towards the plasma membrane by using cycloheximide, a drug that inhibits protein synthesis but does not block transport to the cell surface. Indirect immunofluorescence experiments showed (Fig 4A4C) that treatment of cells for up to 5 hr did not diminish staining of the Golgi. The concentration of the drug used (10 µg/ml) was found to inhibit incorporation of [35S]-methionine into TCA-precipitable proteins by more than 95%. Therefore, the AE2 protein can be considered a resident of the Golgi membranes.
|
AE2a mRNA Is the Predominant mRNA Species in Fibroblasts
Because both the N- and C-terminal antibodies recognize all known AE2 variants (Fig 1), Northern blotting and RT-PCR analyses were necessary to specify more exactly the Golgi-resident AE2 isoform or variant. In three different cell lines that show prominent Golgi staining with antibodies to AE2 (two of which are shown in Fig 2), Northern blotting analyses detected only a 4.4-kb mRNA species with an AE2 cDNA probe that corresponded to a portion of the C-terminal membrane-spanning domain of AE2 (Fig 5A). On the basis of its size, this mRNA species corresponds to the full-length AE2a mRNA transcript. Transcripts of the N-terminally truncated AE2 isoforms in rat (AE2b, 4.2 kb and AE2c, 3.8 kb) and human tissues [AE2b(1) and AE2b(2)] were not detected in these cell lines. It should be noted that the probe used should hybridize with all N-terminally truncated AE2 variants if they were present in these cells.
|
To confirm the identity of the 4.4-kb transcript detected as the full-length AE2a mRNA, we next performed RT-PCR to amplify the complete coding region of the AE2a mRNA as four overlapping fragments. For the RT-PCR, we used total RNA isolated either from cultured human fibroblasts or from human testis tissue. Rat testis is known to express mainly the AE2a mRNA (
Antisense Oligonucleotides Inhibit the Expression of Endogenous AE2 Protein in the Golgi Membranes
To provide additional evidence for the identity of the Golgi-associated AE as the full-length AE2a isoform, we treated COS-7 cells with AE2a-specific antisense oligonucleotides (25 µg/ml) that corresponded to the translational initiation site and flanking regions of the AE2a mRNA. Treatment for 3 days under normal cell culture conditions was sufficient to cause a marked inhibition (80%, based on fluorescent intensity measurements) of the expression of endogenous AE2 polypeptide in the Golgi in COS-7 cells (Fig 5E), as evidenced by indirect immunofluorescence staining using the C-terminal anti-AE2 antibody. The effect was specific because nonsense oligonucleotides did not decrease the expression of the Golgi-localized AE2 protein (Fig 5D). Unfortunately, we have been unable to quantitate the inhibition by immunoblotting, most probably because the C-terminal epitope has been reported to be sensitive to SDS (
Transient Expression of GFP-tagged AE2a Fusion Protein
Indirect immunofluorescence with the anti-AE2 antibodies showed consistent staining of the Golgi membranes in COS-7 cells (Fig 5D). These cells therefore express an endogenous AE2 protein in the Golgi membranes. To enable detection and localization of transiently expressed human AE2a protein in these monkey cells, we inserted a cDNA encoding the green fluorescent protein (GFP) into the N-terminus of the human AE2a cDNA cloned from the testicular tissue. This does not affect membrane insertion of the fusion protein because AE polypeptides are known to have an internal signal sequence.
Transient expression of the GFP-tagged full-length human AE2a protein in COS-7 cells showed (Fig 6A) that the fusion protein accumulated in juxtanuclear structures that most probably represented the Golgi membranes. A similar expression pattern was also seen in transfected CHO-K1 cells (data not shown). Accumulation of the GFPAE2a fusion protein predominantly in the Golgi membranes was confirmed by using both nocodazole and co-localization experiments with antibodies to known Golgi-markers. First, we treated transfected cells for the last 4 hr during the 20-hr transfection with nocodazole, a drug that severs microtubule polymerization. In drug-treated cells, the AE2a fusion protein was found in small punctate structures that were scattered throughout the cytoplasm (Fig 6B). Staining of the transfected cells with antibodies to either giantin (Fig 6D) or ß-COP (Fig 6G) also showed that the GFPAE2a fusion protein co-localizes with the Golgi markers used. However, non-overlapping areas were also detected in transfected cells (Fig 6E and Fig 6H).
