Kidney AE1 Research Articles

Glyceraldehyde 3-phosphate dehydrogenase is required for band 3 (anion exchanger 1) membrane residency in the mammalian kidney

The mammalian kidney isoform of the essential chloride-bicarbonate exchanger AE1 differs from its erythrocyte counterpart, being shorter at its N terminus. It has previously been reported that the glycolytic enzyme GAPDH interacts only with erythrocyte AE1, by binding to the portion not found in the kidney isoform. (Chu H, Low PS. Biochem J 400:143–151, 2006). We have identified GAPDH as a candidate binding partner for the C terminus of both AE1 and AE2. We show that full-length AE1 and GAPDH coimmunoprecipitated from both human and rat kidney as well as from Madin-Darby canine kidney (MDCK) cells stably expressing kidney AE1, while in human liver, AE2 coprecipitated with GAPDH. ELISA and glutathione S-transferase (GST) pull-down assays using GST-tagged C-terminal AE1 fusion protein confirmed that the interaction is direct; fluorescence titration revealed saturable binding kinetics with Kd 2.3 ± 0.2 μM. Further GST precipitation assays demonstrated that the D902EY residues in the D902EYDE motif located within the C terminus of AE1 are important for GAPDH binding. In vitro GAPDH activity was unaffected by C-terminal AE1 binding, unlike in erythrocytes. Also, differently from red cell N-terminal binding, GAPDH-AE1 C-terminal binding was not disrupted by phosphorylation of AE1 in kidney AE1-expressing MDCK cells. Importantly, small interfering RNA knockdown of GAPDH in these cells resulted in significant intracellular retention of AE1, with a concomitant reduction in AE1 at the cell membrane. These results indicate differences between kidney and erythrocyte AE1/GAPDH behavior and show that in the kidney, GAPDH is required for kidney AE1 to achieve stable basolateral residency.

Impaired trafficking and intracellular retention of mutant kidney anion exchanger 1 proteins (G701D and A858D) associated with distal renal tubular acidosis

Novel compound heterozygous mutations, G701D, a recessive mutation, and A858D, a mild dominant mutation, of human solute carrier family 4, anion exchanger, member 1 (SLC4A1) were identified in two pediatric patients with distal renal tubular acidosis (dRTA). To examine the interaction, trafficking, and cellular localization of the wild-type and two mutant kidney AE1 (kAE1) proteins, we expressed the proteins alone or together in human embryonic kidney (HEK) 293T and Madin-Darby canine kidney (MDCK) epithelial cells. In individual expressions, wild-type kAE1 was localized at the cell surface of HEK 293T and the basolateral membrane of MDCK cells. In contrast, kAE1 G701D was mainly retained intracellularly, while kAE1 A858D was observed intracellularly and at the cell surface. In co-expression experiments, wild-type kAE1 formed heterodimers with kAE1 G701D and kAE1 A858D, and promoted the cell surface expression of the mutant proteins. The co-expressed kAE1 G701D and A858D could also form heterodimers but showed predominant intracellular retention in HEK 293T and MDCK cells. Thus impaired trafficking of the kAE1 G701D and A858D mutants would lead to a profound decrease in functional kAE1 at the basolateral membrane of alpha-intercalated cells in the distal nephron of the patients with dRTA.

Structural Characterization of the Cytosolic Domain of Kidney Chloride/Bicarbonate Anion Exchanger 1 (kAE1)

Kidney anion exchanger 1 (kAE1) is a membrane glycoprotein expressed in alpha-intercalated cells in the collecting ducts of the kidney where it mediates electroneutral chloride/bicarbonate exchange. Human kAE1 is a truncated form of erythroid AE1 missing the first 65 residues of the N-terminal cytosolic domain, which includes a disordered acidic region (residues 1-54) and the first beta-strand (residues 55-65) of the folded region. Unlike erythroid AE1, kAE1 does not bind deoxyhemoglobin, glycolytic enzymes, or cytoskeletal components. To understand the effect of the N-terminal deletion on the structure of the cytosolic domain, we performed an extensive biophysical analysis on His 6 tagged cytosolic domains of erythroid AE1 (cdAE1), kidney AE1 (cdkAE1), and a novel truncation mutant (cdDelta54AE1) missing the first 54 residues, but retaining the beta-strand. Circular dichroism did not detect any major differences in secondary structure, and sedimentation analyses showed that all three proteins were dimeric. Differential scanning calorimetry revealed that cdAE1 and cdDelta54AE1 had similar thermal stabilities with midpoints of transition higher than cdkAE1. cdAE1 and cdDelta54AE1 underwent similar pH-dependent fluorescence changes, while cdkAE1 exhibited a higher intrinsic fluorescence at neutral and acidic pH. Urea denaturation resulted in dequenching of tryptophan fluorescence in cdAE1, while tryptophans in cdkAE1 were already dequenched in the native state. We conclude that the absence of the central beta-strand in cdkAE1 results in a less stable and more open structure than cdAE1. This structural change, in addition to the loss of the acidic amino-terminal region, may account for the altered protein binding properties of kAE1.

