Molecular Biology of the Anion Exchanger Gene Family

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 123

Molecular Biology of the Anion Exchanger Gene Family RONR. KOPITO Department of Biological Sciences, Stanford Universiv, Stanford, California 94305

I. Introduction The erythrocyte anion exchanger, band 3, is one of the most thoroughly studied plasma membrane proteins. The major integral membrane glycoprotein of erythrocytes, band 3 is present at ~ 1 . x2 106 and 8 x 105 copies per cell in mammalian (Steck, 1978) and avian (Jay, 1983) erythrocytes, respectively. This protein is, in essence, a molecular chimera in which two distinct functional domains are segregated into discrete NH2 and COOH-terminal domains. The 50-kDa NH2-terminal domain is disposed to the cytoplasm, where it interacts with several cytosolic and cytoskeletal proteins (Low, 1986). Most prominent among these interactions is the association with ankyrin (Bennett and Stenbuck, 1979), which forms the link between the meshlike spectrin-actin cytoskeleton and the plasma membrane (Bennett, 1985). The capacity to associate with ankyrin appears to be highly conserved, despite the relatively low overall sequence homology among the corresponding NH2-terminal domains of band 3 from various species (see later). The amino-terminal domain of human erythrocyte band 3 also contains binding sites for several glycolytic enzymes and hemoglobin, and is a substrate for endogenous and exogenous tyrosine kinases (Mohamed and Steck, 1986; Low, 1986). The cytoplasmic domain of band 3 may play a role in red blood cell senescence (Kay, 1984; Low et al., 1985). The binding of the NH2-terminal domain of human band 3 to denatured hemoglobin leads to clustering of the protein (Low et al., 1985). Such band 3 clusters are thought to be recognized by circulating autoantibodies, serving as a signal for elimination of senescent erythrocytes from the bloodstream. The 65-kDa COOH-terminal fragment of band 3 remains tightly associated with the plasma membrane following limited proteolysis of erythrocyte ghosts. This fragment defines a domain that is essential, if not sufficient, for catalyzing the 1 : 1, electroneutral, and reversible exchange of chloride and bicarbonate across the erythrocyte plasma membrane. Such activity serves to regulate intracellular pH and to facilitate the transport of C02 in the circulation. The kinetics and pharmacology of band 3-mediated anion exchange are highly conserved among erythrocytes from different species and are the subject of several comprehensive reviews (Passow, 1986; Jay and Cantley, 1986; Knauf, 1986; Brahm, 1988). 177

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RON R. KOPITO

The two functionally distinct domains of band 3 are reflected in the protein’s primary and predicted secondary structures, deduced from the nucleotide sequences of full-length cDNA clones for mouse (Kopito and Lodish, 1985a), chicken (Cox and Lazarides, 1988; Kim et al., 1988) and human (Tanner et af., 1988; Lux et al.. 1989) erythrocyte band 3. The sequence of the NH2-terminal =400 amino acids predicts an extremely polar domain with low overall a-helical content and no hydrophobic stretches of sufficient length to span the lipid bilayer. The COOH-terminal, membrane-associated, 400 amino acid domain has 10 stretches of highly hydrophobic residues that are predicted to form as many as 12 membrane-spanning hydrophobic or amphipathic a-helices (Kopito and Lodish, 1985a). Detailed discussion of band 3 topology, structure, and transport kinetics have been reviewed (Passow, 1986; Jay and Cantley, 1986; Knauf, 1986; Jennings, 1984) and will not be discussed further here. The recent cloning of several genes that are highly homologous to erythrocyte band 3 defines a new gene family. This article will review data on the structure and function of anion exchangers in the context of this newly identified family. The first section will focus on the structure and organization of the anion exchanger genes, and the conservation of function among the polypeptides they encode. The second section will discuss the expression of the band 3 gene in erythroid cells, and the last will review some of the biochemical, pharmacological, and immunological evidence for expression of the anion exchanger gene family in nonerythroid tissues. 11. The Anion Exchanger (AE) Gene Family

A. IDENTIFICATIONOF THE AE GENEFAMILY the cloning of band 3 cDNA (Kopito and Lodish, 1985a) from mouse erythropoietic spleen cells paved the way for the identification of the anion exchanger (AE) gene family. Throughout this review the following conventions will be used. The genetic locus encoding each anion exchanger homolog will be designated “AEn.” The transcripts of AEn will be designated “AEn mRNA’ and the protein product, “AEn polypeptide.” The one exception is “band 3,” which I use to refer explicitly to the protein product of the AEl gene expressed in erythroid tissues. That the AEI cDNA encodes the erythrocyte anion exchanger, band 3, was confirmed by comparison of its deduced amino acid sequence with the sequences of several protein fragments purified from human erythrocyte ghosts (Kopito and Lodish, 1985a). Subsequently, sequences for AEI from human (Tanner et al., 1988; Lux et al., 1989) and chicken (Cox and Lazarides, 1988; Kim et al., 1988) erythroid precursors have been published, exhibiting a high

ANION EXCHANGER GENE FAMILY

179

degree of overall sequence homology. Three other genes have been subsequently cloned by hybridization at reduced stringency to AEl cDNA. A partial clone for AE2 was originally isolated from human erythroleukemia cell line K562 (Demuth et al., 1986). The sequence of the full-length AE2 cDNA, which is considerably longer than AE1, was determined from cDNAs isolated from renal tissue and lymphoid cells (Alper et al., 1988), choroid plexus (Lindsey et al., 1990), and gastric mucosa (Kudrycki et al., 1990). A novel member of this family, AE3, has been cloned from brain (Kopito et al., 1989; Kudrycki et al., 1990). Comparison of the sequences of these three cDNAs clearly indicates that they are all encoded by distinct genes, thus constituting a bona-fide gene family. A fourth member has been cloned as a 7-kb genomic EcoRI restriction fragment, whose sequence indicates that it is also a unique member, AE4, of this family (R. R. Kopito, unpublished results). However, no tissue has yet been shown to express AE4, nor has any AE4 cDNA clone been isolated.

