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J Biol Chem, Vol. 274, Issue 33, 23617-23626, August 13, 1999
From the Department of Pharmacology, University of Minnesota
Medical School, Minneapolis, Minnesota 55455
Three major types of opioid receptors, µ (MOR),
Opioids induce pharmacological as well as other physiological and
cellular effects via opioid receptors
(ORs)1 (1). Three major types
of opioid receptors have been identified and cloned, namely the µ (MOR), In general, the localization of the ORs coincides with the
pharmacological actions of the opioids (4). In the case of DOR, there
is also a good correlation between the presence of DOR mRNA and
We previously reported isolation of mouse genomic clones of DOR, from
which we determined two major transcription initiation sites, the
exon-intron structure and 1.3-kb 5'-flanking sequence (14). The
isolation and partial analysis of mouse genomic DNA of DOR has made it
possible to study regulatory elements of the DOR gene. A 1.3-kb DNA
fragment upstream from the translation start site ( Here, we report a functional analysis of the DOR promoter region in
NS20Y, a mouse neuroblastoma cell line that constitutively expresses
DOR. Using in vivo functional assays and in vitro
protein-DNA binding assays, we have defined a minimal DOR promoter. We
also demonstrate that the functional necessity of a putative Sp1
binding site as well as an E box for the transcription activation of
DOR. We show that the E box and GC box, as well as the simultaneous binding and functional synergy between their associated factors, are
crucial for the promoter activity of the DOR gene.
Plasmid Construction--
Luciferase fusion plasmids were
constructed containing 1300 bp of upstream regulatory sequence (pD1300
construct; Cell Culture--
Mouse neuroblastoma NS20Y cells were grown in
DMEM medium with 10% heat-inactivated fetal calf serum in an
atmosphere of 10% CO2 and 90% air at 37 °C.
Schneider's Drosophila line 2 (SL2) cells were grown at
22-24 °C in Schneider's Drosophila medium (Life
Technologies, Inc.) containing 10% heat-inactivated fetal calf serum.
Transient Transfection and Reporter Gene Activity
Assay--
NS20Y cells were transfected using the DOTAP (Roche
Molecular Biochemicals) lipofection method as described previously
(22). Briefly, cells at approximately 40% confluence were transfected with an equimolar amount of each test plasmid. Forty-eight hours after
transfection, cells grown to confluence were washed and lysed with
lysis buffer (Promega). To control for differences in transfection
efficiency from dish to dish, a one-fifth molar ratio of pCH110 plasmid
(Amersham Pharmacia Biotech) containing the Nuclear Extract Preparation--
Nuclear extracts were prepared
from NS20Y cells using the method described by Johnson et. al (25).
Briefly, cells were grown to confluence, harvested, and washed with
phosphate-buffered saline. All of the following steps were performed at
4 °C. The cells were resuspended in sucrose buffer (0.32 M sucrose, 3 mM CaCl2, 2 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl, pH 8.0, 1 mM dithiothreitol (DTT),
0.5 mM PMSF, and 0.5% Nonidet P-40). The lysate was
microcentrifuged at 500 × g for 5 min to pellet the
nuclei, which were washed with sucrose buffer without Nonidet P-40. The
nuclei were resuspended in low salt buffer (20 mM Hepes, pH
7.9, 25% glycerol, 0.02 M KCl, 1.5 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and
0.5 mM PMSF), followed by addition of high salt buffer to
extract the nuclei, with incubation for 20 min on a rotary platform.
Diluent (2.5 vol. of 25 mM Hepes, pH 7.6, 25% glycerol,
0.1 mM EDTA, 0.5 mM DTT, and 0.5 mM
PMSF) was added and the sample was microcentrifuged at 13,690 × g for 15 min. Aliquots of the supernatant (nuclear extract)
were stored at Electrophoretic Mobility Shift Assay (EMSA)--
EMSA was
performed with 32P-labeled double-stranded oligonucleotides
that were incubated with nuclear extract in EMSA buffer (10 mM Tris, pH 7.5, 5% glycerol, 1 mM EDTA, pH
7.1, 50 mM NaCl, 1 mM DTT, 1 mM
EDTA, and 0.1 mg/ml poly(dI-dC)). For oligonucleotide competition
analysis, a 100-fold (or as indicated in figures) molar excess of
competitor oligonucleotides was also added to the mixture. After
incubation at 22 °C for 30 min, the mixture was analyzed on 5%
nondenaturing polyacrylamide gels. For antibody supershift assays, 1 µl of monoclonal antibodies to Sp1, Sp2, Sp3, Sp4, USF1, USF2, or
c-Myc (Santa Cruz Biotechnology, Inc.) was added to the mixture. The
reaction was then incubated on ice for 1 h. Protein-DNA complexes
and free DNA were fractionated on 5% polyacrylamide gels in 1×
Tris-glycine EDTA buffer (50 mM Tris, pH 8.3, 380 mM glycine, and 2 mM EDTA) at 4 °C and were visualized by autoradiography.
Functional Analysis of Promoter Activity of the Mouse DOR
Gene--
In order to identify the functional DOR promoter and
elucidate the regulatory elements of the DOR gene, serial deletional analyses were performed with the 1.3-kb DNA fragment from upstream regulatory region of the DOR gene (Fig.
1A). A primary reporter construct and its serial deletion constructs were prepared from this
1.3-kb DNA fragment as described under "Materials and Methods." This primary construct, designated as pD1300, and its serial
5'-deletion constructs, designated as pD890, pD675, pD262, pD210,
pD182, and pD150, are illustrated in Fig. 1B. The pGL3-basic
plasmid (designated as basic), containing no promoter and no
enhancer, was included as a negative control, while the pGL3-control
plasmid (designated as control) containing the SV40 promoter
and SV40 enhancer was used as the positive control. The promoter
activity of each construct was tested by transient transfection assays
in NS20Y cells, a DOR-expressing neuronal cell line.