|
Detection of GFP-tagged AE2a Fusion Protein by Immunoblotting
To show that the transfected cDNA encodes a full-size AE2a protein, we next performed immunoblotting experiments with the total cell lysates from transfected COS-7 cells (Fig 7A) by using the anti-GFP monoclonal antibody (Fig 7, Lane 3) and the C- and N-terminal anti-AE2 antibodies (Fig 7, Lanes 2 and 4). All antibodies recognized one major protein of about 200 kD. In untransfected cells, this band was not seen (Fig 7, Lane 1). The size of this band corresponds well with the predicted molecular size of the fully glycosylated AE2a protein (165 kD), given that GFP itself has a molecular size of about 27 kD. The identity of an additional 165-kD band that was detected variably only with the anti-GFP monoclonal antibody (Fig 7A, Lane 3) is not known. The calculated molecular weight of the AE2a protein based on its amino acid composition is 137 kD, so the 165-kD band may represent an unglycosylated form of the GFP-tagged AE2 polypeptide (27 kD + 137 kD) or its proteolytic product. It is not precisely known if and to what extent the AE2 protein is glycosylated, but it has four potential N-glycosylation sites, three of which reside in its extensive third extracellular loop.
|
Quantitation of the Cell Surface Expression of the GFPAE2 Fusion Protein
Because all AE polypeptides are usually considered to be plasma membrane proteins, it was necessary to quantitate the amount of the GFPAE2a fusion protein that was transported to the plasma membrane in transfected COS cells. Cells transfected with the GFP-tagged AE2a cDNA were subjected on plates to extensive enzymatic digestion with trypsin at 4C or at 37C for 3060 min. At 37C, this resulted in detachment of the cells from the plates. Subsequent analyses by SDS-PAGE, immunoblotting, and quantitation of the 200-kD fusion protein (Fig 7B) and its potential degradation products showed that it was not degraded by this extracellularly added enzyme. Nevertheless, AE2 contains a number of putative cleavage sites for trypsin, e.g., in its third extracellular domain. On the other hand, permeabilization of the cells with saponin during trypsin digestion resulted in complete degradation of the fusion protein (data not shown). As an alternative approach, we used biotinylation of the cell surface proteins on plates with sulfoNHSbiotin. We then affinity-purified biotinylated proteins with immobilized streptavidin before SDS-PAGE. Immunoblotting with the anti-GFP antibody showed that the GFPAE2a fusion protein was not accessible to biotinylation in transfected cells (Fig 7C, Lanes 13). In contrast, a number of other cell proteins bound to streptavidin beads and could be detected on the same blots with peroxidase-conjugated streptavidin (Fig 7, Lane 4). These findings indicate that only negligible amounts of the AE2a fusion protein are transported to the cell surface in transfected COS-7 cells.
