Molecular Cloning, Genomic Organization, and Functional
Expression of Na+/H+ Exchanger Isoform 5 (NHE5)
from Human Brain*
Nancy R.
Baird
,
John
Orlowski§,
Elöd Z.
Szabó§,
Hans C.
Zaun
,
Patrick J.
Schultheis¶,
Anil G.
Menon¶, and
Gary E.
Shull¶
From ¶ the Department of Molecular Genetics, Biochemistry, and
Microbiology and the
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 |
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 |
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.
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.
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 |
-
Sardet, C.,
Franchi, A,
and Pouyssegur, J.
(1989)
Cell
86,
271-280
-
Orlowski, J,
Kandasamy, R. A.,
and Shull, G. E.
(1992)
J. Biol. Chem.
267,
9331-9339[Abstract/Free Full Text]
-
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]
-
Wang, Z.,
Orlowski, J.,
and Shull, G. E.
(1993)
J. Biol. Chem.
268,
11925-11928[Abstract/Free Full Text]
-
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]
-
Numata, M.,
Petrecca, K.,
Lake, N.,
and Orlowski, J.
(1998)
J. Biol. Chem.
273,
6951-6959[Abstract/Free Full Text]
-
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]
-
Orlowski, J.,
and Grinstein, S.
(1997)
J. Biol. Chem.
272,
22373-22376[Free Full Text]
-
Wakabayashi, S.,
Shigekawa, M.,
and Pouyssegur, J.
(1997)
Physiol. Rev.
77,
51-74[Abstract/Free Full Text]
-
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]
-
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]
-
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]
-
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]
-
Kandasamy, R. A.,
and Orlowski, J.
(1996)
J. Biol. Chem.
271,
10551-10559[Abstract/Free Full Text]
-
Mount, S. M.
(1982)
Nucleic Acids Res.
10,
459-472[Abstract]
-
Kozak, M.
(1984)
Nucleic Acids Res.
12,
857-872[Abstract]
-
Orlowski, J.
(1993)
J. Biol. Chem.
268,
16369-16377[Abstract/Free Full Text]
-
Chen, C.,
and Okayama, H.
(1987)
Mol. Cell. Biol.
7,
2745-2752[Medline]
[Order article via Infotrieve]
-
Franchi, A.,
Perucca Lostanlen, D.,
and Pouyssegur, J.
(1986)
Proc. Natl. Acad. Sci. U. S. A.
83,
9388-9392[Abstract]
-
Yu, F. H.,
Shull, G. E.,
and Orlowski, J.
(1993)
J. Biol. Chem.
268,
25536-25541[Abstract/Free Full Text]
-
Attaphitaya, S.,
Park, K.,
and Melvin, J. E.
(1999)
J. Biol. Chem.
274,
4383-4388[Abstract/Free Full Text]
-
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]
-
Pathak, B. P.,
Shull, G. E.,
Jenkins, N. A.,
and Copeland, N. G.
(1996)
Genomics
31,
261-263[CrossRef][Medline]
[Order article via Infotrieve]
-
Jackson, I. J.
(1991)
Nucleic Acids Res.
14,
3795-3798
-
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]
-
Muller, R. M.,
Taguchi, H.,
and Shibahara, S.
(1987)
J. Biol. Chem.
262,
6795-6802[Abstract/Free Full Text]
-
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]
-
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.