B. STRUCTUREAND CHROMOSOMAL LOCATION OF THE AE GENES AE1 is encoded by a single-copy gene in the mouse (Kopito et al., 1987b) and human (Showe et al., 1987; Stewart et al., 1989) and chicken (Cox et al., 1985; Kim et al., 1989) genomes. The chromosomal location for human AE1 has been mapped by in situ hybridization to chromosome 17q21 -) qter (Showe et al., 1987), and has subsequently been shown to be tightly linked to the gene for nerve growth factor (Stewart et al., 1989). The single human AE2 gene has been mapped by to region 7q35 + 7q36 by in situ hybridization (Palumbo et al., 1986). The chromosomal location for AE3 has not been determined. The mouse AE1 gene (Kopito et al., 1987b) encompasses a minimum of 17 kb of DNA, atthough there may be unmapped upstream exons. The AEl gene is interrupted by 19 introns, which range in length from 79 to 3900 bp, and whose positions correlate with certain gross structural features of the protein’s predicted secondary structure. In particular, the 9 introns that interrupt the membrane-associated anion exchange domain all occur at positions encoding the putative hydrophilic ‘‘loops,’’ which connect each of the 10 hydrophobic regions of the AE1 polypeptide that are proposed to span the lipid bilayer as a-helices. The location of three regions of pronounced @-turncharacter within the polar aminoterminal domain of the protein also correlate well with individual exons, although the significance of these observations, if any, is unclear. It is interesting to note that exon #11, which contains the first putative membrane-spanning a-helix (and the internal signal sequence) also contains a ~ 1 1 5 - bhighly ~ conserved sequence that corresponds to the junction between the membraneassociated and cytoplasmic domains. The presence of multiple introns allows for a large number of potential combinatorial patterns for alternative splicing, although such transcripts have not been reported. The mouse AEl gene contains

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several repetitive DNA elements. A mouse B1-type alu sequence is found within the 5’-flanking region (Kopito et al., 1987a), and a B2-type element within intron #13 (Kopito et al., 1987b). This intron also has several unusual DNA sequence elements, including tandem duplications and a 29-fold repeat of the dinucleotide GT. Interestingly, the recently cloned chicken AE 1 gene (Kim et al., 1989)-which is of comparable size to the mouse gene (=28 kb)-also has several prominent repeat sequences including several iterations of the repeated GT motif. The significance of these sequences, if any, is obscure. Several introdexon boundaries are conserved between the mouse AE1 and AE3 genes. The only such junctions not conserved are found within the cytoplasmic domain (C. W. Morgans and R. R. Kopito, unpublished observations). C. SEQUENCE HOMOLOGY AMONG THE AE POLYPEPTIDES Comparison of the sequence identity among the anion exchanger gene family members, quantified in Table I, indicates that both structural domains of band 3 are conserved both between species and between individual isoforms. Homology is highest within the COOH-terminal domains, ranging between 80 and 98% and lowest among the NH2-terminal domains, ranging between 59 and 84%. Chicken AE1 is only slightly more akin to mammalian AEl than it is to mammalian AE2 or AE3. However, similarity is greatest among AE genes of the same class across species (e.g., compare mouse and human AE2). These results are consistent with the interpretation that AE 1-3 evolved from a common ancestor prior to the divergence of mammals and rodents. The amino acid compositions of the murine AEI-3 polypeptides, deduced from the cDNA sequences, are shown in Table 11. The overall composition of both major structural domains of the three AE homologs are very similar to each other. These compositions are also in close agreement with the experimental data reported for human band 3 by Steck (1978). Consistent with the high degree of sequence identity, the membrane domains of all three homologs also share the greatest uniformity in amino acid composition. This domain bears an overwhelming excess of hydrophobic amino acids and a large net positive charge. Considerably greater variation in composition is found among the cytoplasmic domains. These domains are enriched in proline and glutamate, consistent with data for band 3 indicating a lack of significant a-helical content and a large net negative charge. The cytoplasmic domains of AE2 and AE3 also have a large proportion of proline and almost identical proportions of acidic residues, but differ from AEl most dramatically, in their quota of basic residues. This difference results in a large discrepancy in net charge on the cytoplasmic domain, ranging from -19 (AE1) to +5 (AE3). Such a difference may have significant implications for the function of the cytoplasmic domains of the three homologous proteins.

181

ANION EXCHANGER GENE FAMILY TABLE I

SEQUENCE bENl'lTY WITHIN THE ANION EXCHANGER GENE FAMILY~

AEI HWIan

AEI

Human Mouse Chicken AE2 Humanb Mouse Rat AE3 Mouse

Mouse

AE2

Chicken

Human

Mouse

NH2

COOH

NH2

COOH

NH2

COOH

NH2

COOH

NH2

-

95 86

-

85

-

-

-

-

84 68

61

*

-

-

-

-

-

COOH NH2 COOH -

-

-

64 64

85 86 85

64 64

82 83 82

* *

97 97

99

-

67 67

86 87 85

62

82

59

81

63

80

*

82

70

*

*

Rat

-

-

-

98

-

82

69

83

-

-

Comparison of percentage sequence identity between the amino- and carboxy-terminal domains of the cloned members of the anion exchanger gene family. Sequences were aligned, pairwise, using the GAP program (Devereux er al., 1984) and a unitary comparison matrix. bThe published sequence for human AE2 was corrected for frameshift errors as described in the legend to Fig. 1. This sequence is also lacking the 5' end; the partial sequence for the amino-terminal domain was therefore not used in the comparison (asterisks).

1. The CytoplasmicDomain In addition to their greater length, the cytoplasmic domains of AE2 and AE3 are more similar in composition to each other than to AE1 (Table 11, Fig. 1). The NH2-terminal275 residues of both AE2 and AE3 are extremely polar, with a net charge of -1 7 and -16, respectively. Other conserved features of this region include a histidine-rich motif at position 79 and a similarity in the distribution of the abundant proline and charged residues. A major difference between AE2 and AE3 is presence, in the latter protein, of continuous runs of basic (positions 107, 321) and acidic (position 135) residues. There are also several regions of the cytoplasmic domain that are conserved among all of the AE genes: positions 336-343,433442,536554,622431, and 690-725. Note that the residues that are conserved in all the AEl genes (capital letters) are almost always conserved in AE2 and AE3. It is tempting to speculate that, like AEI, AE2 and AE3 also participate in ankyrin binding, but the absence of a consensus sequence for an ankyrin-binding site on AE1, and the relatively low overall homology in this domain, preclude such an interpretation. Subsequent studies suggest that accessibility of ankyrin to its binding site on band 3 is highly dependent on the folding of the cytoplasmic domain, suggesting that this interaction may be formed from several noncontiguous segments of the protein (Davis et al., 1989; Thevenin et