Detergent Extraction of the Golgi-associated Anion Exchanger
To evaluate whether the Golgi-localized AE2a might be associated with the recently identified Golgi membrane skeleton, we utilized the nonionic detergent extraction protocol (Triton X-100 at 4C) and the poor solubilization properties of many cytoskeleton-associated proteins (
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this study, we have identified the full-length AE2a isoform as the Golgi-associated anion exchanger in fibroblasts. The evidence supporting this conclusion is several-fold and includes immunological data (N- and C-terminal anti-AE2 antibodies), mRNA analyses, the use of AE2a-specific antisense oligonucleotides to inhibit endogenous AE2 expression in the Golgi and, finally, the demonstration that the transiently expressed GFP-tagged AE2a fusion protein also accumulated in the Golgi membranes in transfected cells. These data are compatible with our earlier immunological data obtained with the C-terminal anti-AE1 antiserum and with our preliminary sequencing data in rat osteosarcoma cells (
Overexpression of proteins may lead to formation of aggresomes around the microtubule organizing center (MTOC) if the protein degradation capacity of cytosolic proteasomes is exceeded (
Identification of the AE2a isoform as the Golgi-associated AE raises important questions concerning both its functioning and the apparently variable positioning of different AE polypeptides either in the basolateral or apical plasma membrane in some epithelial cells and, on the other hand, in the Golgi membranes, e.g., in fibroblasts. The vast number of transcripts known to encode AE polypeptides in rat chicken and human tissues (
Alternatively, localization may depend on the cell type in which AE polypeptides are expressed. Studies with kidney AE1 have indicated that the same polypeptide can be sorted either basolaterally or apically and that sorting in this case is guided by external cues (
The positioning of the AE2a isoform in the Golgi membranes in fibroblasts apparently involves specific sorting signals that may cause its oligomerization and oligomerization-dependent retention in the Golgi membranes (
![]() |
Acknowledgments |
---|
Supported by a grant from the University of Oulu.
We wish to thank Drs R. Baron and W. Horne, D. Anstee, and M. Czerwinsky for kindly providing antibodies used in this study.
Received for publication June 1, 2000; accepted September 25, 2000.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alper SL, Kopito RR, Libresco SM, Lodish HF (1988) Cloning and characterization of a murine band 3-related cDNA from kidney and from a lymphoid cell line. J Biol Chem 263:17092-17099
Alper S, Rossman H, Wilhelm S, StuartTilley AK, Shmukler BE, Seidler U (1999) Expression of AE2 anion exchanger in mouse intestine. Am J Physiol 277:G321-332
Alper SL, StuartTilley AK, Biemesderfer D, Shmukler BE, Brown D (1997) The fodrin-ankyrin cytoskeleton of choroid plexus preferentially colocalizes with apical Na+/K+-ATPase rather than with basolateral anion exchanger AE2. Am J Physiol 273:F601-614
Beck KA, Buchanan JA, Malhotra V, Nelson WJ (1994) Golgi spectrin: identification of an erythroid ß-spectrin homolog associated with the Golgi complex. J Cell Biol 127:707-723[Abstract]
Beck KA, Buchanan JA, Nelson WJ (1997) Golgi membrane skeleton: identification, localization and oligomerization of a 195 kDa ankyrin isoform associated with the Golgi complex. J Cell Sci 110:1239-1249
Bennett V (1992) Ankyrins. Adaptors between diverse plasma membrane proteins and the cytoplasm. J Biol Chem 267:8703-8706
Chow A, Dobbins JW, Aaronson PS, Igarashi P (1992) cDNA cloning and localization of a band 3-related protein from ileum. Am J Physiol 263:G345-352
Cox KH, AdairKirk TL, Cox JV (1995) Four variant chicken erythroid AE1 anion exchangers. J Biol Chem 270:19752-19760
Cox JV, Moon RT, Lazarides E (1985) Anion transporter: highly cell-type-specific expression of distinct polypeptides and transcripts in erythroid and nonerythroid cells. J Cell Biol 100:1548-1557[Abstract]
