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INTRODUCTION |
Familial neurohypophyseal diabetes insipidus
(FNDI)1 (1) is caused by a
deficiency of arginine vasopressin (AVP), a hormone that controls serum
osmolality by altering renal free water clearance (1). Diabetes
insipidus is transmitted as an autosomal dominant trait in these
families. A large number of distinct mutations have been found in the
AVP gene (2-15).
The AVP gene encodes polypeptide precursors consisting of a signal
peptide, AVP, neurophysin (NP), and glycoprotein domains (16).
Prepro-AVP is synthesized in the magnocellular neurons of the
hypothalamus and is converted to pro-AVP by the removal of the signal
peptide. Pro-AVP undergoes several post-translational processing steps,
including the addition of carbohydrate side chains and proteolytic
cleavage to yield AVP, NP, and the glycoprotein. These products are
stored within neurosecretory vesicles in the axonal terminals of the
posterior pituitary gland and are secreted into the blood in response
to osmotic stimuli (17).
Most of the mutations in individuals with FNDI have been found within
the signal peptide and the NP domains (18). A substitution of Thr for
Ala at the carboxyl terminus of the signal peptide (A(
1)T) is the
most commonly found mutation, and it has been identified in various
ethnic groups, suggesting independent mutational events. Mutations
within the NP domain include amino acid substitutions, a single amino
acid deletion, and premature protein termination. Despite the presence
of a normal allele, FNDI patients develop diabetes insipidus, although
the symptoms are not usually manifest until several months or years
after birth. These features have raised questions concerning the
molecular pathogenesis of the disorder. A limited number of autopsy
studies have demonstrated a paucity of AVP-producing neurons in the
hypothalamus of patients with FNDI (19-22). Consistent with these
pathologic findings, magnetic resonance imaging has revealed an absence
of the bright spot that characterizes the posterior pituitary gland in
a subset of patients with FNDI (12). Based on these observations, it
has been postulated that mutant AVP precursors might be cytotoxic to
AVP-producing neurons.
In a previous study (4), the A(
1)T mutation was shown to cause
inefficient cleavage of the signal peptide, giving rise to aberrant
precursors that were glycosylated, but not cleaved, by signal
peptidase. This finding raised the possibility that the aberrant
precursors might accumulate and lead to cellular toxicity. In support
of this idea, the expression of several different FNDI mutants was
shown to impair the intracellular trafficking of mutant AVP precursors
and the viability of neuroblastoma cells (23).
The cytotoxicity of mutant AVP precursors may be sufficient to account
for the autosomal dominant mode of inheritance of FNDI. However, in
some autosomal dominant diseases, the mutant protein exerts
dominant-negative activity to alter the function of the normal allele
(24). AVP precursors have been shown to physically interact with each
other in vitro (25). It is therefore possible that mutant
AVP precursors could form heterodimers with wild-type (WT) protein
products to alter their function and contribute to the pathogenesis of
FNDI. In this study, we examined the physical and functional
interactions between WT and mutant AVP precursors by expressing
epitope-tagged precursors in cultured cells.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructions--
Expression vectors for the WT and
mutant (G57S (2), A(
1)T (4),
E47 (5), and C67X (9)) AVP
precursors (Fig. 1A) have been
described previously (23). For epitope tagging (Fig. 1B),
restriction sites for ClaI, SpeI, and
XbaI (ATCGAT ACTAGT TCTAGA) were introduced by polymerase
chain reaction immediately after the last codon of the glycoprotein
domain (WT, G57S, A(
1)T, and
E47) or the 66th codon of the NP
domain (C67X), and the resulting vectors were digested with
ClaI and XbaI. Annealed oligonucleotides containing the Myc-His tag or the influenza hemagglutinin (HA) tag,
along with sequence overhangs for the ClaI and
XbaI sites, were ligated into the same restriction sites in
the plasmid vectors (Fig. 1B). The Myc-His tag contains the
c-Myc epitope (EQKLISEEDL), the intervening amino acid sequence
(NSAVD), and polyhistidine sequence (His6) from the
pcDNA3.1MycHis expression vector (Invitrogen, San Diego, CA). The
His6 sequence is added to allow protein purification. The
amino acid sequence of the HA tag is YPYDVPDYA. After polymerase chain
reaction and subcloning, the entire cDNA sequence was verified by
the dideoxy-mediated chain termination method (26). All the cDNAs
were introduced into the pRc/RSV vector (Invitrogen). The vector
without a cDNA insert was used as a control in some
experiments.

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Fig. 1.