As shown in Fig. 1B, the luciferase reporter constructs with
different 5'-ends from A Minimum DOR Promoter Sequence Confers Promoter Activity in Both
Orientations but of Different Magnitude--
In addition to 5'-serial
deletional analysis of the DOR promoter, a 3'-serial deletional
analysis was also carried out to define the minimum core promoter of
the DOR gene. As shown in Fig. 2, a
construct containing a 3'-deletion of Identification of the GC Box and Its Associated Factors of the DOR
Promoter--
In order to localize the important cis
elements for regulation of the DOR promoter, a series of linker scan
mutations throughout the minimum DOR promoter region (
By comparing the sequences of the mutated region from the mutant
constructs exhibiting reduced promoter activities to the consensus
sequences of known transcription factors, two putative binding sites of
known transcription factors were identified from three mutant
constructs that showed more than 60% decrease in promoter activities
(Table I, pDm226, pDm190, and pDm184). One of these binding sites was a
GC box (shown in Fig. 3A,
underlined), which is the putative binding site for
transcription factors of the Sp1 family (19-21). Mutation of this GC
box (pDm226) resulted in about 65% decrease in promoter activity of
that of the wild type DOR promoter in NS20Y cells (Fig.
3A).
In order to identify the transcription factors bound to this GC box of
the DOR promoter, EMSAs were performed using nuclear extracts prepared
from NS20Y cells. The oligonucleotide D232/215, representing the DOR
promoter sequence from
At least four transcription factors, Sp1, Sp2, Sp3, and Sp4, are known
to belong to the Sp family (19-21). In order to identify those members
binding to the DOR GC box, immunosupershift assays were carried out
with Sp1, Sp2, Sp3, or Sp4 antibodies (Abs). As shown in Fig.
3C, the protein-DNA complex band c was retarded by anti-Sp1
Ab to the position of band b (Fig. 3C, lane
11, arrow b). The anti-Sp3 Ab could
recognize both bands d and e, shifting both bands to a higher position,
band a (Fig. 3C, lane 13,
broken arrow a). Furthermore, all
three bands, c, d, and e, could be converted to bands a and b in the
presence of both anti-Sp1 and anti-Sp3 Abs (Fig. 3C,
lane 15). It was of interest that the amount of
band b, the Sp1-D232/215 complex, recognized by anti-Sp1 Ab was more
abundant than that of band a, the Sp3-D232/215 complex detected by
anti-Sp3 Ab (Fig. 3C, lanes 11,
13, and 15, bands a and
b). However, none of the bands was recognized by either anti-Sp2 or anti-Sp4 Ab (Fig. 3C, lanes
12 and 14). Taken together, these results
demonstrate that the GC box of the DOR promoter is the binding site for
the Sp1 and Sp3 transcription factors. Mutation of this site resulted
in eliminating the binding of both Sp1 and Sp3 to the DOR promoter and
diminished the DOR promoter activity.
Sp1 Transcription Factor trans-Activates the DOR Promoter in
Vivo--
It has been reported that the Sp1 transcription factor plays
a critical role in the basal transcription of several genes with TATA-less promoters (16, 21). In order to demonstrate that the Sp1
factor activates the DOR promoter via direct interaction with the DOR
GC box in vivo, we performed cotransfection assays in
Drosophila SL2 cells, which do not express endogenous Sp1
protein (26). As shown in Fig. 4,
co-transfection assays using the Sp1 expression vector and pD262 DOR
promoter construct in Drosophila SL2 cells demonstrated that
Sp1 can specifically activate the promoter of the pD262 construct in a
dose-dependent manner. Mutation of the DOR GC box
(construct pDm226) diminished this trans-activation of the
DOR promoter by Sp1 (Fig. 4). Thus, we concluded that Sp1 not only
binds to the GC box of the DOR promoter in vitro but also in
this manner trans-activates the DOR promoter in
vivo. However, mutation of the GC box did not completely eliminate
DOR promoter activity in SL2 cells, which might be due to the binding of overexpressed Sp1 protein to unidentified low affinity sites within
the DOR promoter region.
Identification of the E Box and Its Associated Factors of the DOR
Promoter--
As noted earlier, two putative binding sites of known
transcription factors were identified in the DOR promoter region by linker scanning and sequence comparison. In addition to the GC box, a
second binding site was an E box (Fig.
5A, underlined), which is a putative binding site for the transcription factors of the
basic helix-loop-helix leucine zipper (bHLH/LZ) family (27). In NS20Y
cells, mutations of the E box in the DOR promoter region (pDm190,
pDm184) abolished more than 80% of the promoter activity exhibited by
the wild type construct pD262 (Fig. 5A). In contrast, a
promoter construct (pDm178) retained most of the promoter activity
while its E box was intact (Fig. 5A). Several members of the
bHLH/LZ family are able to bind to the sequence (-CACGTG-) of the E box
(26). Therefore, by using EMSA, we tested several members of this
family as candidates for binding to the promoter E box of DOR gene.
These included c-Myc (28-29), which plays a role in cell proliferation
and differentiation that could be relevant to the limited expression
pattern of DOR, as well as upstream stimulatory factor (USF), USF1 and
USF2, because of their ubiquitous expression.
As illustrated in Fig. 5B, an oligonucleotide (D198/169)
containing the E box and its flanking sequence of the DOR promoter was
used as the probe; and two consensus oligonucleotides, an E box and a
c-Myc binding sequence, were used as the unlabeled competitors. Results
show in Fig. 5C that nuclear proteins of NS20Y cells formed
two major protein-DNA complexes with D198/169 (lane
1, arrowheads a and b).
Formation of these two major protein-DNA complexes, bands a and b,
could be blocked by either the c-Myc (Fig. 5C,
lane 3) or the E box consensus oligonucleotide
(Fig. 5C, lane 5). The anti-USF1 Ab
shifted both bands a and b to higher positions (Fig. 5C,
lane 6, marked by broken
arrows on the left), while the anti-USF2 Ab also
retarded portions of these bands (Fig. 5C, lane
7, marked by arrows on the right).
However, the anti-c-Myc Ab did not recognize any of the protein-DNA
complexes formed (Fig. 5C, lane 4).
Thus, it appears that USF1 and USF2, but not c-Myc, were the nuclear
factors bound to the DOR E box in vitro. Moreover, it was
obvious that both of the protein-DNA complexes represented by bands a
and b contain USF1, because the anti-USF1 Ab could shift both bands to
higher positions (Fig. 5C, lane 6). In
contrast, only a portion of the protein-DNA complexes was recognized by anti-USF2 Ab (Fig. 5C, lane 7). Thus,
we conclude that in NS20Y cells, USF1 homodimers (Fig. 5C,
lane 7, band c) and
USF1/USF2 heterodimers, but not USF2 homodimers, were present and bound to the DOR E box.
