Molecular Cloning, Genomic Organization, and Functional Expression of Na+/H+ Exchanger Isoform 5 (NHE5) from Human Brain*

Nancy R. BairdDagger , John Orlowski§, Elöd Z. Szabó§, Hans C. ZaunDagger , Patrick J. Schultheis, Anil G. Menon, and Gary E. Shullparallel

From   the Department of Molecular Genetics, Biochemistry, and Microbiology and the Dagger  Department of Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524 and the § Department of Physiology, McGill University, H3G 1Y6 Montreal, Quebec, Canada

    ABSTRACT
Top
Abstract
Introduction
References

To isolate a cDNA encoding Na+/H+ exchanger isoform 5 (NHE5), we screened a human spleen library using exon sequences of the NHE5 gene. Clones spanning 2.9 kilobase pairs were isolated; however, they contained several introns and were missing coding sequences at both the 5' and 3' ends. The missing 5' sequences were obtained by 5'-rapid amplification of cDNA ends and by analysis of an NHE5 genomic clone, and the missing 3' sequences were obtained by 3'-rapid amplification of cDNA ends. Polymerase chain reaction amplification of brain cDNA yielded products in which each of the introns had been correctly excised, whereas the introns were retained in products from spleen and testis, suggesting that the NHE5 transcripts expressed in these organs do not encode a functional transporter. The intron/exon organization of the NHE5 gene was analyzed and found to be very similar to that of the NHE3 gene. The NHE5 cDNA, which encodes an 896-amino acid protein that is most closely related to NHE3, was expressed in Na+/H+ exchanger-deficient fibroblasts and shown to mediate Na+/H+ exchange activity. Northern blot analysis demonstrated that the mRNA encoding NHE5 is expressed in multiple regions of the brain, including hippocampus, consistent with the possibility that it regulates intracellular pH in hippocampal and other neurons.

    INTRODUCTION
Top
Abstract
Introduction
References

Six members of the mammalian Na+/H+ exchanger (NHE)1 family, including five plasma membrane (NHE1-5) and one mitochondrial (NHE6) exchanger, have been identified by molecular cloning studies (1-7). Full-length cDNAs encoding NHE1-4 (1-5) and NHE6 (6) have been characterized, thereby providing their amino acid sequences and allowing the development of expression constructs and isoform-specific nucleic acid and antibody probes. The use of these reagents has led to a substantial body of information about the expression patterns of these isoforms and their functional characteristics (reviewed in Refs. 8 and 9). In recent studies, mice carrying mutations in the genes encoding NHE1, -2, and -3 have been developed, and their phenotypes have been analyzed to determine their specific physiological functions in vivo (10-12). In contrast, the complete primary structure of NHE5 has not been determined, and nothing is known about its functional characteristics and physiological roles.

The human NHE5 gene was identified several years ago by low stringency screening of a cosmid library using an NHE2 cDNA probe and mapped to chromosome 16q22.1 (7). Northern blot analysis demonstrated that NHE5 transcripts were expressed at easily detected levels in brain, spleen, and testis and at trace levels in skeletal muscle and revealed differences in the sizes of the transcripts in each tissue. Unlike the other isoforms, expression of NHE5 was not observed in epithelial tissues. Based on the partial coding sequences obtained and limited analysis of the structure of the gene, NHE5 appeared to be more closely related to the amiloride-resistant NHE3 isoform than to other members of the family. Its abundant expression in brain and apparent similarity to NHE3 raised the possibility that NHE5 might be the amiloride-resistant Na+/H+ exchanger that was described in hippocampal neurons (13), particularly since NHE3 expression is negligible in brain (2). To begin a systematic analysis of the functional characteristics and physiological roles of NHE5, we have cloned and functionally expressed a cDNA encoding the complete amino acid sequence of human NHE5, determined the intron-exon organization of its gene, and demonstrated that its mRNA is expressed in hippocampus and other structures of the brain.