Strategy for epitope tagging of the AVP
precursors. A, the structure of the AVP precursors is
shown along with the four AVP mutants analyzed in this study. Mutations
include a substitution of Ser for Gly at amino acid 57 (G57S), a
replacement of Ala at the carboxyl terminus of the signal peptide
(position 1) with Thr (A( 1)T), a deletion of Glu at position 47 ( E47), and a premature termination at position 67 (C67X)
in the NP domain. SP, signal peptide; VP,
vasopressin; GP, glycoprotein. B, restriction
sites for ClaI and XbaI were introduced
immediately after the last codon of the WT and mutant AVP precursors.
DNA cassettes encoding epitopes were ligated into the ClaI
and XbaI sites, giving rise to expression vectors for
epitope-tagged WT and mutant AVP precursors.
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Cell Culture and Transfection--
Human embryonic kidney tsa
201 cells (27) were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum in a 5% CO2
atmosphere at 37 °C. Cells were transfected by the calcium phosphate
method as described previously (28). In precursor interaction assays
(see below), cells were treated with 10 µg/ml brefeldin A (BFA)
(Sigma) for 12 h to inhibit protein transport from the endoplasmic
reticulum (ER) to the Golgi apparatus (29).
Metabolic Labeling and Immunoprecipitation--
Continuous
metabolic labeling and immunoprecipitation were performed as described
previously (23). Briefly, transiently transfected cells were labeled
for 12 h in Dulbecco's modified Eagle's medium containing 100 µCi of Expre35S35S protein labeling mixture
(DuPont). Cell extracts and culture medium were subjected to
immunoprecipitation using polyclonal anti-AVP or anti-NP antibodies
(ICN, Costa Mesa, CA). Immunoprecipitates were separated by 16.5%
SDS-polyacrylamide gel electrophoresis (PAGE) followed by
autoradiography. For pulse-chase analyses, transfected cells were
labeled for 15 min with 100 µCi of
Expre35S35S protein labeling mixture in 0.5 ml
of methionine- and cysteine-free Dulbecco's modified Eagle's medium
and chased using 1 ml of complete Dulbecco's modified Eagle's medium
containing 100 µg/ml cycloheximide (Sigma). Cycloheximide was added
to completely inhibit further synthesis of labeled precursors after the
pulse labeling. In some experiments, immunoprecipitates were treated
with endoglycosidase H (Endo H) (New England Biolabs Inc., Beverly, MA)
according to the instructions of the manufacturer. Densitometric
analyses were performed using a GS-700 imaging densitometer (Bio-Rad).
Quantitative analyses were performed with film exposures in the linear range.
Precursor Interaction Assays--
Cells transfected with
expression vectors for precursors, with or without the Myc-His epitope
tag, were labeled for 12 h in the presence of 10 µg/ml BFA.
After labeling, cells were lysed in buffer A (20 mM Tris
(pH 8.0), 150 mM NaCl, 1% Triton X-100, 5 mM
imidazole, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A). Cell
extracts were incubated with the Talon metal affinity resin
(CLONTECH, Palo Alto, CA) in the presence of 10 mM imidazole for 30 min at 4 °C with gentle rocking.
After extensive washing in buffer A containing 15 mM
imidazole, bound proteins were eluted from the resin by boiling in
SDS-PAGE sample buffer containing dithiothreitol and subjected to
16.5% SDS-PAGE followed by autoradiography. For the in
vitro precursor interaction assay, WT precursors with and without the Myc-His tag were translated in vitro using the TNT
reticulocyte lysate system (Promega, Madison, WI) in the presence of
[35S]methionine (DuPont) and canine microsomal membranes
(Promega). The [35S]methionine-labeled proteins were
subjected to the binding reaction with metal affinity resin as
described above. Extensive washing and the inclusion of imidazole
throughout the assay are necessary to reduce nonspecific protein
interactions. Under these conditions, <10% of the total input protein
is typically bound to the affinity resin (30).
In Vivo Cross-linking and Western Blot Analysis--
A
non-cleavable cross-linker, disuccinimidyl suberate (DSS) (Pierce), was
dissolved in dimethyl sulfoxide at a concentration of 100 mM. For the detection of homodimers, cells expressing
precursors with the HA tag were collected in phosphate-buffered saline
and then incubated in either phosphate-buffered saline containing Me2SO (1:100 dilution) or phosphate-buffered saline
containing 1 mM DSS for 30 min at room temperature.
Immediately after performing the cross-linking reaction, cells were
lysed in a buffer containing 20 mM Hepes (pH 7.9), 420 mM NaCl, 20% glycerol, 1.5 mM
MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. Whole cell extracts were subjected to 15% reducing SDS-PAGE followed
by electrotransfer to polyvinylidene difluoride membranes (Boehringer
Mannheim). Membranes were probed with horseradish peroxidase-conjugated
anti-HA antibody (Boehringer Mannheim) according to the instructions of
the manufacturer. Subsequently, proteins were detected using the
enhanced chemiluminescence detection system (Boehringer Mannheim). For
the detection of heterodimers as well as homodimers, cells expressing
precursors with the Myc-His tag and those with the HA tag were
subjected to an in vivo cross-linking reaction as described
above. After the reaction, cells were lysed in buffer A, and cell
extracts were incubated with the metal affinity resin as described
above for the precursor interaction assay. Bound proteins were eluted
from the resin and separated by 15% SDS-PAGE under reducing
conditions. After Western blot transfer, membranes were probed with the
anti-HA antibody.