Aelcon Ae2con Mode3

p. . y . . GAGLEP

183

ANION EXCHANGER GENE FAMILY TABLE I1

PREDICTED AMINO ACIDCOMFQSITIONOF THE ANION

Whole sequence

EXCHANGER GENEFAMILY MEMBERS“

Membrane domainb

Cytoplasmic domainb

Residue

AEl

AE2

AE3

AEl

AE2

AE3

AEl

AE2

AE3

Ala (A) CYS (C) Asp (D) Glu (E) Phe (F) Gly (G) His (H) Ile (I) LYS(K) Leu (L) Met (M) Asn (N) h o (P) Gin (Q) h g (R) Ser (S) Thr (TI Val (V) Trp (W) ‘M (Y)

6.1 0.7 4.2 6.6 5.2 7.3

8.3 0.8 4.0 8.6 5.0 7.1 2.8 4.2 3.6 11.9 2.5 1.6 7.5 3.8 6.8 6.9 5.3

6.3 0.6 2.6 3.9

6.8 1.4 2.7 5.2

7.3 1.o 7.7 4.3 15.9 3.1 1.6 6.1 3.3 3.5 6.9 4.9 9.6 1.4 2.6

7.1 1.5 6.6 4.1 14.5 4.1 2.1

6.0 0.7 6.2 9.8 2.4 7.4 2.1 3.3 2.6 15.7 1.7 2.6 7.9 3.8 6.7 6.0 5.7 5.7 1.o 2.9

6.9 0.6 6.4 10.0 2.4 7.4 3.8 2.4

2.9 3.9 7.0 5.6 8.9 1.4 1.7

7.7 1.o 2.7 4.6 7.1 7.5 1.5 6.3 2.9 16.9 2.1 1.3 6.7 3.3 4.4 5.6 6.3 8.5 1.3 2.1

9.4 0.4 4.9 11.1 3.2 7.1 3.8 2.5 3.3 10.0 1.4 1.3 9.3 4.4 8.9 6.8 5.0

9.8 1.6 1.1 10.7 3.0 8.2 9.2 4.0

0.8 1.4

7.3 0.7 4.8 7.7 4.4 7.4 2.9 4.1 4.2 12.8 1.8 1.2 9.0 3.1 6.6 7.7 5.0 6.8 1.1 1.5

0.4 1.1

0.9 1.o

12.6 13.3 28.1

12.5 13.6 28.1

6.5 8.8 40.7

7.9 9.5 37.4

7.3 8.8 38.8

16.0 11.4 27.1

16.0 16.0 21.4

1.5

5.7 3.6 15.8 2.5 2.0 6.9 3.6 5.0 6.5 5.3 7.9 1.2 2.7

10.8 D+E H + K + R 10.0 I + L + F + V 34.5

7.0

1.5

1.5

5 .O

5.1

5.1

5.8

16.4 17.1 20.3

-19 +8 0 +5 +13 +8 +12 +8 -7 Net chargec Rankorde~dLVGPE LEAPG LPESG LVIFG LVFGS LVAGF LEPGR ELAPR PELSR #Numbers refer to the mole percentage of each amino acid. *Sequences are divided into membrane and cytoplasmic domain as shown in Fig. 1. cThe net charge was calculated from the sum of the actual numbers of acidic (E, D) and basic (R, K, H) residues in each domain multiplied by their assumed charge (+1 for basic, -1 for acidic). &‘Rankorder” ranks the five most abundant residues (based on mole percentage) in each domain.

FIG.1. Alignment of the sequences of the members of the anion exchanger gene family. Shown are the consensus (plurality) sequences of the members of AEl (Aelcon) and AE2 (Ae2con) compared with the sequence of mouse AE3 (Kopito et al., 1989). Consensus sequences were generated using the programs GAP and LINEUP (Devereux ef al., 1984) to align the published sequences for mouse (Kopito and Lodish, 1985a),chicken (Kim el al., 1988), and human (Luxef al., 1989) AEl, and those for human (Demuth ef al., 1986). mouse (Alper er al., 1988), and rat (Lindsey er al., 1990) AE2. The human AE2 sequence (Demuth er al., 1986) was corrected for frameshift errors as noted by Alper et al. (1988).

Aelcon Ae2con m0de3 Aelcon Ae2con m0ae3 Aelcon Ae2con m0de3

Aelcon Ae2con m0de3 Aelcon Ae2con m0de3 Aelcon Ae2con Mode3

ANION EXCHANGER GENE FAMILY

185

al., 1989; Willardson et al., 1989). Alternatively, it is possible that the ankyrinbinding site is a linear sequence of contiguous residues, but may be masked in a conformationally dependent fashion. Among the most potent antibodies at blocking ankyrin-band 3 interaction are those directed against a “central” region (position 433489) (Davis er al., 1989; Willardson et al., 1989). This “central” region also contains a trypsin site that is protected from digestion by ankyrin (Davis et al., 1989) and a cystine residue (Cys201 in human band 3) that must be reduced for ankyrin binding to occur (Thevenin et al., 1989). Interestingly, this particular region is poorly conserved, even among band 3 proteins from different species, although its flanking sequences are highly conserved. Considerably more data are needed to elucidate the ankyrin-binding site on AE1, and to determine the function of the corresponding domains of AE2 and AE3. 2. The Membrane Domain The consensus sequences of the various AE family members are aligned in Fig. 2, illustrating the extensive identity among the proteins within the COOHterminal domain. Such a high degree of sequence identity predicts that the proteins have similar biological activities. Recent studies in which AE2 (Lindsey et al., 1990) and AE3 (Kopito er al., 1989) have been expressed in mammalian cells confirm these predictions, demonstrating that these gene products, like band 3, catalyze electroneutral, sodium-independent Cl/HC03 exchange. This conclusion is also supported by the recent finding of increased 36C1-influx into Xenopus oocytes injected with AE2 mRNA (Alper et al., 1989b). A hallmark of band 3-mediated anion exchange is its sensitivity to competitive and noncompetitive inhibition by 4,4’-diisothiocyanostilbenedisulfonate (DIDS) (Cabantchik er al., 1978). This inhibition covalently modifies a single lysine residue (Jennings and Nicknish, 1984) on band 3, which has been proposed to be either Lys860 or Lys863 (Fig. 2) corresponding to positions 539 and 542 of the mouse AE1 sequence (Kopito and Lodish, 1985a). The observation that Cl/HC03 exchange by AE2 (Lindsey et al., 1990) and AE3 (Kopito et al., 1989) can also be irreversibly blocked by DIDS lends support to the assignment (Kopito and Lodish, 1985b) of the DIDS-binding site to either of the two aforementioned, highly conserved lysine residues. Replacement of both of these Lys residues with Arg does not alter the anion exchange capacity of the protein, nor its ability to be competitively inhibited by DIDS, but does eliminate covalent binding

FIG. 1. (continued) Capital letters indicate complete sequence identity among all members in a given alignment. Lowercase letters indicate a plurality (i.e., two out of three match). A unitary matrix was used in all comparisons. Therefore, this figure indicates sequence identify, not similarity. Dashes denote the presence of a gap to achieve optimum alignment, and dots indicate a lack of plurality at that position. The shaded portion identifies the COOH-terminal membrane-associated domain.