    EXPERIMENTAL PROCEDURES

Isolation and Characterization of cDNA Clones-- A human spleen 5' stretch cDNA library (CLONTECH) was screened under high stringency conditions using a 32P-labeled probe corresponding to exons 2 and 3 of the human NHE5 gene (7). Two partial cDNAs (clone 11 and clone 26) that gave strong hybridization signals were identified and plaque-purified. 5'-RACE was performed using 5'-RACE-ready spleen cDNA (CLONTECH) and gene-specific primers complementary to nts 365-383 (for the primary PCR amplification) and 334-358 (for the secondary PCR amplification). 3'-RACE was performed using human brain Marathon cDNA (CLONTECH) and a gene-specific PCR primer corresponding to nts 2339-2365. 5'-RACE products were subcloned into the TA cloning vector (Invitrogen), and 3'-RACE products were subcloned into the PCR II vector (Invitrogen). Coding sequences from exon 1 that were not included in the 5'-RACE product were obtained by partial sequence analysis of a pBluescript subclone of a 13.5-kb HindIII genomic fragment containing exon 1 of the human NHE5 gene. DNA sequence analysis was performed manually by the chain termination procedure or automatically using Perkin-Elmer ABI Prism dye-termination technology.

Determination of Intron/Exon Boundaries-- Multiple pairs of upstream (sense) and downstream (antisense) oligonucleotide primers, based on the cDNA sequence and designed to allow the generation of overlapping fragments spanning the entire length of the gene, were synthesized. When designing the primers we considered the location of introns in the rat nhe3 gene (14), to which the NHE5 gene seemed to be most closely related. Each set of primers was used in a PCR reaction with the previously isolated human NHE5 cosmid clone HP 7-1 (7), which contained the entire gene, serving as a template. The amplified genomic DNA fragments, which began and ended with exon sequences, were subcloned into the PCR II plasmid vector (Invitrogen) and sequenced from each end. Intron/exon boundaries were identified by both the presence of consensus sequences for intron/exon boundaries (15) and by discontinuities in the nucleotide sequence when compared with the cDNA sequence. Intron sizes were estimated by agarose gel electrophoresis of the intron-containing fragments.

Human NHE5 cDNA Reconstruction-- To reconstruct a complete human NHE5 cDNA lacking intron sequences, six contiguous overlapping regions that spanned the coding region were amplified by PCR using the isolated cDNAs and RACE products as templates. Adjacent fragments were joined by PCR until the coding region was reassembled into a single cDNA fragment containing unique KpnI and XbaI restriction endonuclease sites at the 5' and 3' ends, respectively. By using PCR mutagenesis, an influenza virus hemagglutinin epitope YPYDVPDYAS was inserted at the C-terminal end to allow for immunological detection of the protein, and a more optimal translation initiation sequence (16) was engineered at the 5' end of the cDNA (CCCCGCCACCATGC, with ATG as the initiation methionine codon; C in the +4 position was retained because using the more optimal G would have altered an amino acid). The entire PCR product was sequenced to confirm the fidelity of the coding region. The modified human pNHE5HA cDNA was inserted into a mammalian expression vector under the control of the enhancer/promoter region of the immediate early gene of human cytomegalovirus as described previously (17).

Stable Expression of NHE5 in Cultured Cells and 22Na+ Influx Measurements-- Na+/H+ exchanger-deficient Chinese hamster ovary cells (AP-1), cultured as described previously (17), were transfected with pNHE5HA by the calcium phosphate-DNA coprecipitation technique (18). Colonies were selected for stable expression of Na+/H+ exchanger activity by their survival during repeated acute NH4Cl-induced acid loads (19). Cells stably expressing NHE5 were grown to confluence in 24-well plates and loaded with H+ using the NH4+ prepulse technique (17, 19), and 22Na+ influx assays were performed in the presence and absence of 1 mM amiloride as described in earlier studies (4, 20). Intracellular pH following acid loading was ~6.2 and uptake assays were performed using carrier-free 22NaCl.

Northern Blot Analysis-- A human brain Northern blot was obtained from CLONTECH. Each lane contained approximately 2 µg of poly(A)+ RNA from hippocampus and 7 other anatomical structures of the brain. The blot was analyzed using a probe corresponding to exons 2 and 3 of the NHE5 gene. Both the probe and the high stringency hybridization and washing conditions were the same as those used previously to determine the distribution of NHE5 mRNA in human tissues; in that study (7), no hybridization signals were detected in kidney, small intestine, or colon, demonstrating that under high stringency conditions the NHE5 probe does not cross-hybridize with mRNAs encoding NHE3 or any of the other isoforms.