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RESULTS |
Processing of WT AVP Precursors--
Human embryonic kidney tsa
201 cells were transiently transfected with an empty vector or with
expression vectors for WT AVP precursors with or without the Myc-His
epitope tag to allow studies of precursor expression and processing.
After continuous metabolic labeling, cell extracts and medium were
harvested and subjected to immunoprecipitation. A 14-kDa protein (Fig.
2A, lanes 3,
5, 9, and 11) was detected in cells
transfected with an empty vector (lanes 1 and 7),
suggesting that this band is nonspecific. Using the anti-NP antibody, a
similar series of precursors were identified, independent of the
presence of the Myc-His epitope tag. The WT AVP precursor was 21 kDa
(lane 3), whereas the intracellular precursor containing the
Myc-His epitope tag was 25 kDa (lane 5). The expression levels of the 21- and 25-kDa precursors for the WT and Myc-His-tagged AVP precursors were similar. The intracellular precursors consisted of
doublets (lanes 3 and 5), presumably reflecting
different states of glycosylation. In the medium, the 22-kDa (WT)
(lane 4) and 26-kDa (Myc-His) (lane 6) precursors
as well as a 12-kDa protein were detected. The sizes of the precursors
in the medium were increased relative to those in the cell extracts,
reflecting the addition of carbohydrate moieties prior to secretion
(Fig. 2B). Each of the precursor forms and the 12-kDa
protein were also immunoprecipitated with the anti-AVP antibody (Fig.
2A, lanes 9-12), indicating that they contain
both the AVP and NP domains. The 12-kDa protein corresponds to the
intermediate form observed in neuro2A cells (Fig. 2B) (23). These experiments demonstrate that the sizes of the processed AVP
precursor products in this cell line and indicate that the presence of
the Myc-His epitope tag does not alter the level or the efficiency of
precursor processing or secretion into the medium.

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Fig. 2.
Processing of WT AVP precursors.
A, proteins produced in transiently transfected tsa 201 cells were continuously labeled with [35S]methionine and
cysteine. Cell extracts (C) and media (M) were
subjected to immunoprecipitation using either anti-NP or anti-AVP
antibodies, and the proteins were analyzed by 16.5% SDS-PAGE and
autoradiography. A 14-kDa nonspecific band is denoted by an
asterisk. Molecular mass markers are indicated to the left.
Bands corresponding to WT or Myc-His-tagged precursors are indicated by
arrows. B, shown is a schematic representation of
post-translational processing and intracellular trafficking of AVP
precursors in tsa 201 cells. Prepro-AVP is converted to pro-AVP by the
removal of the signal peptide and by the addition of carbohydrate
within the ER. Pro-AVP that is partially glycosylated within the ER (21 kDa) is glycosylated further within the Golgi apparatus (shown by the
underline). Most of the terminally glycosylated pro-AVP (22 kDa) is constitutively exported into the medium. A small fraction of
the glycosylated precursors undergo proteolytic processing, yielding
the intermediate form, consisting of the AVP and NP domains (12 kDa).
Inefficient cleavage of the A( 1)T precursor by signal peptidase
results in a 23-kDa aberrant precursor that is glycosylated, but is not
cleaved by signal peptidase. SP, signal peptide;
V, AVP; GP, glycoprotein.
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Digestion of the immunoprecipitated proteins with Endo H (Fig.
3A) demonstrated that the WT
intracellular precursors (21 and 25 kDa) (lanes 1 and
11) were sensitive to Endo H, yielding 17- and 21-kDa
digested products (lanes 21 and 31). In contrast,
the products in the medium (lanes 2 and 12) were
resistant to Endo H digestion (lanes 22 and 32).
The Endo H resistance of the secreted precursors is consistent with
their increased molecular size, likely reflecting glycosylation within
the Golgi apparatus. Little or no Endo H-resistant precursors were
detected within cells (lanes 21 and 31),
suggesting that AVP precursors are rapidly exported into the medium or
undergo further processing once they are glycosylated within the Golgi
apparatus (Fig. 2B).

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Fig. 3.
Metabolic labeling of cells expressing WT or
mutant AVP precursors. A, cells expressing WT or mutant
precursors with or without the Myc-His tag were continuously labeled
for 12 h. Cell extracts (C) and media (M)
were subjected to immunoprecipitation using the anti-NP antibody.