186

RON R.KOPITO

of the inhibitor (Bartel et al., 1989). The lack of effect of mutations of Lys860 on covalent DJDS inhibition of anion exchange (Garcia and Lodish, 1989) suggests, by elimination, that the site of covalent DIDS binding is Lys863. However, this residue is not conserved in the chicken band 3 sequence (Cox and Lazarides, 1988; Kim er al., 1988), even though this protein is covalently modified by [3H2]-DIDS (Jay, 1983). The most likely explanation for these results is that either lysine is capable of binding to the inhibitor, consistent with a model for band 3 structure in which both lysines are present on the same face of an amphipathic a-helix (Kopito and Lodish, 198Sb). Such a conclusion could be tested by expression of band 3 mRNAs in which Lys863 has been mutated individually. Expression in COS cells of mutant versions of AEI (A. Lindsey and R. R. Kopito, unpublished), AE2 (Lindsey et al., 1990), and AE3 (Kopito et al., 1989), which lack almost the entire cytoplasmic domain, also leads to enhanced CI/HCO3 exchange, confirming that the COOH-terminal 400 amino acids of these proteins are sufficient for correct insertion into the plasma membrane and for Cl/HCO, exchange. 111. Anion Exchanger Gene Expression in Erythroid Cells

A. PROPERTIES OF THE BAND3 POLYPEPnDE On SDS-PAGE, human and mouse band 3 migrates as a diffuse band of M, 90,000-105,OOo (Steck, 1978). This apparent heterogeneity is generally considered to reflect variability in the structure of the single N-linked oligosaccharide chain. Anion exchanger synthesized by induced murine erythroleukemia (MEL) cells (Patel and Lodish, 1987) or mouse spleen cells (Braell and Lodish, 1981) migrates with essentially the same broad electrophoretic mobility observed in erythrocytes, arguing that AEI heterogeneity is the consequence of variable biosynthesis of the core oligosaccharide and not a consequence of modification of the protein during its lifetime in the circulation, as has been previously suggested (Sabban el al., 1980). vigorous treatment of erythrocyte band 3 with endoglycosidase F results in considerable sharpening of the band (M. M. Jennings, personal communication). Despite these results, it has never been rigorously shown that mammalian erythrocyte band 3 is composed of a single polypeptide chain. Indeed, the possibility of alternate forms of the polypeptide is supported by the recent observation that extensive digestion of the 35-kDa COOH-terminal AEl chymotryptic fragment with endoglycosidase F yields two distinct bands that differ in mobility by 1-2 kDa (M. M. Jennings, personal communication). Chicken band 3, in contrast to its mammalian counterpart, is composed of two polypeptides with apparent M, lOS,OOO and 100,OOo (Jay, 1983).Both polypeptides

ANION EXCHANGER GENE FAMILY

187

can be labeled covalently with the anion transport inhibitor [3H2]DIDS, exhibit nearly identical peptide maps and are immunologically indistinguishable (Jay, 1983). The conclusion that both polypeptides are encoded by the same gene is strengthened by genomic Southern blot analysis using chicken AEl cDNA clones (Cox et al., 1985; Kim et al., 1988). Several explanations are possible to account for the origin of the band 3 doublet on SDS gels of mature chicken erythrocyte ghosts. The two polypeptides may arise from a single common primary translation product, which could then be modified posttranslationally. Such modifications could include differential glycosylation or proteolytic processing. Alternatively, the two proteins may be derived from two distinct mRNAs. Cell-free translation of mRNA from anemic adult hen reticulocytes in the absence of microsomal membranes results in the synthesis, in approximately equal proportion, of two AE1 polypeptides of M, ~95,000,consistent with the existence of two distinct AEI mRNAs (Kim el al., 1988). Endoglycosidase H treatment of AE1 polypeptides from pulse-labeled chicken embryos also suggests that the mature proteins are derived from two precursors of MI ~95,000and ~97,000(Cox ef al., 1987). These results indicate that two distinct mRNAs are encoded by a single chicken AE1 gene, which are translated and posttranslationallyprocessed to produce the doublet that is observed upon SDS-PAGE of mature erythrocyte membranes. Complementary DNAs representing chicken AE1 mRNA have been independently isolated by immunological screening of phage expression libraries (Cox ef al., 1985; Cox and Lazarides, 1988; Kim ef al., 1988). Kim ef al. (1988) identified two cDNAs, pBIIIC1 and pBIIIC2, which differ at their 5’ ends. Conceptual translation of these two cDNAs predicts two polypeptides, C1 and C2, of M, 92,000 and 99,000. The predicted polypeptides differ only in the translational start site, with one protein being shorter by 33 amino acids at the NH2 terminus. Both the molecular weights and isoelectric points predicted for C1 and C2 are in close agreement with actual electrophoretic measurements of the chicken AE1 polypeptides (Jay, 1983). Cell-free translation of mRNA transcribed from clone pBIIIC1 produces a major polypeptide that is immunoreactive with a band 3 monoclonal antibody and migrates on SDS-PAGE with mobility indistinguishable from the unglycosylated protein synthesized in v i m from hen reticulocyte mRNA (Kim et al., 1988). It is, therefore, probable that the two clones pBIIIC1 and pBIIIC2 represent the major mRNA species that encode the two forms of chicken AE1. Formal proof, such as direct NH2-terminal sequence of the polypeptides, or binding of antibodies to specific NH2-terminal peptides is, however, still lacking. B. TRANSCRIPTIONAL INITIATION OF AE 1 The transcriptional start sites of chicken (Kim et al., 1989) and murine (Kopito et al., 1987a) AE1 have been mapped by primer extension and nuclease