    RESULTS

Isolation and Characterization of the Human NHE5 cDNA-- Because a previous study demonstrated that NHE5 mRNA was expressed in spleen, testis, and brain, we initially screened a human spleen cDNA library in an attempt to isolate a full-length cDNA. Two overlapping cDNAs, spanning 2.9 kb, were isolated and their nucleotide sequences determined. After aligning the deduced amino acid sequence with rat NHE3, it was apparent that the 5' and 3' ends of the coding sequence were missing and that the open reading frames of both clones were interrupted by possible intronic sequences. The composite sequence spanned nts 163-2496 of the open reading frame shown in Fig. 1 and included a 281-nt insertion following nt 733, a 221-nt insertion following nt 1132, and an 80-nt insertion following nt 1842. The first two insertions, but not the third, were flanked by consensus splice donor and acceptor sequences. PCR analysis of brain cDNA and subsequent analyses of the gene (discussed below) demonstrated that these insertions correspond to intron 4, intron 6, and part of intron 12, respectively.


View larger version (80K):
[in this window]
[in a new window]
 
Fig. 1.   Nucleotide sequence of the NHE5 cDNA and deduced amino acid sequence of the protein. Nucleotides are numbered at the right, with nucleotide 1 corresponding to the apparent translation initiation codon. Amino acids are numbered below the sequence.

To obtain the N-terminal coding sequence, 5'-RACE was performed using spleen cDNA. The longest RACE product extended the sequence to nt 36, but the translation initiation codon was not included. Because the 5'-most sequence in the RACE product corresponded to sequences within exon 1 of the NHE1 and nhe3 genes, we subcloned and analyzed a genomic fragment containing this exon of the NHE5 gene, and we obtained the remaining 5'-coding sequence and part of the 5'-untranslated sequence. To obtain additional 3' sequences, 3'-RACE was performed using brain cDNA. This yielded a 1.4-kb product containing the rest of the C-terminal coding sequence, the 3'-untranslated region, and an extensive poly(A) tract.

To determine whether the three sequences that interrupted the open reading frame were the result of incomplete or aberrant processing of the primary transcript, we first performed PCR analysis of spleen, testis, and brain cDNA using primers flanking the sequences in question. With spleen cDNA we obtained one product in which intron 6 was correctly excised, but all other spleen and testis products were apparently derived from processing intermediates or incorrectly spliced mRNAs. PCR amplification of brain cDNA yielded products in which each of the three intron sequences that were identified in the spleen cDNAs were correctly excised. However, we also identified a brain PCR product in which the donor site of exon 4 (nt 733) was spliced to a cryptic acceptor site (nt 820) in exon 5.

The composite nucleotide sequence of the NHE5 cDNA is shown in Fig. 1. It contains a 2688-nucleotide open reading frame, which encodes a protein of 896 amino acids with a molecular mass of ~99 kilodaltons, a 984-nt 3'-untranslated sequence, and a 71-nt poly(A) tract. The apparent translation initiation codon is in an acceptable context (16), and its position correlates well with that of the other isoforms (see Fig. 2 and Ref. 4). At the 5' end of the composite sequence we have included 68 nts that were immediately 5' of the initiation codon in the genomic sequence. Because this GC-rich sequence does not contain a potential splice acceptor site and the estimated size of the NHE5 mRNA in brain is 4.2 kb (compared with 3.7 kb for the coding and 3'-untranslated regions), it seems likely that all of this sequence is included in the 5'-untranslated region of the mRNA.


View larger version (68K):
[in this window]
[in a new window]
 
Fig. 2.   Amino acid similarity comparison. The amino acid sequences of human NHE5 is compared with rat NHE3 (2). Asterisks indicate sequence identity. Potential transmembrane domains, identified by the hydropathy program of Eisenberg et al. (28) are underlined. The positions of exon boundaries in the human NHE5 and rat nhe3 (14) genes are indicated by arrowheads.