Immunoprecipitates were treated with or without Endo H to remove
carbohydrates. Samples were separated by 16.5% SDS-PAGE followed by
autoradiography. The band corresponding to the C67X
precursors with the Myc-His tag (14 kDa) overlaps a nonspecific band
(*). Molecular mass markers are indicated, and bands corresponding to
WT or Myc-His-tagged precursors are shown by arrows.
B, cells expressing WT or mutant precursors were labeled for
15 min and chased for the indicated times. Cell extracts and media were
subjected to immunoprecipitation using the anti-NP antibody. The
left panel shows data from films with exposures in the
linear range. The amount of precursors in cells and the medium 1 h
after the pulse labeling is expressed as a percentage of the total
radiolabeled precursors present after the labeling. The right
panel shows data from films with a longer exposure. The
asterisks indicate a 14-kDa nonspecific band.
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Post-translational Processing and Intracellular Trafficking of
Mutant AVP Precursors--
The expression and processing of the G57S,
A(
1)T,
E47, and C67X mutant AVP precursors were
examined in parallel with the WT precursors described above. Under
conditions of continuous labeling (Fig. 3A), the mutant AVP
precursors with and without the Myc-His tag were readily detected
within cells (lanes 1, 3, 5,
7, and 9 and lanes 11, 13,
15, 17, and 19). In fact, in most cases, the level of mutant precursor expression was greater than that
of WT precursor expression (lanes 1 and 11). The
migration of the G57S precursors (lanes 3 and 13)
was indistinguishable from that of the WT precursors, whereas the
A(
1)T (lanes 5 and 15) and
E47 (lanes
7 and 17) precursors migrated differently. The slower
migration of the A(
1)T mutant likely reflects the formation of
aberrant precursors that are glycosylated, but not cleaved by signal
peptidase (Fig. 2B) (4, 23). The slightly faster migration
of the
E47 mutant may reflect the deletion of a single amino acid.
The C67X mutant, with and without the Myc-His tag (14 and 10 kDa) (Fig. 3A, lanes 9 and 19), was
also expressed well within cells. In the medium, 22- and 26-kDa
precursors as well as 12-kDa intermediate forms were detected for the
WT, G57S, and A(
1)T mutants (lanes 2, 4, and
6 and lanes 12, 14, and
16). The sizes of the secreted WT and A(
1)T mutant
precursors in the medium were the same because their products are
identical once the signal peptide is removed. The amount of the G57S
and A(
1)T proteins detected in the medium was reduced compared with
that of the WT protein. Little or no precursors were detectable in the
medium for the
47E and C67X mutants (lanes 8 and 10 and lanes 18 and 20).
The effects of Endo H treatment were similar for the mutant and WT
precursors (Fig. 3A). Digestion of intracellular G57S, A(
1)T, and
E47 AVP precursor proteins with and without the Myc-His tag (lanes 3, 5, and 7 and lanes
13, 15, and 17) gave rise to 17- and 21-kDa
proteins (lanes 23, 25, and 27 and
lanes 33, 35, and 37). The G57S and
A(
1)T precursors in the medium (lanes 4 and 6 and lanes 14 and 16) were resistant to Endo H
digestion (lanes 24 and 26 and lanes
34 and 36). No Endo H-resistant precursors were
detected within the cells, indicating that like the WT precursors, most
of the mutant precursors are readily secreted into the medium after
glycosylation within the Golgi apparatus. As expected, Endo H had no
effect on the C67X mutant precursors (lanes 9 and
19 and lanes 29 and 39).
Pulse-chase analyses were performed to further evaluate the kinetics of
precursor processing and secretion. AVP precursors recovered in cells
and in the medium 1 h after the pulse labeling were normalized to
the amount of labeled products present after labeling (Fig.
3B, left panel). The recovery of WT precursors in
the medium was 31%, whereas that of the mutants ranged between 0 and
7%, indicating reduced secretion of mutant precursors. The total
recovery of WT precursors in cells and the medium was 71%, whereas the
recovery of the C67X mutant precursor was only 35%. In the
case of other mutant precursors, the recovery was between 50 and 64%.
These results suggest that intracellular degradation is involved in the
inefficient secretion of mutant precursors.
Pulse-chase analyses also revealed that mutant precursors were still
retained within cells 8 h after pulse labeling (Fig. 3B, right panel). By comparison, most of the WT
precursors were secreted into the medium by 8 h, indicating that
the mutant AVP precursors are secreted into the medium less efficiently
than the WT precursors. Because precursors appear to be rapidly
secreted into the medium after glycosylation (Fig. 3A),
these findings suggest that the G57S, A(
1)T, and
E47 mutant
precursors are not transported from the ER to the Golgi apparatus as
effectively as the WT precursors. The levels of the C67X
mutant were relatively low due to intracellular degradation (see
above), but it, too, appears to be retained within the intracellular
pool, consistent with previous studies in which ER retention of the
C67X precursors was demonstrated using immunofluorescence
staining in neuro2A cells (23). Taken together, these results show that
both intracellular degradation and retention are responsible for the
reduced secretion of mutant precursors.