Demuth RR, Showe LC, Ballantine M, Palumbo A, Frazer PJ, Cioe L, Rovera G, Curtis PJ (1986) Cloning and structural characterization of a human non-erythroid band 3-like protein. EMBO J 5:1205-1214[Abstract]
Devarajan P, Stabach PR, Mann AS, Ardito T, Kashgarian M, Morrow JS (1996) Identification of a small cytoplasmic ankyrin (AnkG119) in the kidney and muscle that binds beta I sigma spectrin and associates with the Golgi apparatus. J Cell Biol 133:819-830[Abstract]
Green J, Kleeman CR (1992) Role of calcium and cAMP messenger systems in intracellular pH regulation of osteoblastic cells. Am J Physiol 262:C111-121
Holappa K, Mustonen M, Parvinen M, Vihko P, Rajaniemi H, Kellokumpu S (1999) Primary structure of a sperm cell anion exchanger and its messenger ribonucleic acid expression during spermatogenesis. Biol Reprod 61:981-986
Johnston JA, Ward CL, Kopito RR (1998) Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143:1883-1898
Jons T, Drenckhahn D (1998) Anion exchanger 2 (AE2) binds to erythrocyte ankyrin and is colocalized with ankyrin along the basolateral plasma membrane of human gastric parietal cells. Eur J Cell Biol 75:232-236[Medline]
Kellokumpu S, Neff L, Kopito R, Baron R (1988) A 115-kD polypeptide immunologically related to erythrocyte band 3 is present in Golgi membranes. Science 242:1308-1311[Medline]
Kellokumpu S, Sormunen R, Väänänen HK, Metsikkö K (1995) Defective maturation of a viral glycoprotein and partial loss of the Golgi stack structure during in vitro myogenesis. Exp Cell Res 220:101-111[Medline]
Kopito RR (1990) Molecular biology of the anion exchanger gene family. Int Rev Cytol 123:177-199[Medline]
Kopito RR, Lee BS, Simmons DM, Lindsey AE, Morgans CW, Schneider K (1989) Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger. Cell 59:927-937[Medline]
Kopito RR, Lodish H (1985) Primary structure and transmembrane orientation of the murine anion exchange protein. Nature 316:234-238[Medline]
Kudrycki KE, Newman PR, Schull GE (1990) cDNA cloning and tissue distribution of mRNAs for two proteins that are related to the band 3 Cl-/HCO-3 exchanger. J Biol Chem 265:462-471
Linn SC, Kudrycki KE, Schull GE (1992) The predicted translation product of a cardiac AE3 mRNA contains an N terminus distinct from that of the brain AE3 Cl-/HCO-3 exchanger. J Biol Chem 267:7927-7935
Machamer CE (1991) Golgi retention signals: do membranes hold the key? Trends Cell Biol 1:141-144
MartinezAnzo E, Castillo JE, Diez J, Medina JF, Prieto J (1993) Immunohistochemical detection of chloride/bicarbonate anion exchangers in human liver. Hepatology 19:1400-1406
Medina JF, Lecanda J, Acin A, Ciesielczyk P, Prieto J (2000) Tissue-specific N-terminal isoforms from overlapping alternate promoters of the human AE2 anion exchanger gene. Biochem Biophys Res Commun 267:228-235[Medline]
Ostedgaard LS, Jennings ML, Karniski LP, Schuster VL (1991) A 45-kDa protein antigenically related to band 3 is selectively expressed in kidney mitochondria. Proc Natl Acad Sci USA 88:981-985[Abstract]
Parkkila S, Rajaniemi H, Kellokumpu S (1993) Polarized expression of a Band3-related protein in mammalian sperm cells. Biol Reprod 49:326-331[Abstract]
Peters LL, Shivdasani RA, Liu S-C, Hanspal M, John KM, Gonzalez JM, Brugnara C, Gwun B, Mohandas N, Alper S, Orkin ST, Lux S (1996) Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton. Cell 86:917-927[Medline]
Stankewich MC, Tse WT, Peters LL, Ch'ng Y, John KM, Stabach PR, Devarajan P, Morrow JS, Lux SE (1998) A widely expressed ßIII spectrin associated with Golgi and cytoplasmic vesicles. Proc Natl Acad Sci USA 95:14158-14163
Van Adelsberg JS, Edwards JC, Al-Awqati Q (1993) The apical Cl/HCO3 exchanger of beta intercalated cells. J Biol Chem 268:11283-11289
Van Adelsberg J, Edwards JC, Takito J, Kiss B, Al-Awqati Q (1994) An induced extracellular matrix protein reverses the polarity of band 3 in intercalated epithelial cells. Cell 76:1053-1061[Medline]
Wang Z, Schultheis PJ, Schull GE (1996) Three N-terminal variants of the AE2 Cl-/HCO-3 exchanger are encoded by mRNAs transcribed from alternative promoters. J Biol Chem 271:7835-7843