Intron/Exon Organization of the Gene-- A major difficulty in characterizing the cDNA was the presence of three sequences in the spleen clones that interrupted the open reading frame. To confirm that these sequences were derived from introns, and to evaluate the relationship between NHE5 and NHE3, we determined the intron/exon organization of the NHE5 gene (Table I). This analysis revealed that the first two sequences interrupting the open reading frame in the original cDNAs corresponded to introns 4 and 6. The position of the third sequence (80 nts), which lacked consensus donor and acceptor sites, corresponded to that of intron 12; it most likely arose from the use of cryptic acceptor and donor sites within this intron, although we have not sequenced the entire intron to confirm this. The NHE5 gene spans ~18 kb and contains 16 exons and 15 introns, whereas there are 17 exons and 16 introns in the rat nhe3 gene (14). Most of the splice donor and acceptor sites matched the established consensus sequences (15); however, the donor sites for exons 13 and 14 contained GC rather than GT dinucleotides at the beginning of the intron. With the exception of a single nucleotide shift for intron 5, the positions of the first 12 introns precisely match those of the nhe3 gene (see Fig. 2), and the positions of introns 13-15 are also very similar in the two genes. The nhe5 gene does not have an intron corresponding to that of intron 16 of the nhe3 gene.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Exon/intron boundaries of the human NHE5 gene

Amino Acid Sequence Comparisons-- Hydropathy analysis showed that NHE5 has the same membrane topology as the other plasma membrane Na+/H+ exchangers, with multiple membrane spanning domains in the N-terminal half of the protein (underlined in Fig. 2) and an extensive cytoplasmic domain in the C-terminal half. Pairwise comparisons of amino acids 41-543 of NHE5 with the corresponding regions of NHE1-4 (Table II) demonstrated that it is most closely related to NHE3. (This region was chosen because it is clearly homologous in all five plasma membrane NHE isoforms and can be aligned unambiguously and with few gaps.) The overall amino acid similarity between NHE5 and NHE3 (Fig. 2), with all gaps in the alignment considered a mismatch, is 48% (64% in the N-terminal membrane spanning region and 31% in the C-terminal cytoplasmic region). The similarity between these two isoforms is clear throughout much of the cytoplasmic domain and, with the inclusion of one large gap in NHE3, seems to extend almost to the extreme C terminus.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Percentage amino acid identity in pairwise comparisons of NHE isoforms 1-5
Amino acids 47-543 of human NHE5 were compared with the corresponding regions of NHE1-4. The extreme N-terminal and the more C-terminal regions, which do not align well and exhibit only limited similarity among the five isoforms, were not included in this comparison.

Functional Expression of NHE5-- To determine whether NHE5 is a functional Na+/H+ exchanger, we expressed the cDNA in Na+/H+ exchanger-deficient cells and measured H+-dependent Na+ influx activity. As shown in Fig. 3, the initial rates of 22Na+ influx in the parental AP-1 cells was very low and could not be inhibited by addition of 1 mM amiloride, a concentration that inhibits even the relatively amiloride-resistant NHE3. In contrast, 22Na+ influx activity in cells expressing NHE5 was very high relative to background levels (~25-fold) and was fully inhibited by addition of 1 mM amiloride.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3.   Stable expression of human NHE5 in Na+/H+ exchanger-deficient Chinese hamster ovary (AP-1) cells. The human NHE5 expression construct was transfected into Na+/H+ exchanger-deficient Chinese hamster ovary (AP-1) cells, and stably transfected cells were selected by repeated acute intracellular acid loads. Isolated colonies were assayed for Na+/H+ exchange activity in 24-well plates. Initial rates of H+-activated 22Na+ influx were measured in untransfected AP-1 cells and in a clonal isolate expressing NHE5 (AP-1NHE5-C3) in the absence or presence of amiloride (1 mM). Values represent the mean ± S.D. of four determinations.

Distribution of the NHE5 mRNA in Brain-- To examine the distribution of NHE5 in human brain, a multiple tissue Northern blot containing RNA from a number of anatomical structures, including hippocampus, was hybridized with an NHE5 probe. A 4.2-kb NHE5 mRNA was identified in the hippocampus and in the other structures examined (Fig. 4). The highest levels of expression were in the caudate nucleus, and only trace levels were detected in corpus callosum and substantia nigra.