Physical Interaction of AVP Precursors--
AVP precursors were
previously shown to interact with each other in vitro using
synthetic peptides and immobilized NP (25). We used a protein
"pull-down" assay to assess interactions among the precursor
proteins. Cells were treated with BFA, which blocks protein transport
from the ER to the Golgi apparatus, to allow a similar degree of
retention of both mutant and WT AVP precursors. Treatment of cells with
BFA completely eliminated the release of precursors and intermediate
forms into the medium, resulting in a comparable degree of WT and
mutant precursor expression (data not shown). Cells expressing
precursors with and without the Myc-His tag were labeled in the
presence of BFA prior to lysis. Cell extracts were incubated with metal
affinity resin to bind AVP precursors containing the Myc-His tag to the
resin. After extensive washing, bound proteins were separated on 16.5%
SDS-polyacrylamide gels followed by autoradiography (Fig.
4A). AVP precursors with the Myc-His tag were retained by the metal affinity resin (lane
1), but those without the Myc-His tag were not retained
(lane 2), demonstrating a specific interaction of the
His6-tagged precursors with the resin.

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Fig. 4.
Detection of AVP precursor interactions using
a metal affinity resin. A, cells transfected with
expression vectors for precursors with or without the Myc-His tag were
continuously labeled with [35S]methionine and cysteine.
After labeling, cell extracts were incubated with the metal affinity
resin. Bound proteins were separated by 16.5% SDS-PAGE followed by
autoradiography. B, WT precursors with or without the
Myc-His tag were synthesized by in vitro translation
in the presence of microsomal membranes. The labeled precursors (5 µl) were subjected to the interaction assay as described under
"Experimental Procedures."
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WT and mutant precursors with the Myc-His tag interacted with their
respective precursors without the tag (Fig. 4A, lanes 3-7), as reflected by the fact that the precursors without the Myc-His tag were also retained by the affinity column. These results suggest that WT and mutant precursors form homodimers. WT precursors with the Myc-His tag also interacted with the G57S, A(
1)T,
E47, and C67X mutant precursors (lanes 8-11). And in
the reverse format, the G57S, A(
1)T,
E47, and C67X
mutant precursors with the Myc-His tag interacted with the WT
precursors (lanes 12-15). These results indicate that the
WT and mutant precursors form both homo- and heterodimers.
Precursor proteins were also labeled during in vitro
translation to provide an estimate of the fraction of input proteins that interact with the metal affinity resin in this assay (Fig. 4B). WT precursors with and without the Myc-His tag were
translated in vitro in the presence of microsomal membranes
to produce prohormones (4). WT precursors with the Myc-His tag (25 kDa)
(lane 2) interacted with metal affinity resin (lane
4). WT precursors (21 kDa) (lane 1) without the tag did
not interact with the resin (lane 3), but were retained in
the presence of WT precursors with the Myc-His tag (lane 5).
In comparison with the input proteins (lanes 2 and 4), ~8% of the WT precursors with the Myc-His tag
were bound, which is similar to the interactions of previously
characterized protein dimers in this type of assay (30). Approximately
7% of the total input of WT precursors without the tag was associated with WT precursors with the Myc-His tag (lanes 1 and 5), suggesting that the majority of WT precursors
without the tag interact with WT precursors with the epitope tag.
Homodimerization of WT and Mutant AVP
Precursors--
Cross-linking studies were used to further assess the
formation of AVP precursor homodimers within the cellular environment. WT or mutant precursors with the HA tag were expressed, and the cells
were subjected to an in vivo cross-linking reaction with DSS
prior to lysis. After Western blot transfer, membranes were probed with
an anti-HA antibody (Fig. 5). The
monomeric form of the WT, G57S, A(
1)T, and
E47 precursors was 23 kDa, whereas the C67X monomer was 12 kDa. In the absence of
DSS treatment, homodimerization of the G57S and
E47 precursors (46 kDa) was detected (lanes 3 and 7), but
little or no homodimerization was seen with the WT, A(
1)T, and
C67X (24 kDa) precursors (lanes 1,
5, and 9). After treatment with DSS, all of the
WT and mutant precursors formed homodimers (lanes
2, 4, 6, 8, and
10). The ratio of dimer to monomer for the C67X
mutant was greater than that for the WT, G57S, A(
1)T, and
E47
precursors.

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Fig. 5.