188

RON R . KOPITO

protection analysis. Transcription of the chicken AE 1 gene in erythroid cells is initiated from two different promoters residing on separate 5’ exons (Fig. 2). The C2 mRNA (which encodes the longer of the two polypeptides) is generated by splicing the exon transcribed from the 5’-most promoter (P2) to the first common exon, as shown in Fig. 2A. The C1 transcript is generated from the downstream promoter (Pl), and contains an additional exon not present in the C2 transcript (Fig. 2B). This latter exon contains the AUG at which translation of the larger AEI polypeptide is initiated. The C2 transcript, lacking this exon and, hence, this AUG, is presumed to initiate at the first Met codon, within the first common exon. Both chicken AE 1 promoter sequences contain features common to most polymerase I1 genes, including TATA, SPl, APl, AP2, and CCAAT binding sites, in marked contrast to the corresponding region of the murine gene (see later). Furthermore, the first intron of chicken AE1 contains a 4 0 0 - b p repeat sequence, RI, whose motif, GGGA(T/C)AGA resembles the consensus binding site for the erythroid cell-specific nuclear enhancer-promoter binding factor, NF-El (Kim et al., 1989). Thus the single chicken AEl gene is transcribed from two distinct promoter elements giving rise to two mRNA species (which differ only at their 5’ termini) by alternative RNA splicing. These mRNAs are present in erythroid cells in roughly equal proportions and are translated into the two chicken AEI polypeptides. In contrast, the single mouse erythrocyte AEl protein is translated from a family of at least five mRNAs that differ only in their 5’ untranslated regions (Kopito et al., 1987a) (Fig. 3). Transcriptional initiation of the mouse AE1 gene in erythroid cells occurs at some five sites within a span of 43 bp of the same exon (Kopito et al., 1987a). Alternative mRNA splicing is not apparently involved in the generation of these messengers. Examination of the genomic sequence upstream to these cap sites reveals no elements characteristic of most eukaryotic polymerase I1 promoters, again contrasting with the chicken AE 1 promoters. The sequence of this region also lacks homology with the regulatory regions of other mammalian erythroid-specific genes, such as globin.

c. EXPRESSION OF AEl DURfNG ERYTHROID DIFFERENTIATION AEI mRNA is translated on membrane-bound polysomes, and the polypeptide is cotranslationally inserted into the membrane of the endoplasmic reticulum (Braell and Lodish, 1981, 1982). This process is mediated by an internal signal sequence (Braell and Lodish, 1982; Kopito and Lodish, 1985a) and requires signal recognition particle (SRP) (R. R. Kopito, unpublished results). Regulation of AEl gene expression and protein processing have been studied in both the avian and murine systems. In chickens (Chan, 1977) and mice (Chang er al., 1976), erythrocyte membrane proteins are made asynchronously. AE1 mRNA is detectable at all stages of erythroid development in chicken embryos,

189

ANION EXCHANGER GENE FAMILY

A

1 kb P2

B

100 bp

A

x c2

U G

c1 FIG.2. Transcriptional initiation of the chicken AEl gene (Kim et al., 1989). (A) Structure of the 5' end of the chicken AEl gene. pl and p2 refer to promoter elements. Shaded boxes denote exons, unshaded regions introns. RI refers to the repetitive sequence element identified within intron # l . (B) Representation of the 5' end of the two major chicken erythroid AEl transcripts showing pattern of alternative splicing. AUG refers to the site of putative translational initiation within the two mRNAs.

increasing sharply between day 4 (primitive cells) and day 10 (mostly immature definitive cells) (Cox et af., 1985). Nuclease protection analysis indicates that, during this period of erythroid development, both major AE1 transcripts are expressed in roughly equal quantities, suggesting that the two AE1 promoters are equally efficient in the same cells throughout development (Kim et al., 1988). Chicken erythroblasts transformed with temperature-sensitive (ts) mutants of the oncogene, v-erbB (ts-v-erbB) express erythroid-specific genes such as AE 1 and globin, and synchronously differentiate into erythrocytes within 3-4 days following shifting to the nonpermissive temperature (Kahn et af., 1986). Coexpression of another oncogene, v-erbA in these cells, inhibits transcription of certain erythroid-specific genes and blocks erythroid differentiation. The verbA polypeptide represents a virally transduced cellular receptor for thyroid hormone that no longer binds to the hormone but retains DNA-binding activity (Weinberger et al., 1986). Zenke et af. (1988) have demonstrated that v-erbA expression in ts-v-erbB-transformed erythroblasts blocks expression of AE 1, but not of other erythroid-specific genes such as globin and band 4.1. They conclude that this oncogene specifically suppresses AE1 transcription, which is somehow causally related to the block in differentiation, perhaps by interfering with the regulation of intracellular pH. Interestingly, no binding site for erbA has been identified within either the chicken or mouse AE1 gene sequence. Expression of the chicken AEl gene under the control of a non-erbA-responsive promoter

190

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should help to elucidate the role of this proto-oncogene of the anion exchanger in erythroid differentiation. In mice, AEl synthesis begins only after completion of the synthesis of the bulk of cytoskeletal proteins such as spectrin and actin (Chang et af., 1976).This finding is consistent with the observation that AEl associates with a preassembled membrane cytoskeletal network in embryonic chicken erythroid cells (Cox et al., 1987). In MEL cells, AEl protein synthesis is maximal 3-4 days following dimethyl sulfoxide (DMS0)-induced differentiation (Sabban et al., 1980),paralleled by a rise in steady-state AEl mRNA (Kopito and Lodish, 1985b Kopito et al., 1987a).This increase parallels a rise in AE1 gene transcription that is maximal at day 2-3 (Fraser and Cums, 1987). In contrast, steady-state levels of the mature AE1 protein continue to rise beyond 4 days of induction as the protein accumulates at the plasma membrane (Pate1 and Lodish, 1987). These observations suggest the existence of regulatory elements associated with the AEl gene that direct its expression coordinately-but not synchronously- with other erythroid-specific genes.

IV. Anion Exchanger Expression in Nonerythroid Cells There is abundant physiological and pharmacological evidence for anion exchange activity in many, if not all, mammalian cell types. Similarly, tissue-specific homologs of the major structural components of the erythrocyte membrane skeleton such as specmn (fodrin) and ankyrin, appear to be ubiquitous. These observations argue for the existence of proteins functionally, if not structurally, related to band 3. The similarity among the AE family members implies that they share antigenic cross-reactivity. For this reason, and because of the possibility that multiple polypeptides can be derived from a single gene (by alternative mRNA processing), it is nearly impossible to infer patterns of gene expression from immunological data. Antibodies to erythrocyte band 3 recognize epitopes from a host of nonerythroid cells including platelets, hepatocytes, neutrophils, and neuroblastoma (Kay et al., 1983), smooth muscle (Drenckhahn et al., 1984), lymphocytes and fibroblasts (Kay et af., 1983; Drenckhahn et al., 1984), kidney (Kay et al., 1983; Cox et al., 1985; Drenckhahn et al., 1985; Jennings er al., 1985; Kopito et al., 1988; Alper et al., 1989a), stomach (Kellokumpu et al., 1988; Thomas er al., 1989), and turtle bladder (Drenckhahn et af.,1987). In the following discussion I will focus on the three best-characterized systems for which there is data at both the protein and mRNA level.