USF Binding Contributes to the Promoter Activity of the DOR
Gene--
The data in Fig. 5A suggest that a functional E
box (
As shown in Fig. 6B, the probe D198/169 formed two distinct
protein-DNA complex bands with NS20Y nuclear proteins (lane
2, arrowheads a and b).
Formation of these bands could be blocked by the probe itself and by
Dm178 (Fig. 6B, lanes 3 and
6), but not by either Dm190 or Dm184 (Fig. 6B,
lanes 4 and 5). Moreover, when using
Dm190 and Dm184 as the probe, there was no specific complex formed
(Fig. 6B, lanes 7 and 8).
However, when Dm178 was used as the probe, it displayed USF binding
ability similar to that of the D198/169 (Fig. 6B,
lane 9). The intact E box binding ability of
oligonucleotide Dm178 was consistent with the strong promoter activity
of the construct pDm178 (Fig. 5A). In contrast, abolished E
box binding abilities of Dm190 and Dm184 were in agreement with
diminished promoter activities of pDm190 and pDm184, respectively (Fig.
5A). Thus, USF binding to the DOR E box reflects its
trans-activation effect on the promoter activity of the DOR gene.
USF2 trans-Activates the DOR Promoter in Vivo--
The data in
Fig. 5C indicate that USF2 could only be detected in the
form of heterodimers. Since the heterodimer of USF1/2 is most likely
acting as a transcription activator in vivo (30), this
suggests that USF2 may be critical for the DOR expression. In order to
evaluate the functional role of USF2 in DOR promoter activity in
vivo, co-transfection assays were carried out in NS20Y cells with
the promoter construct pD262 and the USF2 expression vector.
As shown in Fig. 7, the promoter activity
of pD262 was elevated more than 2-fold when it was co-transfected with
wild type USF2. As noted earlier, USF belongs to the bHLH/LZ family.
USF1 and USF2 are capable of binding to DNA in the form of either
homodimers or heterodimers with each other, through their leucine
zipper dimerization domain (31). The basic region (b) of these proteins provides their DNA binding affinity and specificity, with deletion of
this motif abolishing DNA binding. To evaluate the role of USF2 in DOR
promoter activity, further experiments were carried out with USF2 Functional Interaction of the DOR GC Box and E Box--
Our data
presented earlier demonstrate that both E box and GC box are important
for the DOR promoter activity and are located 40-90 bp upstream of one
major transcription initiation site (position
The results shown in Fig. 8 demonstrated
that simultaneous mutation of the GC box and E box (pD262Sp1*/E*)
almost completely abolished the DOR promoter activity of pD262,
reducing its luciferase activity to the same level as that of the empty
reporter gene vector (pGL3-basic). However, comparison of the
luciferase activities of the constructs pD262, pDm226 (Sp1*), pDm184
(E*), and pD262Sp1*/E* suggests a cooperative relationship between the
GC box and E box to the promoter activity. First, the promoter activity
of pD262Sp1*/E* (construct with both the E box and GC box mutated) is
arbitrarily set as 1-fold (Fig. 8, fold
activation). Then, a 20-fold activation is observed with
pD262 (construct with both the E box and GC box intact). When this is
compared with the 7.6-fold activation of pDm226 (intact E box and
mutated GC box) and 1.6-fold activation of pDm184 (intact GC box and
mutated E box), it is clear that the effect is substantially more than
additive. Thus, it suggests a functional synergy between the GC box and
E box for the DOR promoter activity. Therefore, both the GC box and E
box are required for the maximum promoter activity of the DOR gene,
although the E box obviously played the major role and contributed more
than the GC box did to the promoter activity.
Physical Interaction of USF and Sp1/Sp3 on the DOR
Promoter--
In addition to functional synergy, the proximity of the
DOR GC box and E box also implies the possible physical interaction between their associated factors. This was investigated by EMSA competition assay with NS20Y nuclear extracts. As shown in Fig. 9A, all three probes featured
the sequence from the same promoter region of the DOR gene (
As shown in Fig. 9B, nuclear factors and the probe
D232/169E* formed bands with the characteristics of Sp-DNA complexes
(Fig. 9B, lanes 1 and 2,
bands b and c), since formation of
these bands was blocked by the presence of Sp1 consensus
oligonucleotide. Likewise, the bands formed using the probe
D232/169Sp1* did not appear in the presence of E box consensus
oligonucleotide (Fig. 9B, lanes 3 and
4, band d). Thus, mutation of the E
box abolishing USF binding and mutation of the GC box blocking Sp1/Sp3
binding to the DOR promoter region were consistent with the data shown earlier. Interestingly, the probe D232/169, which contained both an
intact GC box and E box, formed an additional band of larger size (Fig.
9B, lane 6, band
a) than the bands corresponding to the Sp-D232/169 complexes
and USF-D232/169 complexes (Fig. 9B, lane
6, bands b-d). Formation of this band
could be abolished by oligonucleotides containing either the DOR E box,
D198/169, or the DOR GC box, D232/215 (Fig. 9B,
lanes 7 and 9) but not by the mutated
DOR promoter sequences, Dm184 and Dm226 (Fig. 9B, lane 8). Taken together, these results
demonstrate that binding of either USF or Sp1/Sp3 to the DOR promoter
sequence did not affect binding of the other factor. However, formation
of USF-Sp-D232/169 complexes indicates that simultaneous binding of USF
and Sp factors to the DOR promoter sequence was highly favored since
almost no binding of USF alone could be seen when both the GC box and E box were intact (Fig. 9B, lane 6,
band d). This suggests that a direct or
additional factor-mediated physical interaction between USF and Sp
factors occurred when both the E box and GC box were present in the DOR
promoter region. The mechanism of physical interaction between USF, Sp,
and/or additional factors on the DOR promoter needs to be further investigated.
It was also observed that much lesser amounts of USF-D232/169 complexes
were formed in the presence of USF-Sp physical interaction (Fig.
