ABSTRACT
The retinoid X receptors
[alpha]
-,
[beta]
- and
[gamma]
- (RXRs) share a highly conserved `C' region or DNA binding domain (DBD).
The conserved `DE' region or ligand binding domain (LBD) of the RXRs is
functionally complex, mediating dimerization and a ligand-dependent activation function (AF-2). The AB or N-terminal region of the RXRs is poorly conserved and encodes a
ligand-independent activation function (AF-1). RXR
[gamma]
mRNA is preferentially expressed in skeletal and cardiac muscle, however, cell-specific steroid receptor-mediated
trans
-activation is a poorly understood phenomenon. We utilized the GAL4 hybrid
assay system and have demonstrated that RXR
[gamma]
contains two functional domains in the AB and DE regions that activate
transcription in a ligand-independent and -dependent manner respectively. The functions of the AB (AF-1) and DE (AF-2) domains were regulated by cAMP-dependent protein kinases, furthermore, the
function of AF-2 in the LBD was activated by 8-Br-cAMP, independent of 9-
cis
-retinoic acid treatment. Deletion analysis demonstrated that the AF-1 of RXR
[gamma]
, is located between amino acids 1 and 103 and contained multiple motifs that
were targets of cAMP-dependent protein kinases. Transfection analyses in non-muscle and myogenic cells clearly demonstrated that: (i) the AF-1 of RXR
[gamma]
functions in a muscle-specific manner and is required for optimal ligand-dependent
trans
-activation from an RXRE; (ii) RXR
[gamma]
trans
-activates more efficiently in a myogenic background.
Retinoids play an important and fundamental role in development, differentiation
and homeostasis (
1
-
3
). The effects of retinoids are mediated by two subgroups of the steroid
receptor superfamily of nuclear receptors that bind specific DNA sequences,
termed hormone response elements (HREs), and act as ligand-inducible transcriptional regulators (
4
,
5
). The two subgroups that mediate the effects of retinoids are the retinoic acid
receptors (RAR[alpha], [beta], [gamma] and various isoforms) that mediate transcriptional
activation in response to both all-
trans
-retinoic acid (RA) or 9-
cis
-RA and retinoid X receptors (RXR[alpha], [beta] and [gamma]), that mediate transcriptional activation in response
to 9-
cis
-RA (
3
,
6
).
The RXRs share similar structural domains with other members of the steroid
receptor superfamily based on amino acid similarity (
3
,
5
). RXRs and other members of the steroid/thyroid nuclear receptor family share a
highly conserved C region or DNA binding domain (DBD) and DE region or ligand
binding domain (LBD) (
3
). The LBD of the three RXRs characterized to date is functionally complex,
mediating ligand binding, dimerization and a ligand-dependent activation function (AF-2) responsible for ligand-mediated transactivation (
7
-
11
). The AB regions of the three RXRs show <40% homology (
6
), with RXR[alpha] and RXR[gamma] containing an activation function (AF-1) responsible for ligand-independent transactivation (
8
). The functional properties of the N-terminal AB region of the steroid/thyroid receptors has developed into an
area of increasing interest. Recent studies on the members of the steroid
receptor gene family indicate that this region, depending on the receptor, may
play an important role in DNA binding, transactivation, cell type- and/or promoter-specific regulation or interaction with the general transcription
factor TFIIB (
12
, and references therein). In terms of the retinoid receptors transactivation
and promoter-specific regulation has been shown to be mediated by the different N-terminal regions of RXRs and RARs when linked to the DBD of the
estrogen receptor (
8
,
13
) or GAL4 (
14
).
Many of the nuclear receptors identified to date have been found to be
phosphoproteins (
15
), including RAR[alpha], RAR[beta] and RAR[gamma] (
16
-
18
). The protein kinase C- and cAMP-dependant protein kinase pathways have been implicated in regulation
of retinoid-mediated transcription, which suggest that phosphorylation processes may
be involved in regulating the function of retinoid receptors (
19
-
21
). In recent studies with RXRs and RARs it has been demonstrated that in both
the presence and absence of ligand manipulation of the phosphorylation state of
the cell with okadaic acid (OA), which inhibits protein phosphatases PP1 and
PP2A (
22
), led to increases in transactivation of RXR- or RAR-responsive reporter genes in transient transfection experiments (
23
,
24
). In an effort to further investigate the role of phosphorylation in relation
to RXR[gamma]-mediated transcriptional activation we have studied the effects of
8-Br-cAMP [an activator of protein kinase A (PKA)] and OA on the AF-1 and AF-2 domains of RXR[gamma].