A. KIDNEY The isolation and sequencing of AEl cDNA clones from kidney (Kudrycki and Shull, 1989; Brosius et af., 1989) represent the only unambiguous demon-

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stration of expression of the AE1 genes in a nonerythroid tissue. The major AE1 transcript present in mouse kidney is 4.2 kb, ~ 0 . 2kb shorter than its erythroid counterpart (Alper et al., 1987). This transcript can be detected on Northern blots of mouse kidney mRNA with an AE1 cDNA probe corresponding to exons 10-12 but not with a probe corresponding to exons 1-3 (Brosius er al., 1989), suggesting that the erythroid and renal transcripts differ at their 5’ ends. Nuclease protection and primer extension studies have mapped.the 5’ end of the major mouse (Brosius et al., 1989) and rat (Kudrycki and Shull, 1989) kidney AEI transcripts to be upstream of the 5’ terminus of exon 4 (Fig. 3). The AE1 protein predicted from such a transcript would be =9 kDa shorter than its erythroid counterpart, probably initiating at Met80 of the mouse erythroid sequence. Such a polypeptide would contain all of the transmembrane domain necessary for anion transport, but would lack sequences from the extreme NH, terminus of the erythroid protein. This missing region in human band 3 contains many acidic residues that are believed to participate in the binding of glycolytic enzymes (Low, 1986). It is not clear what effect, if any, such truncation would have on ankyrin binding or on anion exchange activity. Several AE1-hybridizing species of different size have also been detected by Northern blotting of kidney mRNA from mouse (Alper et al., 1987; Brosius et al., 1989) and rat (Kudrycki and Shull, 1989). These may represent additional minor AE1 transcripts, or transcripts of other members of the AE gene family, cross-hybridizing, even at the elevated stringencies under which the studies were conducted. Possibly, one of these minor bands represents the AEl transcript containing all of exon 1, previously identified by nuclease protection of kidney RNA (Kopito et al., 1987a). Kudrycki and Shull have reported that the 5’ end of rat kidney AE1 mRNA contains sequences highly homologous to sequences within intron 3 from mouse, and speculate that, in rat, the AE1 gene is transcribed from a promoter residing within the rat equivalent of this mouse intron (Kudrycki and Shull, 1989). The situation appears to be different in mouse, however, where sequences from this intron are apparently lacking from the major kidney transcript (Brosius et al., 1989). Further studies will be necessary to identify the mechanisms and pattern of transcription of the AE1 gene in kidney. Anion exchanger gene expression in the kidney has been extensively studied by immunocytochemical techniques. Polyclonal (Drenckhahn et al., 1985; Wagner et al., 1987; Drenckhahn and Merte, 1987; Alper er al., 1989a; Kopito et al., 1988) and monoclonal (Schuster et al., 1986; Wainwright et al., 1989; Wagner et al., 1987) antibodies to erythrocyte band 3 or to synthetic peptides derived from the mouse AEI sequence (Kopito er al., 1988; Alper er al., 1989a), all stain exclusively the basolateral membranes of the intercalated cells of the collecting duct in mammalian kidney. Staining with all available antibodies in mouse, rat, rabbit, and human kidney is undetectable in proximal tubule, thick and thin limbs of Henle, and glomerulus. Antibodies to human band 3, which

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A

B 100 bD A

G U

RBC A U G

Kidney FIG.3. Transcription of the murine A E I gene. ( A ) Suucture of the 5‘ end of the murine AEI gene (Kopito ef nl.. 1987b) showing the first six exons (shaded) to scale. (9) Representation of the major AEI transcripts in mouse erythroid cells (RBC) (Kopito ef nl., 1987a) and mouse and rat kidney (Kudrycki and Shull. 1989; Brosius et 01.. 1989). Locations of putative translational initiation sites for erythroid transcripts (at the 3’ end of exon #2) and renal transcripts (at the 5 ’ end of exon #4) are indicated (AUG).

cross-react with the avian erythroid protein, also stain intercalated cells of the chicken kidney collecting tubule (Schuster et al., 1986). In contrast, Cox et al. (1985) have reported that antibodies to chicken band 3 stain a subset of cells in the chicken proximal tubule. Because of the close homology among AE1-3, particularly within the membrane domain, and because of the fact that all three AE genes are expressed in kidney (see later), these immunological data must be interpreted with caution. Monoclonal antibodies (Schuster et al., 1986; Wagner et al., 1987) that recognize epitopes within the cytoplasmic domain of band 3 support the conclusion that the intercalated cell antigen is, indeed, AE1. Human collecting duct band 3positive cells lack at least three epitopes present in the cytoplasmic domain of the erythroid protein, consistent with RNA analyses suggesting that kidney AE 1 lacks sequences from exons 1-3 (Wagner et al., 1987). An identical staining pattern was obtained with an antipeptide antibody to band 3 residues 214-228, which are in the middle of the cytoplasmic domain in a region that is not conserved among AE1-3 (corresponding to positions 494-508 in Fig. 1) (Alper et al., 1989a).