9B, lanes 6 and 8,
band d) than in the absence of this interaction (Fig. 9B, lane 9, band
d). In contrast, the amount of Sp-D232/169 complexes was
largely unaffected by the presence or absence of the USF-Sp physical
interaction (Fig. 9B, lanes 6-8,
bands b and c). It suggests the
presence of excess amount of Sp-D232/169 complexes and limited amount
of USF-D232/169 complexes in the EMSA competition assays. Thus, binding
of USF to the DOR promoter sequence is more critical than that of
Sp1/Sp3 in NS20Y cells. This conclusion is also in agreement with the
results of functional assays noted earlier, that the E box contributes
more than the GC box does to the promoter activity of the DOR gene.
The goal of this study was to identify the cis-elements
and trans-acting factors critical to transcriptional
regulation of the DOR gene. Based on the results of 5'- and 3'-end
serial deletion analyses, we have localized the core promoter
controlling this expression to a minimal region situated between Based on our results, two cis-elements were identified to be
crucial for the DOR promoter activity. The first cis-element is a GC box. Sp1 and Sp3 were shown bound to this element (Fig. 3C).
Sp1 often acts as an activator of Sp1 binding sites (34), and also
plays an essential role in transcription by tethering preinitiation
complexes to the promoter through interaction with TFIID at both TATA
and TATA-less promoters (21, 35-36). We demonstrated that Sp1 is an
activator of DOR expression by showing that Sp1 is the major binding
factor on the DOR GC box in NS20Y cells (Fig. 3C,
lanes 11, 13, and 15), and
that it activates the DOR promoter in Drosophila SL2 cells
(Fig. 4). Sp3, in contrast, was defined originally as an inhibitor of
Sp1-mediated activation, based on its competition with Sp1 for the
Sp1-binding site (19, 37-38). Recently, however, Sp3 has been shown to
function as a dual-function regulatory factor, also possessing
stimulatory activity in some circumstances (39-40). In any case, the
relative levels of Sp1 and Sp3 proteins are likely to be important
determinants of transcription activity in cells that express both
factors (41-42). While both Sp1 and Sp3 were present and bound to the
DOR promoter GC box in NS20Y cells, the amount of Sp1-containing
complexes was much greater than that of the Sp3-containing complexes
in vitro (Fig. 3C, lane 11,
band a and lane 13,
band b). Thus, further studies will be needed to
determine whether Sp3 can activate DOR expression, or whether it simply
acts as an inhibitor to modulate Sp1-mediated expression of the DOR
in vivo.
The E box is another crucial cis-element for the DOR
promoter activity (Fig. 5). Two ubiquitous transcription factors, USF1 and USF2, were identified bound to this E box (Fig. 5C).
USF2 was also demonstrated to trans-activate the DOR
promoter in vivo (Fig. 7) and found binding to the DOR E box
only in the form of heterodimer (Fig. 5C). As with Sp1 and
Sp3, different ratios of these homo- and heterodimers of USF are found
in different cell types (30, 43). In the case of NS20Y cells, we found
that heterodimers of USF1/2 comprised about 80% of the USF binding activity to the DOR E box, with the remaining 20% of binding activity contributed by USF1 homodimers. We observed no binding of USF2 homodimers to the DOR promoter E box (Fig. 5C). The
functional roles of USF1/2 heterodimers and USF1 homodimers in NS20Y
cells are not clear. However, USF1/2 heterodimer is most likely acting as a transcription activator in vivo (30), while USF1
homodimer may play a dual-function role for the DOR promoter (44). Thus the ratio between homo- and heterodimers of USF1 and USF2 may also
determine the magnitude of the DOR promoter activity. In any case, our
results suggest that both USF1 and USF2 are playing a critical role in
the regulation of DOR expression.
Substantial evidence indicates that USF interacts with other
transcription factors, such as TFIID, AP1, ETS-1, and Stat-1 (33,
45-47). In this report, we demonstrate a physical interaction between
USF and Sp1/Sp3 (Fig. 9). In NS20Y cells, several observations suggest
that the formation of USF-D232/169 complexes is the rate-limiting step
in the formation of USF-Sp-D232/169 complexes. First, only trace
amounts of USF-D232/169 complexes were detected (Fig. 9, lanes 6 and 8, band
d) under conditions in which USF-Sp-D232/169 complexes were
observed (Fig. 9, lanes 6 and 8,
band a), while excess amounts of the
Sp1/Sp3-D232/169 complexes were present (Fig. 9, lanes
6 and 8, bands b and
c). Second, there was always substantial Sp1/Sp3 binding to
D232/169 (Fig. 9, lanes 1, 6,
7, and 8, bands b and
c). Finally, the complex of USF-D232/169 increased dramatically when formation of USF-Sp-D232/169 complex was disrupted by
the Sp1 site-containing oligonucleotide, D232/215 (Fig. 9, lanes 6 and 9, band
d). Thus, almost all of the USF bound to D232/169, forming
higher molecular weight complexes with Sp1/Sp3, while excess amounts of
the Sp1/Sp3-D232/169 complexes were left. It indicates that the USF
binding determines the extent of simultaneous binding of USF and Sp
factors to the DOR promoter. In addition to these EMSA studies
indicating that USF binding to the E box plays a decisive role in DOR
promoter activation, a similar conclusion is consistent with the
results of our mutation experiments. Thus, mutation of the DOR E box
abolished promoter activity much more effectively than mutation of the
DOR GC box. Furthermore, since the DOR promoter is a TATA-less
promoter, the formation of the preinitiation complex may rely on both
the GC box and E box in the DOR promoter region, especially the close
location of them to the transcription initiation site. The physical
interaction between the DOR E box and GC box reported here, as well as
their interactions with TAFII55 (33), make it possible that
both the E box and GC box are involved in the basal transcription of
the TATA-less DOR promoter.
As mentioned earlier, the expression of the DOR is restricted (5). The
5'- promoter region of the DOR gene displays high expression activity
in DOR-expressing neuronal cell lines, including NS20Y (Fig.