The expression plasmids pGALO (
25
), pSG5 (Stratagene) and pSG5RXR[gamma] (
26
) and reporter plasmids pBLCAT2 (ptkCAT) (
27
), pG18
2
-tkCAT (
28
) and G5E1b-CAT (
29
) have been described elsewhere. Generation of mRXR[gamma] DNA fragments was performed by PCR with Pfu DNA polymerase, using the
manufacturer's buffer. All PCR products were cloned into the
Sma
I site of pBS (Stratagene) and then isolated after
Eco
RI digestion. GAL-RXR[gamma] was constructed by excising mRXR[gamma]1 DNA from the pSG5-RXR[gamma] vector, end filling with Klenow fragment and
ligating into an end-filled
Sal
I site in pGALO. GAL-RXR[gamma]AB was constructed by PCR using oligonucleotides 173, 5'-GCGGAATTCACCATGTATGGAAATTATTCCCAC-3', and 225, 5'-GCGGAATTCATAAGATGTGTTTCACCAGAGAC-3', to generate
the RXR[gamma] AB region, which was ligated into the
Eco
RI site of pGALO. GAL-RXR[gamma]DE was generated by PCR using oligonucleotides 223, 5'-GCGGAATTCACCAAGCGGGAAGCTGTGC-3', and 176, 5'-GCGGAATTC
CTCAGGTGATCTGCAGTGGGGTCT-3', to generate the RXR[gamma] DE region, which was cloned into the
Eco
RI site of pGALO. GAL-RXR[gamma]ABDE was constructed by PCR using oligonucleotides 173 (see above)
and 174, 5'-GCGGAATTCCTCAATGTGTTT CA CCAGAGAC-3', to generate the RXR[gamma] AB region and the RXR[gamma] DE region (as described above). The RXR[gamma] AB and DE regions were then
ligated and cloned into
Eco
RI-digested pBS. Plasmids containing only one copy of the RXR[gamma] AB and DE regions were then sequenced to determine the correct
orientation and reading frame. A plasmid containing the RXR[gamma] AB and DE regions [containing amino acids 1-138 and 205-463 of RXR[gamma] separated by four amino acids (Glu-Glu-Phe-Thr)] in the correct orientation
and reading frame was identified, subjected to partial
Eco
RI digestion and the RXR[gamma] ABDE fragment isolated and ligated into the
Eco
RI site of pGALO. GAL-RXR[gamma]1-103 and GAL-RXR[gamma]1-43 were constructed by digesting the PCR-generated RXR[gamma] AB fragment (see above)
with
Hin
cII or
Alu
I to produce the DNA fragments corresponding to amino acids 1-103 and 1-43 respectively, which were then ligated into
Eco
RI/
Sma
I-digested pGALO. GAL-RXR[gamma]1-77, GAL-RXR[gamma]77-138 and GAL-RXR[gamma]104-138 were
constructed by digesting the PCR-generated RXR[gamma] AB fragment (see above) with
Nco
I or
Hin
cII. The isolated DNA fragments were than end filled with Klenow fragment to
produce the DNA fragments corresponding to amino acids 1-77, 77-138 and 104-138, which were then ligated into the end filled
Nde
I,
Sma
I and end filled
Sal
I sites of pGALO respectively. GAL-RXR[gamma]44-138 was constructed by PCR using oligonucleotides 259, 5'-GCGGAATTCACCAGCTACACAGACACCCCAG-3', and 225 (see above) to generate
the DNA fragment corresponding to RXR[gamma] amino acids 44-138, which was ligated into the
Eco
RI site of pGALO. GAL-RXR[gamma]44-103 was constructed by digesting the PCR-generated RXR[gamma] amino acids 44-138 fragment (see above) with
Hin
cII and cloning the DNA fragment corresponding to RXR[gamma] amino acids 44-103 into
Eco
RI/
Sma
I-digested pGALO. GAL-RXR[gamma]44-77 and GAL-RXR[gamma]77-103 were constructed by digesting
the DNA fragment corresponding to RXR[gamma] amino acids 44-103 with
Nco
I. The isolated DNA fragments were then end filled with Klenow fragment to
produce the DNA fragments corresponding to amino acids 44-77 an 77-103, which were then ligated into the end-filled
Nde
I and
Sma
I sites of pGALO respectively. RXR[gamma][Delta]LBD was constructed by PCR using oligonucleotides 173 (see above)
and 262, 5'-GCGGAATTCCTCAACTGGCACATTCTGCCTCAC-3', to generate the RXR[gamma] DNA fragment corresponding to amino acids 1-229, which was ligated into the
Eco
RI site of pSG5. RXR[gamma][Delta]AB was constructed by PCR using oligonucleotides 263, 5'-GCGGAATTCATGACCAGCCCTGGGTCTCTGGTG-3', and 176 (see above) to generate the
RXR[gamma] DNA fragment corresponding to amino acids 129-463, which was ligated into the
Eco
RI site of pSG5. All GALO/RXR[gamma] constructs were sequenced to confirm the reading frame using the
Pharmacia T7 sequencing kit (Uppsala, Sweden).