Dominant-negative effect of Southeast Asian ovalocytosis anion exchanger 1 in compound heterozygous distal renal tubular acidosis

The human chloride/bicarbonate AE1 (anion exchanger) is a dimeric glycoprotein expressed in the red blood cell membrane,and expressed as an N-terminal (Delta1-65) truncated form, kAE1(kidney AE1), in the basolateral membrane of alpha-intercalated cells in the distal nephron. Mutations in AE1 can cause SAO (Southeast Asian ovalocytosis) or dRTA (distal renal tubular acidosis), an inherited kidney disease resulting in impaired acid secretion. The dominant SAO mutation (Delta400-408) that results in an inactive transporter and altered erythrocyte shape occurs in manydRTA families, but does not itself result in dRTA. Compound heterozygotes of four dRTA mutations (R602H, G701D, DeltaV850 and A858D) with SAO exhibit dRTA and abnormal red blood cell properties. Co-expression of kAE1 and kAE1 SAO with the dRTAmutantswas studied in polarized epithelial MDCK(Madin-Darbycanine kidney) cells. Like SAO, the G701D and DeltaV850 mutants were predominantly retained intracellularly, whereas the R602H and A858D mutants could traffic to the basolateral membrane. When co-expressed in transfected cells, kAE1 WT (wild-type)and kAE1 SAO could interact with the dRTA mutants. MDCK cells co-expressing kAE1 SAO with kAE1 WT, kAE1 R602Hor kAE1 A858D showed a decrease in cell-surface expression of the co-expressed proteins. When co-expressed, kAE1 WT colocalized with the kAE1 R602H, kAE1 G701D, kAE1 DeltaV850 and kAE1 A858D mutants at the basolateral membrane, whereaskAE1 SAO co-localized with kAE1 WT, kAE1 R602H, kAE1 G701D, kAE1 DeltaV850 and kAE1 A858D in MDCK cells. The decrease in cell-surface expression of the dRTAmutants as a result of the interaction with kAE1 SAO would account for the impaired expression of functional kAE1 at the basolateral membrane of alpha-intercalated cells, resulting in dRTA in compound heterozygous patients.

Expression and interaction of two compound heterozygous distal renal tubular acidosis mutants of kidney anion exchanger 1 in epithelial cells

Kidney AE1 (kAE1) is a glycoprotein responsible for the electroneutral exchange of chloride for bicarbonate, promoting the reabsorption of bicarbonate into the blood by alpha-intercalated cells of the collecting tubule. Mutations occurring in the gene encoding kAE1 can induce defects in urinary acidification resulting in distal renal tubular acidosis (dRTA). We expressed two kAE1 dRTA mutants, A858D, a mild dominant mutation, and DeltaV850, a recessive mutation, in epithelial Madin-Darby canine kidney (MDCK) cells. Individuals heterozygous with wild-type (WT) kAE1 either did not display any symptoms of dRTA (DeltaV850/WT) or displayed a mild incomplete form of dRTA (A858D/WT), while compound heterozygotes (DeltaV850/A858D) had dRTA. We found that the A858D mutant was slightly impaired in the endoplasmic reticulum (ER) exit but could target to the basolateral membrane of polarized MDCK cells. Despite an altered binding to an inhibitor affinity resin, anion transport assays showed that the A858D mutant was functional at the cell surface. The DeltaV850 mutant showed altered binding to the affinity resin but was predominantly retained in the ER, resulting in undetectable AE1 expression at the basolateral membrane. When coexpressed in MDCK cells, the WT protein, and to a lesser extent the A858D mutant, enhanced the cell surface expression of the DeltaV850 mutant. The DeltaV850 mutant also affected the cell surface expression of the A858D mutant. Compound heterozygous (A858D/DeltaV850) patients likely possess a decreased amount of functional anion exchangers at the basolateral membrane of their alpha-intercalated cells, resulting in impaired bicarbonate transport into the blood and defective acid transport into the urine.