1B) and NMB (data not shown). Thus luciferase was expressed
with a magnitude of 7-fold greater or more in these cells than in cells
not expressing DOR, including hepatocytes, neuro2A, and Chinese hamster
ovary cells (data not shown). However, the preferential expression of
the DOR promoter constructs for DOR-expressing cell lines disappeared
when the minimum promoter region ( Recently, cross talk has been reported between cell- or tissue-specific
transcription factors and ubiquitous USF or Sp1 (46-48, 53-55). A
combinatorial activity of an associated cell-specific activator and
ubiquitous USF or Sp1 is able to direct the transcription in a
cell-type specific manner (49-51, 55). Moreover, there is evidence
indicating the existence of a family of proteins able to bind the Sp1
consensus-binding motif, and it has been suggested that a complex
interaction of several factors at this site may be involved in tissue
specific gene transcription, e.g. that the Sp1/Sp3 ratios in
some cells and Sp1 activity determined by signal transduction (41-42,
56-57) might modulate the promoter activity in a cell-type specific
manner. Therefore, either the GC box or the E box, or indeed an
association between the relative abundance and state of activity of the
trans-acting factors binding these elements, or their
transcription factor partners with cell-type specificity, may provide
the basis for selective cell-specific transcriptional regulation of the
DOR gene.
We thank Dr. H. C. Towle (University of
Minnesota, Minneapolis, MN) for the generous gift of USF2, USF2 *
This work was supported by National Institutes of Health
Grants DA-00546, DA-01583, DA-11806, and KO5-DA-70554 and by the A. & F. Stark Fund of the Minnesota Medical Foundation.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 abbreviations used are:
OR, opioid receptor;
DOR,
Transcriptional Regulation of Mouse 
-Opioid Receptor Gene*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(DOR), and 
(KOR), have been cloned and characterized. Each
opioid receptor exhibits a distinct pharmacological profile as well as
a distinct pattern of temporal and spatial expression in the brain,
suggesting the critical role of transcription regulatory elements and
their associated factors. Here, we report the identification of a
minimum core promoter, in the 5'-flanking region of the mouse DOR gene, containing an E box and a GC box that are crucial for DOR promoter activity in NS20Y cells, a DOR-expressing mouse neuronal cell line.
In vitro protein-DNA binding assays and in vivo
transient transfection assays indicated that members of both the
upstream stimulatory factor and Sp families of transcription factors
bound to and trans-activated the DOR promoter via the E box
and GC box, respectively. Furthermore, functional and physical
interactions between these factors were critical for the basal as well
as maximum promoter activity of the DOR gene. Thus, the distinct
developmental emergence and brain regional distribution of the opioid receptor appear to be controlled, at least in part, by these two
regulatory elements and their associated factors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(DOR), and (KOR), opioid receptors (2). ORs belong to
the superfamily of G-protein-coupled receptors (3) modulating
endocrine, immune, cardiovascular, and gastrointestinal functions.
While all three ORs mediate opioid-induced analgesia, each receptor
type displays a distinct pharmacological profile and a unique cell-type
specific distribution pattern (4-5). Distinct molecular mechanisms
probably coordinate the temporal and spatial expression of each
receptor, but little is known of the regulatory elements and their
associated transcription factors involved in the restricted expression
of ORs.
-agonist binding sites (6). Although DOR is found in the peripheral
nervous system and also in some immune cells, it is mainly confined to
the central nervous system, in various densities at different regions
of the brain (5). In addition, the expression of DOR is also tightly
controlled during development. DOR appears later than MOR and KOR; in
fact, DOR is not detectable prior to the postnatal stages of
development, and even then its emergence lags behind than that of MOR
and KOR (7). Levels of DOR mRNA or of -agonist binding sites can
also be regulated by certain inducers in some cell lines. For example,
DOR mRNA can be up-regulated by nerve growth factor (8), ethanol
(9), or retinoic acid (10). In contrast, a reduction of DOR mRNA
was also observed in the presence of prostaglandin E1,
forskolin, or cyclic AMP analogue (11). Finally, DOR levels may be
altered in certain disease states, such as inflammation (12) and lung
cancer (13). All of these studies, together with the heterogeneity of
the cell-type specific distribution, suggest that the spatial and
temporal expression of DOR is under strict control, able to respond to
specific physiological and pathological parameters. Therefore, to
understand the molecular mechanism of the restricted expression pattern
of DOR will be helpful to gain insights of its functions corresponding
to different development status and physiological settings.
1300 to +1 bp,
with the translation start site designated as +1) of the DOR
5'-flanking region was sequenced and its sequence analyzed by sequence
comparison with the Transcription Factors Database (15). This analysis
indicated that the DOR gene has the features of a typical
"housekeeping" gene. Thus, it lacks a classical TATA box, and there
is no CCAAT box or a consensus initiator (16-18). However, the
promoter region of the DOR gene is rich in G+C content and possesses
several putative GC boxes. It has been suggested that the GC box is the
binding site for members of the Sp1 transcription factor family
(19-21). It is also known that Sp1 is important for both TATA and
TATA-less promoters by interacting with TFIID (21) and involved in the
transcription regulation of some cell- or tissue-specific genes
(53-55). Therefore, to define the basal promoter of the DOR gene will
be the foundation for elucidating the molecular mechanism of DOR expression.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1300 to +1 bp related to the translation start site as +1)
or shorter upstream regulatory sequences of the mouse DOR gene. The
pD1300 construct was created by ligating the 1300-bp fragment from the DOR promoter region with SacI and NcoI sites into
a promoterless and enhancerless luciferase vector, pGL3-basic
(Promega). The 5'-deletion (pD890, pD675, pD262, pD182, and pD150) and
3'-deletion (pD1300/150, pD1300/182, pD1300/262, and pD1300/675)
constructs were generated by restriction digestion, blunt-ended by
fill-in reaction, and religation. The 5'-deletion construct pD210 and the 3'-deletion construct pD1300/232 were prepared by recombinant polymerase chain reaction (PCR) with all of the upstream primers bearing the KpnI site and the downstream primers bearing the
NcoI site. The PCR products were subcloned into
KpnI and NcoI sites of the pGL3-basic vector, and
the correct clones were confirmed by sequencing. The pD262/141 and
pD141/262 constructs were prepared by PCR amplification of the DOR
promoter fragment 262 to 141 with primers bearing a
HindIII site. Then the PCR products were digested with
HindIII and cloned into the HindIII site of the pGL3-basic vector. The correct clones of pD262/141 and pD141/262 were
determined by sequencing. A HindIII linker mutation was
introduced into the pD262 construct by PCR to generate a series of
linker mutation constructs throughout the DOR promoter region (from
262 to 137). The mutated DOR promoter fragments (262 to +1) of
mutant constructs were sequenced and subcloned into pGL3-basic with
NcoI and KpnI to generate linker scan mutant
constructs pDm262 through pDm142. Each mutation construct in the linker
scan analysis was designated by the position of the 5'-end nucleotide
of its mutated sequence. Thus pDm262 represents the mutation construct
containing the mutated sequence (AAGCTT) at positions 262 to 257;
all other linker scan mutant constructs (pDm) contain the mutated
sequence in the six nucleotides beginning with the indicated number.