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Western blots of kidney membranes from rat (Drenckhahn et al., 1985) and human (Wagner et al., 1987) indicate that the major band 3 immunoreactive species migrates on SDS-PAGE with a slower mobility than the erythroid protein, suggesting an M, of 110,000-120,000. This result is difficult to reconcile with the data of Kudrycki and Shull(1989), which predict a kidney polypeptide 9 kDa smaller than band 3, unless the discrepancy reflects differences in the posttranslational processing of the protein. It is possible that the protein detected on Western blots is not the same as the one identified by immunohistwhemistry, and is actually AE2 or AE3. The situation is not likely to be resolved soon, because of the complexity of the pattern of AE gene expression and the diversity of cell types within the kidney. Taken together, however, the available data suggest that a truncated form of AE1, which lacks the NH2-terminal 79 amino acids, is expressed in mammalian kidney exclusively at the basolateral plasma membrane of intercalated cells. This localization is consistent with physiological evidence for the role of CI/HC03 exchange in HC03 reabsorption by the “A” (acid-secreting) intercalated cells. This interpretation is further supported by the finding that >99% of intercalated cells that express apical H+-ATPase (i.e., “A”-type cells) also express basolateral AEl (Alper et al., 1989a). If AE1 is expressed in intercalated cell basolateral membranes, where are AE2 and AE3 expressed in kidney? Where are the products of the “minor” AE1 transcripts expressed, if at all? The inability to detect apical staining with any band 3 antibody in base-secreting (Btype) intercalated cells, despite evidence supporting a role for Cl/HC03 exchange in these cells, implies that the B-cell anion exchanger is different from AEl. An AE1 C-terminal antibody, a-C, (Thomas et al., 1989), which recognizes AE2 as well as AE1 (Lindsey et af., 1990), also stains only intercalated cell basolateral membranes, even in kidneys isolated from chronically alkalotic rabbits (D. Herzlinger and Q. Al-Awqati, personal communication). Since A E 2 mRNA is unambiguously expressed in kidney (Alper et al., 1988), its a-C,-reactive epitope must be masked, expressed at undetectably low levels, or expressed in precisely the same pattern as AE1. B. STOMACH Immunostaining of mouse or rabbit stomach with the polyclonal antipeptide antibody (a-C,) to mouse band 3 reveals intense reaction with the basolateral plasma membrane of parietal cells (Thomas er al., 1989; Kellokumpu et al., 1988). These acid-secreting epithelial cells are functional analogs of the renal “A”-type intercalated cells. They are rich in mitochondria and carbonic anhydrase, and contain apical proton pumps. No plasma membrane staining was observed with a-C, or with a polyclonal antibody against intact band 3 (Thomas et

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al., 1989; Kellokumpu et al., 1988) in other cell types in the stomach. Strong staining of the Golgi apparatus, however (see later), was observed in chief cells (Kellokumpu et al., 1988) and mucous neck in the glandular region of the stomach and in secretory cells of the antrum and forestomach (L. Neff and R. Baron, personal communication). Thus, in the stomach, as well as kidney, plasma membrane staining with band 3 antibodies is restricted to the basolateral membrane of functionally analogous, acid-secreting cell types. While the immunoreactive protein in kidney appears to be the product of the AE1 gene, the corresponding protein in parietal cells is probably not AEI , since this gene is not expressed at significant levels in stomach (Kudrycki et al., 1990). Kudrycki et al. (1990) have isolated cDNAs corresponding to rat AE2 and AE3 from stomach, and have examined the relative abundance of transcripts of these genes in different regions of the gastrointestinal tract. Their data show the highest levels of AE2 RNA in the antrum and &helowest in forestomach and mucosa. In contrast, hybridization of an AE3 probe to RNA from the same stomach regions exhibited an inverse pattern, with highest expression found in the forestomach and almost undetectable levels in mucosa. Hybridization of AE 1 probes to stomach mRNA indicated that this gene is expressed at extremely low levels, if at all, in this organ. These data have led to the proposal (Kudrycki ef al., 1990) that AE2 is the likely candidate for the basolateral CI/HC03 exchanger in parietal cells. Several lines of evidence suggest that such a conclusion may be premature. Immunoblots of gastric mucosal membranes with the a-C, antibody reveal a major polypeptide of 185 kDa and two minor bands in the range 140-145 kDa (Thomas et al.. 1989). Since AE1 is not expressed in stomach, the immunoreactive proteins detected by immunoblotting must be either products of AE2 or of does not cross-react with AE3 (R. R. Kopito, another, unidentified gene [a-C, unpublished results)]. However, AE2 (like AE3) encodes a polypeptide with predicted M,of ~137,000, which migrates on SDS-PAGE with M, 140,000. The glycosylated form of AE2 migrates at 165,000 (Lindsey et al., 1990). In order for the 185-kDa parietal cell antigen to be an AE2 product, it would have to acquire 20-kDa additional mass posttranslationally. The absence of plasma membrane immunoreactivity with a-C, in all other regions of the stomach except parietal cells, is not consistent with the pattern of expression of AE2 mRNA (Kudrycki et al., 1990). Although this gene is expressed in mucosa, highest levels of transcript are found in antrum, a region devoid of parietal cells. One possible interpretation of these data is that the parietal cell basolateral anion exchanger is encoded by yet another AE gene, which gives rise to an 185-kDa polypeptide, and that AE2 encodes a polypeptide that is expressed in the Golgi apparatus of cells involved in the secretory functions of the stomach. Such an interpretation would be consistent with the observation that the a-C,-immunoreactive Golgi protein is most prominently expressed in cell types frequently in-

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volved in secretion (Kellokumpu et al., 1988). The patterns of AE3 expression in stomach and the high levels found in brain and heart (see later) suggest that this anion exchanger homolog may predominate in excitable cells, which in the stomach, includes smooth muscle and neurons.

c. ANION EXCHANGER EXPRESSION IN THE BRAIN Although expression of the AE1 gene is undetectable in brain, both AE2 and AE3 are expressed in a cell type-specific fashion in this tissue. cDNAs encoding AE2 (Lindsey ef al., 1990) and AE3 (Kopito et al., 1989) have been isolated from brain libraries, establishing that both of these genes are expressed, at least, at the mRNA level. The sequence of AE2 from brain is identical to AE2 isolated from stomach (Kudrycki et al., 1990). There is no evidence for alternative mRNA splicing of brain AE2. Analysis of anion exchanger gene expression in brain has been examined by in situ hybridization using probes specific for each gene on serial sections of mouse brain (Kopito et al., 1989; Lindsey et al., 1990). These data confirm the lack of AE1 mRNA and show that AE2 expression is restricted to the epithelial cells of the choroid plexus. Studies using the a-C, antibody establish that a unique M , 165,000 immunoreactive polypeptide is detectable in choroid plexus preparations, consistent with the predicted M , of AE2, and comigrating with AE2 synthesized by COS cells transfected with AE2 cDNA (Lindsey et al., 1990). Immunocytochemical analysis of choroid plexus with a-C, antibody reveals strong staining of both the basolateral plasma membrane (Lindsey ef al., 1990) and the Golgi stacks (L. Neff and R. Baron, personal communication). These data are consistent with the function of AE2 as an anion exchanger that could participate in transepithelial bicarbonate transport, and with other circumstantial data (see earlier), suggesting that the a-C,-immunoreactive antigen in the Golgi (Kellokumpu et al., 1988) is the product of the AE2 gene. In situ hybridization studies indicate that, in contrast to AE2, brain AE3 is expressed in neurons and is absent from nonneuronal cells including glia and choroid plexus (Kopito et al., 1989). Further localization of the AE3 polypeptide within the CNS has not been possible because of the lack of suitable antisera. Although AE3 mRNA is detectable in all brain neurons, significant variations exist in the levels of messenger present in specific sets of neurons. In particular, AE3 hybridizes most intensely with neurons in the deep pontine gray matter, midbrain, and medulla. Strong hybridization has also been observed in Purkinje cells of the cerebellum and mitral cells of the olfactory bulb. Northern blot analysis (Kopito et al., 1989; Kudrycki et al., 1990) reveals that, in addition to brain, high levels of AE3 mRNA are also present in heart. The heart transcript lacks =1 kb from the 5’ end (Kudrycki et al., 1990). Localization of AE3 to a specific cell type in myocardium has not been reported.