View larger version (66K):
[in this window]
[in a new window]
 
Fig. 4.   Expression of NHE5 mRNA in human brain. A Northern blot containing 2 µg of poly(A)+ RNA from each of the indicated regions of the brain was analyzed by hybridization with an NHE5 cDNA probe. The size (kb) of the NHE5 mRNA is depicted on the right. The autoradiographic exposure time was 48 h.


    DISCUSSION

The major objectives of this study were to determine the primary structure of human NHE5 and to explore the possibility that it corresponds to an amiloride-resistant Na+/H+ exchanger that was identified previously in hippocampal neurons (13). The human NHE5 gene was identified several years ago and was shown to be expressed in brain, spleen, and testis (7). In the present study we were unable to isolate NHE5 cDNAs corresponding to fully processed transcripts in human spleen or testis; however, PCR analysis of brain cDNA did yield products that were correctly processed. These cloning experiments and the previous observation that NHE5 transcripts in human spleen and testis are larger than those in brain suggest that intron sequences, which disrupt the open reading frame, are retained in the spleen and testis mRNAs. While this study was in progress we had frequent discussions with Attaphitaya et al. (21), who were conducting similar studies of rat NHE5. When cloning rat NHE5 from brain, these investigators did not encounter processing intermediates or aberrantly spliced transcripts, and their Northern blot analyses revealed high levels of NHE5 mRNA in brain and only trace levels in spleen, testis, and other tissues. Thus, the results from both laboratories suggest that expression of functional NHE5 mRNA may be restricted to brain, although they do not rule out the possibility of expression in tissues and developmental stages that were not examined in these experiments.

On the basis of the current and previous studies (7, 14), it is apparent that NHE5 and NHE3 are more closely related to each other, in both genomic organization and amino acid sequence, than to the other isoforms. As shown in Fig. 2, the location of exon boundaries relative to the coding sequences are almost identical in the NHE5 and nhe3 genes, and only the last intron of the nhe3 gene lacks a counterpart in the NHE5 gene. In contrast, introns 2, 4, and 10-15 of the NHE5 and nhe3 genes have no counterparts in the NHE1 gene (14, 22), and introns 8-11 of the NHE1 gene have no counterparts in the NHE5 and nhe3 genes. The complete genomic organization of the nhe2 and nhe4 genes, which are tightly linked in the mouse genome (23), have not been determined. However, limited analyses of the mouse nhe2 and nhe4 genes (7) indicate that they are more closely related to NHE1 than to NHE5 and nhe3, a conclusion that is supported by comparisons of amino acid sequences (Table II and see Refs. 4, 5, and 7).

An unusual feature of the NHE5 gene is the presence of GC dinucleotides, rather than GT dinucleotides, at the beginning of introns 13 and 14 (Table I). This departure from the consensus sequence is uncommon, but several dozen examples of GC dinucleotides at the beginning of an intron have been documented (24). For example, the sequences, AAG/GCATGT and CAG/GCAAGC, at the donor sites of exons 13 and 14, respectively, are identical to donor sites observed in the RNA polymerase II (25) and heme oxygenase (26) genes, respectively.

The data shown in Fig. 3 demonstrate that human NHE5 mediates H+-dependent Na+ influx and are consistent with the observation that rat NHE5 mediates Na+-dependent pH recovery from an acid load (21). 22Na+ influx in the clonal isolate shown in Fig. 3 was ~0.2 pmol/min/mg protein. Under comparable assay conditions the average 22Na+ influx values for AP-1 cell isolates expressing NHE1, NHE2, and NHE3 was between 0.25 and 0.60 pmol/min/mg protein (17, 20), with activity in the order NHE1 > NHE2 > NHE3. Because the amount of NHE protein expressed in each of the clonal isolates that have been examined has not been determined, the relative activities of the various isoforms cannot be directly compared; however, the activity of the NHE5 isolate examined here is within the range of values observed for clonal isolates expressing NHE3. Rat NHE5 has been shown to be more resistant than NHE1 to 5-(N-ethyl-N-isopropyl)amiloride (21), and detailed pharmacological and biochemical analyses of human NHE5 that are currently in progress2 indicate that human NHE5 is also quite resistant to pharmacological antagonists, although not to as great an extent as NHE3. Thus, both human and rat NHE5 are, in fact, Na+/H+ exchangers and have pharmacological characteristics that more closely resemble NHE3 than NHE1.