The double mutation construct, pD262Sp*/E*, was created using PCR to
introduce a second HindIII linker into pDm226 and to replace the E box (CACGTG) with AAGCTT sequence. All of the mutation constructs were confirmed by DNA sequencing.
-galactosidase gene
driven by the SV40 promoter was included in each transfection and used
for normalization. Drosophila SL2 cells were transfected
with CellFECTINTM (Life Technologies, Inc.) as described in
our previous report (23). Briefly, for each transfection, test plasmid
and CellFECTIN were mixed and incubated at room temperature for 30 min,
before adding to SL2 cells. Forty-eight hours after transfection, cells were washed and lysed. Normalization of the samples in the SL2 transient transfection followed the method described by Conn et. al. (24). The luciferase and -galactosidase activities of each lysate were determined as described by the manufacturers (Promega and
Tropix, respectively).
80 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Deletional analysis of the promoter of
the -opioid receptor gene. A,
a schematic diagram representing the 5'-regulatory region of the mouse
-opioid receptor gene from nucleotide 1300 to the translation
start site (ATG), which is designated as +1. Arrows indicate
two major transcription initiation sites (TIS).
B, a series of DOR promoter/luciferase constructs were
prepared and introduced into NS20Y cells as described under
"Materials and Methods." The line graph on
the left is a schematic representation of the DOR promoter
regions that were included in each construct. Each construct was named
by the number of the 5'-end nucleotide of the inserted DOR promoter
region. A positive control plasmid (control) containing the
SV40 promoter and SV40 enhancer, as well as a negative control plasmid,
the pGL3-basic vector (basic), were included in transient
transfection assays. Luciferase activity of the transfectants was
determined and normalized to -galactosidase activity, then expressed
for each construct as a percentage of the positive control plasmid
(relative luciferase activity %). The histograms on the
right represent the mean values of relative luciferase
activity (%) from at least four independent transfection experiments
with two different plasmid preparations. Error
bars indicate the range of standard errors.
1300 to 262 showed similar luciferase activities, with relatively minor variation. However, a dramatic decrease (more than 50%) in luciferase activity was seen with the
5'-end deletion construct pD210. Moreover, two deletion constructs with
5'-end deletion down to 182 and 150, respectively, displayed luciferase activity reduced to that of the pGL3-basic. Thus, these data
indicated that the sequence from 262 to +1 is sufficient to express
full promoter activity for the DOR gene.
150 to +1 of the DOR promoter
region (designated as pD1300/150) did not affect activity of the DOR promoter. However, a deletion of up to 182 (pD1300/182) sharply reduced DOR promoter
activity. Further 3'-end deletions of the DOR promoter region
(constructs pD1300/232, pD1300/262, and pD1300/675) resulted in a still
greater and ultimately total loss of DOR promoter activity (Fig. 2).
Based on the combined data from the 3'- and 5'- serial deletional
analyses (Figs. 1B and 2), the DOR promoter sequence from
262 to 150 provided more than 90% of the DOR promoter activity.
Thus the sequence from 262 to 150 appeared to be essential for
displaying the DOR promoter activity. Accordingly, a pair of DOR
promoter constructs was created with the DOR promoter sequence from
262 to 141 in either orientation. The region of 262 to 141
includes a major transcription initiation site (Fig. 1A) and
promoter activity of this region was examined. Surprisingly, both
constructs were active in NS20Y cells (Fig. 2), although the promoter
activity of the construct pD262/141 (in native orientation) was about
3-fold more active than that of the construct pD141/262 (with DOR
promoter sequence in reverse orientation). Taken together, these
results indicated that the promoter sequence between 262 and 141 is
necessary and sufficient to provide the DOR promoter activity in both
orientations but of different magnitude in NS20Y cells.
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Fig. 2.
Identification of the minimum DOR
promoter. A series of DOR promoter/luciferase constructs were
prepared and introduced into NS20Y cells as described under
"Materials and Methods." The line graph on
the left is a schematic representation of the DOR promoter
regions that were included in each construct. Each construct was named
by the numbers of both 5'- and 3'-end nucleotides of inserted DOR
promoter DNA fragments. The pD262/141 construct represents the native
orientation, while pD141/262 represents the reverse orientation of the
DOR promoter sequence from 262 to 141, determined to be the minimum
region expressing full promoter activity, as cloned into pGL3-basic.
The pGL3-basic empty vector (basic) was included as the
negative control. Luciferase activity was normalized to
-galactosidase activity and expressed as a percentage of the
activity of the pD1300 construct. The histograms on the
right represent the mean values of relative luciferase
activity (%) of the pD1300 from four independent transfection
experiments with two different plasmid preparations. Error
bars indicate the range of standard errors.
262 to 141)
of the pD262 construct were performed. This construct was used because,
as discussed earlier, this was the largest 5'-end deletion retaining
full promoter activity. The promoter activities of these linker-mutated
constructs were examined, with several mutant constructs displaying
impaired promoter activities (Table
I).
Linker scan analysis defines two crucial elements of the DOR promoter
262 to 137) was mutated with
HindIII linker (AAGCTT) at 6-bp intervals by using the
PCR-based oligonucleotide-directed mutagenesis with the pD262 as DNA
template. The positions of mutated sequence of each mutant construct
are described under "Materials and Methods." The linker scan mutant
constructs and pD262 were transfected into NS20Y cells, and luciferase
activity of each construct was determined. Luciferase activity was
normalized to -galactosidase activity and expressed as a percentage
of the activity of the pD262 construct. The numbers on the left
represent the mean values of relative luciferase activity (%) of the
pD262 from three independent transfection experiments with two
different plasmid preparations. The numbers on the right represent the
standard errors. Three constructs (pDm226, pDm190 and pDm184),
displaying 35% or lower promoter activities than that of pD262, are
indicated with asterisks and reveal the locations of two crucial
elements for the DOR promoter activity.