Multiple cytoplasmic signals direct the intracellular trafficking of chicken kidney AE1 anion exchangers in MDCK cells.

AE1/F(c) receptor chimeras have been used to define the sequences that direct the basolateral sorting, recycling and cytoskeletal association of the chicken AE1-4 anion exchanger in MDCK cells. These analyses revealed that amino acids 1-63 of AE1-4 were sufficient to redirect a cytoplasmic tailless murine IgG F(c)RII B2 receptor from the apical to the basolateral membrane of MDCK cells, where F(c)1-63 associated with elements of the actin cytoskeleton. In contrast to F(c)1-63, chimeras containing amino acids 1-37 (F(c)1-37) or 38-63 (F(c)38-63) of AE1-4 accumulated in intracellular membrane compartments that overlapped late endosomes and the trans-Golgi network (TGN), respectively. Internalization assays indicated that the patterns of localization observed for F(c)1-37 and F(c)38-63 resulted from the recycling of these chimeras from the cell surface. These assays further indicated that F(c)1-37 and F(c)38-63 each possess a basolateral sorting activity. Mutagenesis studies revealed that the endocytic and basolateral sorting activities in F(c)1-37 are dependent upon serine 25, which is located in a sequence similar to a sorting signal in the polymeric immunoglobulin receptor. In addition, the sorting activities associated with F(c)38-63 were dependent upon tyrosine 47 and leucine 50. These residues resided within the sequence, YVEL, which matches the YXXPhi motif (where X is any amino acid and Phi is a hydrophobic residue) that functions as an endocytic and TGN recycling signal for other membrane proteins. Our data indicate that amino acids 1-63 of AE1-4 contain sorting and cytoskeletal binding activities that account for most of the properties previously associated with AE1-4 in MDCK cells. Furthermore, the alternative localization patterns exhibited by chimeras containing various combinations of these activities suggest that interplay between these cytoplasmic activities is critical for specifying AE1-4 localization in epithelial cells.

Impaired trafficking of human kidney anion exchanger (kAE1) caused by hetero-oligomer formation with a truncated mutant associated with distal renal tubular acidosis.

Autosomal dominant distal renal tubular acidosis (dRTA) has been associated with several mutations in the anion exchanger AE1 gene. The effect of an 11-amino-acid C-terminal dRTA truncation mutation (901 stop) on the expression of kidney AE1 (kAE1) and erythroid AE1 was examined in transiently transfected HEK-293 cells. Unlike the wild-type proteins, kAE1 901 stop and AE1 901 stop mutants exhibited impaired trafficking from the endoplasmic reticulum to the plasma membrane as determined by immunolocalization, cell-surface biotinylation, oligosaccharide processing and pulse-chase experiments. The 901 stop mutants were able to bind to an inhibitor affinity resin, suggesting that these mutant membrane proteins were not grossly misfolded. Co-expression of wild-type and mutant kAE1 or AE1 resulted in intracellular retention of the wild-type proteins in a pre-medial Golgi compartment. This dominant negative effect was due to hetero-oligomer formation of the mutant and wild-type proteins. Intracellular retention of kAE1 in the alpha-intercalated cells of the kidney would account for the impaired acid secretion into the urine characteristic of dRTA.

AE anion exchangers in atrial tumor cells.

Intracellular pH homeostasis and intracellular Cl(-) concentration in cardiac myocytes are regulated by anion exchange mechanisms. In physiological extracellular Cl(-) concentrations, Cl(-)/HCO(3)(-) exchange promotes intracellular acidification and Cl(-) loading sensitive to inhibition by stilbene disulfonates. We investigated the expression of AE anion exchangers in the AT-1 mouse atrial tumor cell line. Cultured AT-1 cells exhibited a substantial basal Na(+)-independent Cl(-)/HCO(3)(-) (but not Cl(-)/OH(-)) exchange activity that was inhibited by DIDS but not by dibenzamidostilbene disulfonic acid (DBDS). AT-1 cell Cl(-)/HCO(3)(-) activity was stimulated two- to threefold by extracellular ATP and ANG II. AE mRNAs detected by RT-PCR in AT-1 cells included brain AE3 (bAE3), cardiac AE3 (cAE3), AE2a, AE2b, AE2c1, AE2c2, and erythroid AE1 (eAE1), but not kidney AE1 (kAE1). Cultured AT-1 cells expressed AE2, cAE3, and bAE3 polypeptides, which were detected by immunoblot and immunocytochemistry. An AE1-like epitope was detected by immunocytochemistry but not by immunoblot. Both bAE3 and cAE3 were present in intact AT-1 tumors. Cultured AT-1 cells provide a useful system for the study of mediators and regulators of Cl(-)/HCO(3)(-) exchange activity in an atrial cell type.