When we began this study we were interested in determining whether NHE5 might be the transporter responsible for the amiloride-resistant Na+/H+ exchange activity identified in rat hippocampal neurons by Raley-Susman et al. (13). Our Northern blot data showing NHE5 expression in hippocampus of human brain and in situ hybridization analysis showing NHE5 expression in rat hippocampus (21) are consistent with this possibility. However, the Na+/H+ exchange activity observed in hippocampal neurons was completely insensitive to 1 mM amiloride, whereas human NHE5 expressed in AP-1 cells is fully inhibited by this concentration of amiloride (Fig. 3). This apparent difference in drug sensitivity could be due to the different conditions under which Na+/H+ exchange activity was measured in the two studies. For example, in the study of Na+/H+ exchange in hippocampal neurons (13), the concentration of extracellular Na+, which is a competitive inhibitor of amiloride binding, was 135 mM, whereas in our own studies only trace levels of 22Na+ were present. NHE4 has been localized to cavi amnoni neurons in rat hippocampus (27), but this isoform also has been shown to exhibit some sensitivity to 1 mM amiloride. Additional studies will be needed to resolve the questions of which isoform(s) mediate control of intracellular pH in hippocampal neurons.

In summary, we have cloned and expressed a fifth member of the plasma membrane Na+/H+ exchanger family and have obtained data indicating that functional expression of this isoform may be restricted to brain. Northern blot analysis showed that it is widely distributed in brain, with significant mRNA expression detected in hippocampus, amygdala, caudate nucleus, hypothalamus, subthalamic nucleus, and thalamus. The detection of NHE5 mRNA in multiple structures of the brain, but the absence of a significant hybridization signal in corpus callosum, which consists primarily of axons and glial cells and has few neuronal cell bodies, raises the possibility that NHE5 may be a neuron-specific isoform. If this proves to be the case, then this isoform would be the most highly cell type-specific member of the Na+/H+ exchanger family.

    ACKNOWLEDGEMENT

We thank James E. Melvin for critical review of the manuscript and for useful discussions during the course of this work.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK50594 (to G. E. S) and DK41496 (to A. G. M.) and Medical Research Council of Canada Grant MT-11221 (to J. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF111173.

parallel To whom correspondence should be addressed: Dept. of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, 231 Bethesda Ave., ML 524, Cincinnati, OH 45267-0524. Tel.: 513-558-0056; Fax: 513-558-8474; E-mail: shullge{at}ucmail.uc.edu.

The abbreviations used are: NHE, Na+/H+ exchanger; NHE1-6, Na+/H+ exchanger isoforms 1-6; nt(s), nucleotides; kb, kilobase; RACE, rapid amplification of cDNA ends; PCR, polymerase chain reaction.