Evidence for homodimer formation of WT and
mutant precursors using protein cross-linking. Cells expressing WT
or mutant precursors with the HA tag were incubated in the presence or
absence of 1 mM DSS for 30 min. After the cross-linking
reaction, whole cell extracts were prepared and loaded onto 15%
SDS-polyacrylamide gels. After Western blot transfer, polyvinylidene
difluoride membranes were probed with the anti-HA antibody. The
asterisk indicates a nonspecific band.
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Heterodimerization of WT and Mutant AVP Precursors--
Cells
coexpressing WT and mutant precursors with the HA tag and the Myc-His
tag were subjected to the cross-linking reaction with DSS prior to
lysis. Cell extracts were prepared and incubated with the metal
affinity resin to isolate Myc-His-tagged complexes. After washing,
bound proteins were subjected to Western blot transfer, and the
membranes were probed with the anti-HA antibody (Fig. 6). As a control, WT precursors with the
Myc-His tag were not recognized by the anti-HA antibody (lane
1). WT precursors with the HA tag were not detected in the absence
of precursors with the Myc-His tag (lane 2), indicating that
the precursors with the HA tag were not retained by the affinity resin.
However, WT precursors with the HA tag were detected when coexpressed
with WT precursors containing the Myc-His tag (lane 3). The
detection of 48-kDa proteins by the anti-HA antibody indicates the
formation of mixed complexes consisting of WT precursors with the
Myc-His tag (25 kDa) and those with the HA tag (23 kDa) (lane
3). Similarly, homodimerization of G57S, A(
1)T, and
E47
precursors was demonstrated by the detection of complexes consisting of
the Myc-His- and HA-tagged precursors (lanes
4-6). Little or no homodimerization was seen with the
C67X precursors (lane 7).

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Fig. 6.
Evidence for heterodimer formation between WT
and mutant AVP precursors. Cells expressing precursors containing
the Myc-His tag and those containing the HA tag were subjected to the
cross-linking reaction in vivo. After the reaction, cell
extracts were prepared and incubated with the metal affinity resin.
Bound proteins were eluted from the resin and separated by 15%
SDS-PAGE. After Western blot transfer, membranes were probed with the
anti-HA antibody.
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WT precursors with the Myc-His tag also formed heterodimers with the
G57S, A(
1)T, and
E47 precursors (Fig. 6, lanes
8-10). Similarly, the G57S, A(
1)T, and
E47 precursors
heterodimerized with the WT precursors (lanes
12-14). Heterodimerization between the WT and the G57S and
E47 mutant precursors was prominent, whereas heterodimer formation
between the WT and A(
1)T precursors was relatively weak. Little or no
heterodimerization was detected between the WT and C67X
precursors (lanes 11 and 15). Most of the monomeric forms of the HA-tagged precursors are likely derived from
a physical interaction with precursors with the Myc-His tag during the
incubation with metal affinity resin. Thus, the physical interaction
between monomeric form of precursors detected in this experiment is
similar to that detected in the precursor interaction assay (Fig.
4A).
Dominant-negative Effect of Mutant AVP Precursors--
Because
mutant precursors are not efficiently exported into the medium, it is
possible that they might also alter the transport and processing of the
WT AVP precursors. WT or mutant precursors with the Myc-His tag were
coexpressed in cells with WT precursors that did not contain an epitope
tag. After continuous labeling, cell extracts and medium were
immunoprecipitated using the anti-NP antibody (Fig.
7). Consistent with previous findings
(Fig. 3A), the amount of secreted G57S, A(
1)T,
E47, and
C67X mutant precursors with the Myc-His tag was reduced
compared with that of the WT precursors with the Myc-His tag,
suggesting retention of mutant precursors (Fig. 7A). The
ratios of secreted to intracellular precursors were 0.23, 0.37, 0.14, and 0.06 for the G57S, A(
1)T,
E47, and C67X precursors,
respectively. By comparison, the ratio for the WT precursors was 1.38 (Fig. 7B). When WT precursors with the Myc-His tag were
coexpressed (Fig. 7C), WT precursors without an epitope tag
were effectively exported into the medium (ratio = 1.30). However,
when coexpressed with the G57S, A(
1)T,
E47, or C67X
mutant precursor containing the Myc-His tag, more WT precursors were
detected within cells, and secretion of the WT precursors was
decreased, resulting in ratios of 0.40, 0.73, 0.48, and 0.85, respectively. The retention of intracellular WT precursors was more
prominent with coexpression of the G57S, A(
1)T, and
E47 mutants.
When the C67X precursors with the Myc-His tag were
coexpressed, the amount of intracellular WT precursors and secreted
precursors was decreased. Taking into account the increased degradation
of the C67X precursors (Fig. 3B), it is possible
that WT precursors retained within the ER through heterodimer formation
with the C67X precursors were degraded along with the mutant
precursors.