Intracellular trafficking of variant chicken kidney AE1 anion exchangers: role Of alternative NH(2) termini in polarized sorting and Golgi recycling.

The variant chicken kidney AE1 anion exchangers differ only at the NH(2) terminus of their cytoplasmic domains. Transfection studies have indicated that the variant chicken AE1-4 anion exchanger accumulates in the basolateral membrane of polarized MDCK kidney epithelial cells, while the AE1-3 variant, which lacks the NH(2)-terminal 63 amino acids of AE1-4, primarily accumulates in the apical membrane. Mutagenesis studies have shown that the basolateral accumulation of AE1-4 is dependent upon two tyrosine residues at amino acids 44 and 47 of the polypeptide. Interestingly, either of these tyrosines is sufficient to direct efficient basolateral sorting of AE1-4. However, in the absence of both tyrosine residues, AE1-4 accumulates in the apical membrane of MDCK cells. Pulse-chase studies have shown that after delivery to the cell surface, newly synthesized AE1-4 is recycled to the Golgi where it acquires additional N-linked sugar modifications. This Golgi recycling activity is dependent upon the same cytoplasmic tyrosine residues that are required for the basolateral sorting of this variant transporter. Furthermore, mutants of AE1-4 that are defective in Golgi recycling are unable to associate with the detergent insoluble actin cytoskeleton and are rapidly turned over. These studies, which represent the first description of tyrosine-dependent cytoplasmic sorting signal for a type III membrane protein, have suggested a critical role for the actin cytoskeleton in regulating AE1 anion exchanger localization and stability in this epithelial cell type.

Autosomal dominant distal renal tubular acidosis and the AE1 gene.

Autosomal dominant distal renal tubular acidosis and the AE1 gene.

Autosomal Dominant Distal Renal Tubular Acidosis Is Associated in Three Families with Heterozygosity for the R589H Mutation in the AE1 (Band 3) Cl−/HCO3−Exchanger

Distal renal tubular acidosis (dRTA) is characterized by defective urinary acidification by the distal nephron. Cl-/HCO3- exchange mediated by the AE1 anion exchanger in the basolateral membrane of type A intercalated cells is thought to be an essential component of lumenal H+ secretion by collecting duct intercalated cells. We evaluated the AE1 gene as a possible candidate gene for familial dRTA. We found in three unrelated families with autosomal dominant dRTA that all clinically affected individuals were heterozygous for a single missense mutation encoding the mutant AE1 polypeptide R589H. Patient red cells showed approximately 20% reduction in sulfate influx of normal 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid sensitivity and pH dependence. Recombinant kidney AE1 R589H expressed in Xenopus oocytes showed 20-50% reduction in Cl-/Cl- and Cl-/HCO3- exchange, but did not display a dominant negative phenotype for anion transport when coexpressed with wild-type AE1. One apparently unaffected individual for whom acid-loading data were unavailable also was heterozygous for the mutation. Thus, in contrast to previously described heterozygous loss-of-function mutations in AE1 associated with red cell abnormalities and apparently normal renal acidification, the heterozygous hypomorphic AE1 mutation R589H is associated with dominant dRTA and normal red cells.

NH4Cl activates AE2 anion exchanger in Xenopus oocytes at acidic pHi.