2 E. Szabo and J. Orlowski, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
References

  1. Sardet, C., Franchi, A, and Pouyssegur, J. (1989) Cell 86, 271-280
  2. Orlowski, J, Kandasamy, R. A., and Shull, G. E. (1992) J. Biol. Chem. 267, 9331-9339[Abstract/Free Full Text]
  3. Tse, C.-M., Brant, S. R., Walker, M. S., Pouyssegur, J., and Donowitz, M. (1992) J. Biol. Chem. 267, 9340-9346[Abstract/Free Full Text]
  4. Wang, Z., Orlowski, J., and Shull, G. E. (1993) J. Biol. Chem. 268, 11925-11928[Abstract/Free Full Text]
  5. Tse, C.-M., Levine, S. A., Yun, C. H. C., Montrose, M. H., Little, P. J., Pouyssegur, J., and Donowitz, M. (1993) J. Biol. Chem. 268, 11917-11924[Abstract/Free Full Text]
  6. Numata, M., Petrecca, K., Lake, N., and Orlowski, J. (1998) J. Biol. Chem. 273, 6951-6959[Abstract/Free Full Text]
  7. Klanke, C. A., Su, Y. R., Callen, D. F., Wang, Z., Meneton, P., Baird, N., Kandasamy, R. A., Orlowski, J., Otterud, B. E., Leppert, M., Shull, G. E., and Menon, A. G. (1995) Genomics 25, 615-622[CrossRef][Medline] [Order article via Infotrieve]
  8. Orlowski, J., and Grinstein, S. (1997) J. Biol. Chem. 272, 22373-22376[Free Full Text]
  9. Wakabayashi, S., Shigekawa, M., and Pouyssegur, J. (1997) Physiol. Rev. 77, 51-74[Abstract/Free Full Text]
  10. Cox, G. A., Lutz, C. M., Yang, C.-L., Biemesderfer, D., Bronson, R. T., Fu, A., Aronson, P. S., Noebels, J. L., and Frankel, W. N. (1997) Cell 91, 139-148[CrossRef][Medline] [Order article via Infotrieve]
  11. Schultheis, P. J., Clarke, L. L., Meneton, P., Harline, M., Boivin, G. P., Stemmermann, G., Duffy, J. J., Doetschman, T., Miller, M. L., and Shull, G. E. (1998) J. Clin. Invest. 101, 1243-1253[Abstract/Free Full Text]
  12. Schultheis, P. J., Clarke, L. L., Meneton, P., Miller, M. L., Soleimani, M., Gawenis, L. R., Riddle, T. M., Duffy, J. J., Doetschman, T., Wang, T., Giebisch, G., Aronson, P. S., Lorenz, J. N., and Shull, G. E. (1998) Nat. Genet. 19, 282-285[CrossRef][Medline] [Order article via Infotrieve]
  13. Raley-Susman, K. M., Cragoe, E. J., Jr., Sapolsky, R. M., and Kopito, R. R. (1991) J. Biol. Chem. 266, 2739-2745[Abstract/Free Full Text]
  14. Kandasamy, R. A., and Orlowski, J. (1996) J. Biol. Chem. 271, 10551-10559[Abstract/Free Full Text]
  15. Mount, S. M. (1982) Nucleic Acids Res. 10, 459-472[Abstract]
  16. Kozak, M. (1984) Nucleic Acids Res. 12, 857-872[Abstract]
  17. Orlowski, J. (1993) J. Biol. Chem. 268, 16369-16377[Abstract/Free Full Text]
  18. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752[Medline] [Order article via Infotrieve]
  19. Franchi, A., Perucca Lostanlen, D., and Pouyssegur, J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9388-9392[Abstract]
  20. Yu, F. H., Shull, G. E., and Orlowski, J. (1993) J. Biol. Chem. 268, 25536-25541[Abstract/Free Full Text]
  21. Attaphitaya, S., Park, K., and Melvin, J. E. (1999) J. Biol. Chem. 274, 4383-4388[Abstract/Free Full Text]
  22. Miller, R. T., Counillon, L., Pages, G., Lifton, R. P., Sardet, C., and Pouyssegur, J. (1991) J. Biol. Chem. 266, 10813-10819[Abstract/Free Full Text]
  23. Pathak, B. P., Shull, G. E., Jenkins, N. A., and Copeland, N. G. (1996) Genomics 31, 261-263[CrossRef][Medline] [Order article via Infotrieve]
  24. Jackson, I. J. (1991) Nucleic Acids Res. 14, 3795-3798
  25. Ahearn, J. M., Jr., Bartolomei, M. S., West, M. L., Cisek, L. J., and Corden, J. L. (1987) J. Biol. Chem. 262, 10695-10705[Abstract/Free Full Text]
  26. Muller, R. M., Taguchi, H., and Shibahara, S. (1987) J. Biol. Chem. 262, 6795-6802[Abstract/Free Full Text]
  27. Bookstein, C., Musch, M. W., DePaoli, A., Xie, Y., Rabenau, K., Villereal, M., Rao, M. C., and Chang, E. B. (1996) Am. J. Physiol. 271, C1629-C1638[Abstract/Free Full Text]
  28. Eisenberg, D., Schwarz, E., Komaromy, M., and Wall, R. (1984) J. Mol. Biol. 179, 125-142[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.