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Several unusual AE3 cDNA clones have been isolated from either rat (Kudrycki et al., 1990) or mouse (C. W. Morgans and R. R. Kopito, unpublished observations) brain libraries, suggesting the possibility that multiple isoforms of this gene product may be produced by alternative mRNA processing. Several AE3 cDNA clones have been identified containing apparently unspliced introns. Kudrycki et al. ( 1990) isolated rat brain AE3 cDNAs containing introns of 358 and 578 bp at nucleotide positions 1138 and 3008, respectively. Since they did not report the sequence of the introns, it is not possible to determine whether or not they encode open reading frames, and could be considered “alternate exons.” C. W. Morgans and R. R. Kopito (unpublished data) have also identified a mouse brain AE3 transcript containing an unspliced intron of 3 11 bp at nucleotide 1 138. S I nuclease analysis of brain mRNA indicates that this species represents up to 30% of the total AE3 mRNA in brain. RNAs containing this intron, which lacks an open reading frame, are predicted to encode a polypeptide that is truncated 12 residues downstream of residue 377. Another class of AE3 mRNA has been identified that arises by alternative RNA splicing, resulting in the insertion of 14 bp. This insertion causes a reading frame shift, and the polypeptide encoded by such a mRNA would terminate at an out-of-frame codon 20 residues downstream of amino acid 486. While there are no data supporting the existence of such truncated polypeptides in vivo,the accumulation of steady-state levels of these RNAs in the cell may turn out to have some physiological relevance. Kudrycki er al. (1990) have also reported the identification of an AE3 clone containing an in-frame deletion of amino acids 907-988, resulting in the elimination of putative transmembrane spans 6 and 7 (Kudrycki et al., 1990). They argue that this clone is likely to be an artifact, since the sequences flanking the deletion do not correspond to splice sites in the AEI gene. A similar observation was made by Cox and Lazarides (1988) of a chicken AE1 clone that contained an in-frame deletion of membrane span 8. However, there is no confirmation that any of these unusual cDNAs represent actual mRNAs that are translated into proteins in vivo.

V. Summary The gene family of anion exchangers consists of at least four or five members, of which three have been characterized at the cDNA level. AE1-3 encode polypeptides that share significant homology with the erythrocyte anion exchanger, band 3 (AEI). Expression of cDNAs encoding these genes in heterologous systems confirms that this sequence similarity is reflected in the capacity to mediate reversible Cl/HC03 exchange. While the NH2-terminal domain of band 3 is known to interact with several cytoplasmic proteins in erythrocytes, the function of the analogous domains of AE2 and AE3 remains unknown.

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The AE1 gene is expressed coordinately with other erythroid genes during erythropoiesis in both avian and mammalian erythroid progenitor cells. In addition, AE1 is expressed at the basolateral plasma membrane of the acid-secreting intercalated cells of the kidney. AE2 is expressed in a number of epithelial and nonepithelial cells; it may be expressed in the Golgi apparatus of some of these cells. AE3 is expressed in excitable tissues, including neurons and muscle. It is likely that these proteins play a role in regulation of intracellular pH and chloride in their respective tissue. Understanding of the physiological roles of these proteins, both for ion transport and for plasma membrane organization, remains a central issue.

ACKNOWLEDGMENTS I would like to thank Drs. M. Jennings, J. D. Engel, and G. Shull for sharing data with me prior to publication. I would especially like to express my gratitude to the members of my laboratory for their constructive contributions to this manuscript, and to Lex Bunten for her expert secretarial assistance. R. R. K. is a Lucille P. Markey Scholar in Biomedical Science and this work is supported in part by a grant from the Lucille P. Markey Charitable Trust. REFERENCES Alper, S . L., Kopito, R. R., andLodish, H. F. (1987). Kidney. Inr., Suppl. 23, S117S133. Alper, S. L., Kopito, R. R., Libresco, S. M., and Lodish, H. F. (1988). J . Biol. Chem. 263, 17092-17099. Alper, S. L., Natale, J., Gluck, S., Lodish, H. F., and Brown, D. (1989a). Proc. Nutl. Acud. Sci. U.S.A.86,5429-5433. Alper, S. L., Brosius, F. C., 111, Garcia, A. M., Gluck, S., Brown, D., and Lodish, H. F.(1989b). Ann. N.Y.Acud. Sci. 574, 102-103. Bartel, D., Lepke, S., Layh-Schmitt, G., Legrum, B., and Passow, H. (1989). EMBO J. 8, 360 1-3609. Bennett, V. (1985). Annu. Rev. Biochem. 54,273-304. Bennett, V., and Stenbuck, P. J. (1979). Nature (London) 280,468-473. Braell, W. A., and Lodish, H. F. (1981). J . Biol. Chem. 256, 11337-11344. Braell, W.A., and Lodish, H. F. (1982). Cell (Cambridge, Muss.) 28,23-31. Brahm, J. (1988). Soc. Gen. Physiol. Ser. 43, 141-150. Brosius, F. C., HI, Alper, S . L., Garcia, A. M., and Lodish, H. F. (1989). J. Biol. Chem. 264, 7784-7787. Cabantchik, Z. I., Knauf, P. A., and Rothstein, A. (1978). Biochim. Biophys. Acfu 515,239-302. Chan, L. L. (1977). Proc. Nufl.Acud. Sci. U S A . 74, 1062-1066. Chang, H., Langer, P. J., and Lodish, H. F. (1976). Proc. Nurl. Acud. Sci. U.S.A.73, 3206-3210. Cox, J. V.,and Lazarides, E. (1988). Mol. Cell. Biol. 8, 1327-1335. Cox, J. V., Moon,R. T., and Lazarides, E. (1985). J. Cell Biol. 100, 1548-1557. Cox, J. V,, Stack, J. H., and Lazarides, E. (1987). J Cell Biol. 105, 1405-1416. Davis, L., Lux, S. E., and Bennett, V. (1989). J . Biol. Chem. 264,9665-9672.

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