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Fig. 3.
Identification of the GC box and its
associated factors of the DOR promoter. A, the effect
of the GC box mutation. The DOR promoter construct, pD262, contains the
wild-type promoter sequence from 262 to +1. The GC box consensus in
pD262 is underscored, and the mutated sequence in pDm226 is
shown in boldface. The activities of promoter constructs
were examined by transient transfection assays using NS20Y cells.
Luciferase activity was normalized to -galactosidase activity and
expressed as a percentage of the activity of the pD262 construct. The
data on the right represent the mean values of relative
luciferase activity (%) of pD262 from four independent transfection
experiments. B, three oligonucleotides were used in EMSAs to
identify the Sp binding site in the DOR promoter region as well as its
binding factors. D232/215 was a portion of the DOR promoter region from
232 to 215 containing the GC box consensus (underlined).
Dm226 contains the same sequence as D232/215 except 6 mutated bp shown
in boldface. Sp1 consensus oligonucleotides were a
commercial product from Santa Cruz Biotechnology, Inc. C,
EMSAs were performed by using D232/215 as the probe in the absence
(lane 1) or presence (lanes
2-15) of nuclear extracts from NS20Y cells.
Lanes 3-10, various amount of different
unlabeled competitors were included as indicated. Lanes
11-15, 1 µl of different anti-Sp Abs were added to each
reaction as indicated. Protein-DNA complexes of the D232/215 probe are
marked by arrowheads c, d, and
e. The anti-Sp1 Ab supershifted band is indicated by
arrow b. The anti-Sp3 Ab supershifted band is
indicated by broken arrow a.
232 to 215, which includes the putative
binding site for the Sp1 family (Fig. 3B, D232/215, underlined), was used as the probe.
Three major bands representing protein-DNA complexes were observed in
the presence of the nuclear extracts (Fig. 3C,
lane 2, arrowheads c-e).
These three complexes were all sequence-specific, because formation of
all of them could be prevented by the presence of unlabeled D232/215 in
a dose-dependent manner (Fig. 3C,
lanes 3-5). Competition with the Sp1 consensus
oligonucleotide (Fig. 3B) also blocked the formation of the
protein-DNA complexes c-e in a dose-dependent manner (Fig.
3C, lanes 6-8). However, formation of
these three bands was not affected by using the Dm226 oligonucleotide,
which contained the same sequence as D232/215 except the GC box was replaced by the linker sequence AAGCTT (Fig. 3B,
Dm226, boldface), as the competitor (Fig.
3C, lanes 9 and 10). These
results suggest that Sp1 or Sp1-like factors bind to the DOR GC box.
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Fig. 4.
Sp1 trans-activates the DOR
promoter via Sp1 binding site. Drosophila SL2 cells
were co-transfected with the indicated amounts of pPacSp1 plasmid and
either pD262 (open circles; the wild type DOR
promoter construct) or pDm226 (closed diamonds;
the mutant construct of the DOR promoter with mutated Sp1 site).
Activation was expressed as a percentage of the activity of each
construct in the presence of the indicated amount of pPacSp1 divided by
the activity of the construct without effector (arbitrarily defined as
100%).
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Fig. 5.
Identification of the E box and its
associated factors of the DOR promoter. A, the effect
of the E box mutation. The E box consensus in pD262 is
underscored, and the mutated sequences in mutant constructs
pDm190, pDm184, and pDm178 are shown in boldface. The
activities of promoter constructs were examined by transient
transfection assays in NS20Y cells. Luciferase activity was
standardized to -galactosidase activity and expressed as a
percentage of the activity of the pD262 construct. The data on the
right represent the mean values of relative luciferase
activity (%) of pD262 from four independent transfection experiments.
B, three oligonucleotides were used in EMSAs to identify the
binding factors of the E box in the DOR promoter region. The
oligonucleotide D198/169 contained the sequence from 198 to 169 of
the DOR promoter. Both c-Myc and E box consensus oligonucleotides used
as unlabeled competitors were commercial products from Santa Cruz
Biotechnology, Inc. C, EMSA was performed by using D198/169
as the probe with NS20Y nuclear extracts (lanes
1-7). Lane 1, control reaction;
lane 2, 1 µl of control serum; lane
3, 100-fold excess of c-Myc competitor; lane 4, 1 µl
of anti-c-Myc Ab; lane 5, 100-fold excess of E box
competitor; lane 6, 1 µl of anti-USF1 Ab; lane
7, 1 µl of anti-USF2 Ab. The USF-D198/169 complexes are marked
by arrowheads a and b. The putative
USF1 homodimer is indicated by arrowhead c. The
USF-D198/169 complexes recognized by anti-USF1 Ab are marked by
broken arrows on the left. The
USF-D198/169 complexes recognized by anti-USF2 Ab are marked by
arrows on the right.
185 to 180) resides in the core promoter of the DOR gene.
Mutations of this site (pDm184 and pDm190) abolished the promoter
activity. In order to provide physical evidence for the impaired
USF-binding ability of the mutated DOR E box and correlate the in
vitro binding activity to the reduced promoter activities of
pDm184 and pDm190 in vivo, an EMSA was performed. In
addition to the oligonucleotide D198/169 described above (Fig.
5B), three oligonucleotides, Dm190, Dm184, and Dm178, were
synthesized, corresponding to the DOR promoter sequence 198 to 169
in mutant constructs, pDm190, pDm184, and pDm178, respectively. Both
D198/169 and Dm178 contained the intact E box, while Dm190 and Dm184
contained the mutated E box (Fig. 6A).
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Fig. 6.
USF-binding is critical for DOR promoter
activity. A, the oligonucleotide D198/169 contained the
DOR promoter sequence from 198 to 169 including the consensus E box
(underscored). Three different oligonucleotides, Dm190,
Dm184, and Dm178, all contained the same sequence as D198/169 except
for a 6-bp region mutated as indicated in boldface.