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Fig. 7.
Mutant precursors impair the transport and
processing of WT AVP precursors. A, cells expressing WT
or mutant precursors containing the Myc-His tag and WT precursors
without the epitope tag were continuously labeled. Cell extracts
(C) and media (M) were immunoprecipitated using
anti-NP antibodies followed by 16.5% SDS-PAGE and autoradiography. The
band corresponding to the C67X precursors with the Myc-His
tag (14 kDa) overlaps a nonspecific band (*). B, the ratio
of secreted precursors containing the Myc-His tag to intracellular
precursors was determined using densitometric analyses. C,
the ratio of secreted to intracellular WT precursors is shown. The S.E.
for different samples in B and C ranged from 0.01 to 0.04.
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DISCUSSION |
The structure-function relationship of the NP molecules has been
intensively studied (for review, see Ref. 31). NP consists of two
highly homologous domains that arose from a partial gene duplication
(Fig. 4B) (32). These structural features underlie the
propensity of NP molecules to self-associate (33) and form larger
aggregates under acidic pH (34). The fully processed hormone AVP binds
to NP (35, 36) and induces a greater degree of self-association (37).
NMR analysis of crystallized NP molecules revealed the residues
involved in NP binding to AVP and indicated that the interface for the
monomer-monomer interaction (codons 32-37 and 77-82) forms a
-sheet structure (38). Using a semisynthetic precursor and
immobilized NP, AVP precursors have been shown to exhibit
characteristics similar to those of NP molecules (25, 39). The
precursors self-associate through the NP domains, and the
intramolecular interaction between the AVP and NP domains enhances
self-association, suggesting that higher order aggregates of precursors
may also be formed. These previous in vitro studies suggested the possibility that mutant AVP precursors might interact with WT precursors and affect their function in vivo.
To study the interactions among different AVP precursors in
vivo, it is necessary to be able to distinguish them from one another. For this reason, we attached two different epitopes to the
precursors. The epitopes were added immediately after the last codon of
the WT and mutant precursors to minimize the potential effects of
epitope tagging on precursor function (Fig. 1B). In addition
to its other features, the Myc-His epitope is large enough that
epitope-tagged precursors can be distinguished from non-epitope-tagged precursors because of differences in molecular mass (~3 kDa greater). The observed migration of precursors without the tag was 21 kDa versus 25 kDa with the Myc-His tag. The slightly slower
migration of the Myc-His-tagged precursors may be caused by the
presence of the unusual stretch of polyhistidine codons. Since
post-translational processing and intracellular trafficking of
precursors with the Myc-His tag were similar to those of precursors
without the epitope tag (Figs. 2A and 3A), the
addition of epitope to the carboxyl-terminal end of the AVP precursors
does not appear to affect the processing or transport of the
precursors. The Myc-His tag also allows selective isolation of AVP
precursors using the metal affinity resin. Precursors with the Myc-His
tag were shown to interact specifically with the resin, whereas
precursors without the tag and those with the HA tag did not bind to
the resin (Figs. 4A and 6). The other epitope, HA, was added
to distinguish precursors using an anti-HA antibody. Precursors without
the HA epitope tag and those with the Myc-His tag were not recognized
by the anti-HA antibody (data not shown). Taken together, these two
strategies for epitope tagging of the AVP precursors provide useful
tools for examining their interactions with each other and potentially
other cellular proteins.
The precursor interaction assay revealed an effective physical
association of AVP precursors (Fig. 4A). In this assay,
dimers formed during the incubation with the metal affinity resin as well as those formed within the ER were detected. Thus, evidence of a
physical interaction in this assay does not necessarily imply that
dimerization occurred within the ER. For this reason, we also used an
in vivo cross-linking reaction that was performed prior to
cell lysis to examine intracellular dimerization of precursors. The use
of a non-cleavable cross-linker prevents the dissociation of dimers
after cell lysis. For the detection of homodimers, cells were incubated
in the absence or presence of cross-linker prior to lysis (Fig. 5).