In the course of experiments to define regulation by intracellular pH (pHi) of the AE2 anion exchanger expressed in Xenopus oocytes, we discovered an unexpected regulation of AE2 by NH4+. Intracellular acidification produced by extracellular acidification or produced by equimolar substitution of NaCl with sodium acetate each inhibited AE2 activity. In contrast, intracellular acidification by equimolar substitution of NaCl with NH4Cl activated AE2-associated, trans-anion-dependent, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive 36Cl- influx and efflux. Regulation by NH4+ was isoform specific, since neither erythroid nor kidney AE1 was activated. AE2 activation was maximal at <5 mM NH4Cl; was not mimicked by extracellular KCl, chloroquine, or polyamines; and was insensitive to amiloride, bumetanide, barium, and gadolinium. Whether NH4Cl acts directly on AE2 or on another target remains to be determined. Activation of AE2 by NH4+ may serve to sustain Cl-/HCO3- exchange activity in the presence of acidic pH in renal medulla, colon, abscesses, and other AE2-expressing acidic locales exposed to elevated NH4+ concentration.

Identification of the Proximal Erythroid Promoter Region of the Mouse Anion Exchanger Gene

The AE1 gene is expressed in erythrocytes and the A-type intercalated cells of the kidney distal collecting duct. Although the 5' end of the principal transcript expressed in murine erythroid cells has previously been mapped to a cluster of transcription start sites located immediately upstream of exon 1, the 5' end of the mouse kidney transcript has not been identified. Using the anchored polymerase chain reaction technique to analyze mouse kidney AE1 mRNA, we identified an internal transcription start site located within erythroid intron 3. This site defines an exon of 37 nucleotides that forms the 5' end of the mouse kidney AE1 transcript. AE1 transcripts beginning at this internal start site could not be detected in RNA isolated from purified erythroid progenitor cells or from erythroid cells undergoing erythropoietin-dependent terminal maturation, although transcripts derived from the upstream site were abundant, indicating that only the upstream promoter is active during erythropoiesis. Transient expression of reporter constructs in erythroid and nonerythroid cell lines identified a proximal upstream region of approximately 135 nucleotides that was active as a basal promoter. However, an additional approximately 200 nucleotides of upstream sequence was required for induced levels of activity in erythroid cells. Although our functional approach does not yet indicate the precise sequences required for erythroid induction, the AE1 gene upstream region contains potential GATA sites at -154, -141, and -60; an E-box at -163; CACCC or GGTGG motifs at -188, -121, and -88; and an AP-1/NF-E2-like site at -42.

Variant chicken kidney AE1 anion exchanger transcripts are derived from a single promoter by alternative splicing

Previous studies have demonstrated that three variant transcripts, AE1–3, AE1–4 and AE1–5, are derived from the AE1 gene in chicken kidney. These variant transcripts encode AE1 anion exchangers that possess alternative N-terminal cytoplasmic domains. To determine the mechanisms involved in generating these transcripts, a genomic clone, containing the unique sequences at the 5′ ends of the AE1–4 and AE1–5 transcripts, was isolated. Characterization of this clone revealed that the sequences at the 5′ ends of the AE1–3, AE1–4 and AE1–5 transcripts were each present within an approx. 1.2-kb BamHI fragment of the chicken AE1 gene. RNA blotting and RNase protection analyses using probes derived from this genomic clone have shown that the AE1–4 variant corresponds to the approx. 4.5-kb chicken kidney AE1 transcript, while the AE1–5 variant corresponds to the approx. 5.1-kb transcript. These studies have shown that the AE1–5 transcript extends further 5′ than had been previously shown from cDNA cloning studies, and contains the sequence present at the 5′ end of the AE1–4 transcript. In addition, primer extension analyses have shown that the variant kidney AE1 transcripts initiate transcription from a common site. This result indicates that the expression of the AE1–3, AE1–4 and AE1–5 transcripts is regulated by a single promoter, P3, that is distinct from the PI and P2 erythroid-specific promoters of the chicken AE1 gene.

Variant chicken AE1 anion exchangers possess divergent NH(2)-terminal cytoplasmic domains