B, EMSAs were performed in the absence (lane
1) or presence (lanes 2-9) of NS20Y
nuclear extracts. Lanes 1-6, D198/169 was used
as the probe in the absence (lanes 1 and
2) or presence of different unlabeled competitors as
indicated (lanes 3-6). Lane
7, Dm190 was the probe. Lane 8, Dm184
was the probe. Lane 9, Dm178 was the probe. Major
protein-DNA complexes are marked by arrowheads a
and b.
b,
in which only the basic region was deleted, and USF2Nb, in which
both the basic region and the N-terminal trans-activation domain were deleted. Since both of these USF2 derivatives retain the
helix-loop-helix and leucine zipper region, however, they should be
able to dimerize with either USF1 or USF2. Thus, they would be
predicted to act as dominant negative forms of USF2, able to form
dimers but unable to bind to the E box (32). As shown in Fig. 7, the
DOR promoter construct, pD262, displayed more than a 50% loss in
promoter activity when it was co-transfected with either pUSF2b or
pUSF2Nb. Together with the significant trans-activation of USF2, these data provide direct evidence
that USF2 does act on the DOR E box and trans-activate the
DOR promoter in vivo.
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Fig. 7.
USF trans-activates the DOR
promoter in vivo. NS20Y cells were transfected
with a total of 3 µg of DNA, including 2 µg of the reporter
construct (pD262 or pGL3-basic as indicated) and 1 µg of a plasmid
expressing USF2 (pUSF2) or a dominant negative form of USF2
(pUSF2 b,
pUSF2Nb) or empty expression
vector (v). Activation was expressed as luciferase
activities relative to the luciferase activity of pGL3-basic,
arbitrarily defined as 1. Results are means of three different
experiments. Error bars indicate the range of
standard errors.
142, Fig.
1A). In addition, their associated factors, USF and Sp1,
have been reported to interact with TAFII55, a
transcription factor involved in the basal transcription machinery
(33). This suggests that both USF and Sp1 might contribute to the basal
transcription of DOR and functionally interact with each other via a
common transcription factor partner. To investigate the possibility of functional interaction between them, a double-mutation construct, pD262Sp1*/E*, was created as described under "Materials and
Methods." This double-mutation construct, featuring alterations in
both the GC box and E box, was tested for promoter activity in NS20Y cells, along with pD262 (wild-type), pDm184 (E*) (E box mutated), pDm226 (Sp1*) (GC box mutated), and the empty reporter gene vector (pGL3-basic).
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Fig. 8.
Functional interaction of the GC box and E
box of the DOR gene. NS20Y cells were transfected with various DOR
promoter/luciferase constructs containing either wild type DOR promoter
(pD262), or DOR promoter with mutated GC box
(pDm226), or DOR promoter with mutated E box
(pDm184), or DOR promoter with both mutated GC box and
mutated E box (pD262Sp1*/E*). Luciferase
activities were normalized to -galactosidase activity and expressed
relative to wild type promoter activity, arbitrarily defined as 100%.
Activation was expressed as luciferase activities relative to the
luciferase activity of pD262Sp1*/E*, arbitrarily defined as 1. The
underlined sequences represent either the GC box or E box,
as indicated. Mutant sequences are shown in boldface.
232 to
169) containing both the GC box and E box. In one of these probes,
designated as D232/169, both the GC box and E box were intact. In the
second probe, designated as D232/169E*, the GC box was intact while the
E box was mutated. The third probe, designated as D232/169Sp1*,
contained an intact E box and mutated GC box.
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Fig. 9.
Physical interaction of the GC box and E box
of the DOR promoter. A, three oligonucleotides were
used as probes in EMSA shown in panel B. D232/169
contained the DOR promoter sequence from 232 to 169, with both the
GC box and E box (underlined). D232/169E* contained the same
sequence as D232/169 except the E box was mutated (in bold).
D232/169Sp1* containing the same sequence as D232/169 except the GC box
was mutated (in bold). B, EMSA was performed with
NS20Y nuclear extracts and indicated probes. Lanes
1, 3, and 6, no competitor;
lane 5, probe only; lane 2,
in the presence of Sp1 consensus oligonucleotide (as shown in Fig.
3B) as the competitor; lane 4, in the
presence of E box consensus (as shown in Fig. 5B) as the
competitor; lane 7, in the presence of D198/169
(as shown in Fig. 5B) as the competitor; lane
8, in the presence of the Dm226 and Dm184 (as shown in Fig.
3B and 6A) as the competitors; lane 9, in the presence of the D232/215 (as shown in Fig. 3B) as the
competitor.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
262
and 141 bp upstream of the DOR gene's translation initiation site
(Fig. 2). Surprisingly, this minimum promoter sequence conferred
promoter activity in both orientations, although with different
magnitudes. The promoter with orientation-independent promoter activity
is uncommon in transcription regulation. The mechanism behind this unusual observation remains to be determined.
262 to 141) was impaired by
deletions. Thus, the DOR minimum promoter region appears to contain an
element or elements controlling the cell-type specificity of these DOR
promoter constructs. A similar phenomenon is also found in the
regulation of the MOR gene (22) and human complement component C4 gene
(52), in that the minimum promoter provides the basal promoter activity as well as preferential cell-type expression. Dissection of the minimum
promoter region at a finer level may be helpful in further defining the
mechanisms underlying cell-type specific expression of DOR.
ACKNOWLEDGEMENTS
b,
and USF2 Nb expression plasmids. We thank Dr. R. Tjian
(University of California, Berkeley, CA) for the kind gift of pPacSp1
plasmid. We thank Dr. Y. Sarne and Dr. Y. Wollman (Tel Aviv University,
Tel Aviv, Israel) for the kind gift of NMB cells. We thank Dr. A. P. Smith for helping with the preparation of the manuscript.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Pharmacology,
University of Minnesota Medical School, 3-249 Millard Hall, 435 Delaware St. S.E., Minneapolis, MN 55455. Tel.: 612-626-6539; Fax:
612-625-8408; E-mail: liuxx018@maroon.tc.umn.edu.
ABBREVIATIONS
-opioid receptor;
MOR, µ-opioid receptor;
KOR, -opioid
receptor;
PCR, polymerase chain reaction;
EMSA, electrophoresis
mobility shift assay;
Ab, antibody;
bHLH/LZ, basic-helix-loop-helix
leucine zipper;
USF, upstream stimulatory factor;
bp, base pair(s);
kb, kilobase pair(s);
PMSF, phenylmethylsulfonyl fluoride;
DTT, dithiothreitol;
DMEM, Dulbecco's modified Eagle's medium.
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DISCUSSION
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