When treated with the cross-linker, apparently all of the WT and mutant
precursors formed homodimers. In the absence of the cross-linker,
little or no homodimers were detected for the WT, A(
1)T, and
C67X precursors. In contrast, the G57S and
E47 precursors
readily formed homodimers. It is notable that the G57S and
E47
homodimers were detected by reducing SDS-PAGE, suggesting that these
homodimers are tightly bound. In the subsequent experiments in which
the in vivo cross-linking and affinity reactions were
combined (Fig. 6), homodimerization of the G57S and
E47 precursors
was confirmed to be relatively strong in comparison with the other
precursors (G57S >
E47 = A(
1)T > WT
C67X). In the same experiment (Fig. 6), heterodimerization
between WT and mutant precursors was analyzed, and the G57S and
E47
mutant precursors were also found to form heterodimers with WT
precursors more effectively than the A(
1)T and C67X
precursors. Most of the A(
1)T precursors detected within the cells
are likely to be aberrant precursors anchored to the ER membranes via
the signal peptide (Fig. 2B). This may explain the reduced
detection of homodimers as well as a smaller amount of heterodimers
with WT precursors (Figs. 5 and 6). The C67X precursors lack
5 out of 14 cysteine residues within the NP domain, but still contain a
single
-sheet structure, which may be involved in the
monomer-monomer interaction. Some degree of C67X
homodimerization was detected in some paradigms (Fig. 5), but not in
others (Fig. 6). In addition, heterodimerization with WT precursors was
not detectable (Fig. 6). The extent of dimerization appears to differ
in the two different assays. However, the intracellular cross-linking
assay using the metal affinity resin (Fig. 6) is probably less
sensitive than the other intracellular cross-linking assay (Fig. 5).
Based on data using the precursor interaction assay (Fig. 4), it is
likely that the C67X precursors heterodimerize, to some
extent, with WT precursors. The lack of the second
-sheet structure
in the C67X mutant may account for reduced homo- and
heterodimer formation in comparison with WT or other mutant precursors.
In summary, these data show that WT and mutant precursors form
homodimers and that mutant precursors can heterodimerize with WT
precursors within the ER.
Coexpression of WT and mutant precursors revealed impaired transport of
WT precursors from the ER to the Golgi apparatus in the presence of the
mutant precursors (Fig. 7). This effect was particularly prominent when
the G57S, A(
1)T, and
E47 precursors were coexpressed with the WT
protein. The C67X precursors had less effect on the
transport of WT precursors. We propose that this dominant-negative
effect is mediated through heterodimer formation of WT precursors with
mutant precursors that are retained within the ER. The observation that
the C67X precursors exhibit less pronounced
dominant-negative activity correlates with the relative lack of
heterodimerization between the WT and C67X mutant precursors
(Fig. 6).
We have previously shown that mutant AVP precursors may induce neuronal
cell death as a result of their accumulation within the ER (23).
Although much of the mutant precursor protein that is accumulated
within the ER may be degraded, part may form aggregates over time,
eventually perturbing cellular function and leading to cell death (Fig.
8). In this study, we provide evidence
that mutant AVP precursors can form heterodimers with WT precursors and
impair the production of AVP from the normal allele. This dominant-negative effect of mutant precursors on the transport of WT
precursors from the ER to the Golgi apparatus may account, in part, for
the autosomal dominant mode of inheritance, but it cannot explain the
delayed onset of the disease. It is therefore more likely that the
cytotoxicity of the mutant precursors is the primary cause of the
disease. The formation of heterodimers between WT and mutant precursors
may also contribute to the cytotoxicity. It is notable that among the
many naturally occurring mutations in the AVP gene, there are no
missense mutations within the
-sheet structures that are involved in
the monomer-monomer interaction. Also, all nonsense mutations reported
to date are located within the carboxyl-terminal region of the NP
domain (C61X, C67X, C79X, E81X, P83X, and E87X). The fact that
the C67X precursors are functional in terms of dimer
formation makes it less likely that mispaired disulfide bonds are
formed in the truncated precursors. Rather, the truncated precursors
may retain the first
-sheet structure. In this study, each of the
four representative mutant precursors, G57S, A(
1)T,
E47, and
C67X, formed dimers with WT precursors. It is likely, but
will require further study, that all the mutant precursors found in
FNDI may form heterodimers with WT precursors. The cytotoxicity that
was observed in a previous study (23) varied among the different
mutants (C67X > A(
1)T > G57S >
E47). On the other hand, the rank order of heterodimer formation determined in this study was the inverse (
E47 = G57S > A(
1)T
C67X). For example, the
E47 mutant, which showed the
least cytotoxic effect among the mutants, interacted with WT precursors
the best, whereas the C67X mutant, which exhibited the
greatest degree of cytotoxicity, formed heterodimers with WT precursors
least effectively. It is likely that both dominant-negative activity
and cytotoxicity are involved in the pathogenesis of FNDI. Because
mutant-WT dimers may also be cytotoxic, these mechanisms are not
mutually exclusive.

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Fig. 8.
Proposed model for the molecular basis of
FNDI. Functional and physical interactions occur between WT and
mutant precursors that are retained within the ER. The mutant
precursors impair the transport and processing of the WT precursors.
The accumulation and aggregation of mutant-mutant and mutant-WT
precursors lead to cellular toxicity (23). Thus, the pathogenesis of
FNDI may involve direct cellular toxicity of the mutant AVP precursors
as well as a dominant-negative effect of the mutant precursors on the
transport and processing of the WT gene product.
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