Immunoblotting analyses have demonstrated that antibodies specific for the chicken erythroid AE1 anion exchanger recognize multiple polypeptides ranging in size from approximately 95 to 112 kDa in chicken kidney. To determine the origin of this diversity, we have cloned and characterized the kidney AE1 anion exchangers. These studies have shown that the kidney AE1 polypeptides are encoded by at least three transcripts, AE1-3, AE1-4, and AE1-5, which differ from the erythroid AE1-1 and AE1-2 transcripts in the sequences present at their 5'-ends. The AE1-3 and AE1-5 transcripts encode predicted polypeptides of approximately 94 kDa, which are identical to the erythroid AE1-1 anion exchanger except for the absence of the 78 NH(2)-terminal amino acids of the AE1-1 polypeptide. In contrast, the AE1-4 transcript encodes a predicted polypeptide of approximately 101 kDa, whose 21 NH(2)-terminal amino acids are unique. Characterization of the AE1 cDNAs has suggested that the AE1-3 and AE1-4 transcripts are generated by alternative splicing of a single primary transcript, while DNA blotting analyses have shown that the putative transcription initiation sites of the variant AE1-4 and AE1-5 transcripts lie several kilobases downstream of the transcription initiation sites of the erythroid AE1-1 and AE1-2 transcripts. These results suggest that the pattern of accumulation of the variant kidney AE1 anion exchangers is regulated by a complex pattern of alternative transcriptional initiation and differential RNA splicing.

The Structure of the Human Red Blood Cell Anion Exchanger (EPB3, AE1, Band 3) Gene

The structure and sequence of the human red blood cell anion exchanger (EPB3, AE1, band 3) gene was determined by analysis of genomic and cDNA clones. The gene extends over 18 kb and consists of 20 exons. The cDNA sequence comprises 4,906 nucleotides [excluding the poly(A) tail]. There is extensive similarity between the human and mouse AE1 gene, although the latter covers 17 kb. The additional sequence present within the human AE1 gene compared with the mouse gene is mainly caused by the presence of 6 Alu repetitive units in the human gene between intron 13 and exon 20, one of which is within the 3' noncoding region of the cDNA. Two potential promoter regions within the human AE1 gene are located so that they could give rise to the different transcripts found in erythroid cells and in the kidney. The kidney transcript would lack exons 1 through 3 of the erythroid transcript. The translation initiator downstream to the human kidney promoter would give rise to a protein with a 20 amino acid section at the N-terminus that is not present in the erythroid human AE1 protein or the rodent kidney AE1 proteins.

The structure of the human red blood cell anion exchanger (EPB3, AE1, band 3) gene

The structure and sequence of the human red blood cell anion exchanger (EPB3, AE1, band 3) gene was determined by analysis of genomic and cDNA clones. The gene extends over 18 kb and consists of 20 exons. The cDNA sequence comprises 4,906 nucleotides [excluding the poly(A) tail]. There is extensive similarity between the human and mouse AE1 gene, although the latter covers 17 kb. The additional sequence present within the human AE1 gene compared with the mouse gene is mainly caused by the presence of 6 Alu repetitive units in the human gene between intron 13 and exon 20, one of which is within the 3′ noncoding region of the cDNA. Two potential promoter regions within the human AE1 gene are located so that they could give rise to the different transcripts found in erythroid cells and in the kidney. The kidney transcript would lack exons 1 through 3 of the erythroid transcript. The translation initiator downstream to the human kidney promoter would give rise to a protein with a 20 amino acid section at the N-terminus that is not present in the erythroid human AE1 protein or the rodent kidney AE1 proteins.

Anion exchanger 1 in human kidney and oncocytoma differs from erythroid AE1 in its NH2 terminus.

Acid-secreting intercalated cells of the kidney collecting duct and tumor cells of renal oncocytoma express an anion exchanger that is immunologically related but not identical to the chloride-bicarbonate anion exchanger of erythrocytes (AE1). In this study, we have mapped the binding site of a monoclonal antibody against erythroid AE1 that does not react with either intercalated cells or oncocytoma. The epitope is located close to the NH2 terminus of AE1, indicating that AE1 in intercalated cells and oncocytoma differs in its NH2 terminus from erythroid AE1. This conclusion was supported by an antibody directed against residues 1-14 of erythroid AE1 that does not react with intercalated cells in oncocytoma. Polymerase chain reaction performed with mRNA from a human kidney revealed that the sequence containing the codons for Met-1 and Met-33 in erythroid mRNA is missing in the kidney transcript, whereas the sequence coding for Met-66 is present. DNA sequence data derived from cloning the 5' end of the human kidney AE1 mRNA clearly showed that the 5' untranslated region comprises part of intron 3, the complete exon 4 that is followed by exon 5 containing Met-66 as the site of translation initiation. Altogether, the results indicate that AE1 in the human kidney is an amino-terminally truncated form of erythroid AE1 that is restricted to the basolateral membrane domain of the acid-secreting intercalated cells of the collecting duct and is also expressed in oncocytoma.