COS-1 cells were cultured for 24 h in Dulbecco's modified Eagle's medium
(DMEM) containing 5% charcoal-stripped fetal calf serum (FCS) prior to transfection. Each 60 mm dish of
COS-1 cells (60-70% confluence) was transiently transfected with 5 [mu]g reporter plasmid DNA (G5E1b-CAT, pG182-tkCAT or ptkCAT) expressing chloramphenicol
acetyltransferase (CAT), mixed with the appropriate amount of expression vector
(1 [mu]g for pSG5-RXR[gamma], -RXR[gamma][Delta]LBD, -RXR[gamma][Delta]AB or 3 [mu]g for GAL/RXR[gamma] chimeras)
or pUC18/carrier plasmid in each transfection experiment by the DOTAP
(Boehringer Mannheim)-mediated procedure as described previously (
28
). The DNA/DOTAP mixture was added to the cells in 6 ml fresh medium. After a
period of 24 h fresh medium with or without 0.5 mM 8-Br-cAMP, 50 nM okadaic and/or 9-
cis
-RA (10
-7
M) was added to the cells. The cells were harvested for assay of CAT activity
24-72 h after the transfection period. Each transfection experiment was
performed at least three times in order to overcome the variability inherent in
transfections.
Mouse myogenic C
2
C
12
cells (
30
,
31
) were grown in DMEM supplemented with 20% (v/v) FCS in 6% CO
2
. Prior to and during transfection this cell line was induced to biochemically
and morphologically differentiate into multinucleated myotubes by serum
withdrawal in ligand-deficient medium [DMEM supplemented with 2% (v/v) charcoal-stripped FCS]. Each 60 mm dish of myogenic C
2
C
12
cells (90-100% confluence) was transiently transfected as described above. After a
period of 24 h fresh medium [DMEM supplemented with 2% (v/v) charcoal-stripped FCS] +- 9-
cis
-RA (10
-7
M) was added to the cells. The cells were harvested for assay of CAT activity
24 h after addition of fresh medium. Each transfection experiment was performed
at least three times in order to overcome the variability inherent in
transfections.
The cells were harvested, normalized for protein concentration and CAT activity
measured as previously described (
32
). Aliquots of the cell extracts were incubated at 37oC with 0.1-0.4 mCi [
14
C]chloramphenicol (ICN) in the presence of 5 mM acetyl CoA and 0.25 M Tris-HCl, pH 7.8. After a 1-4 h incubation period the reaction was stopped by addition of 1 ml
ethyl acetate, which was used to extract the chloramphenicol and its acetylated
forms. The extracted materials were analysed on Silica gel thin layer
chromatography plates as described previously (
32
). Quantitation of CAT assays was performed by an AMBIS [beta]-scanner.
To identify and further characterize the domains of RXR[gamma] involved in transcriptional activation we utilized the GAL4 hybrid
system, whereby putative activation domains are fused to the DBD of the yeast
transcription factor GAL4 (
33
,
34
). If these regions encode modular activation domains they complement the GAL4
DBD (to produce a functional
trans
-activator) and induce transcription of the GAL-responsive reporter construct G5E1b-CAT, containing an E1b TATA box with five 17mer GAL4 binding
sites linked to the CAT reporter (
29
). The system utilized an SV40 promoter expression vector pGALO (
25
) that contains a multiple cloning site downstream of the GAL4 DBD. We fused RXR[gamma] and various domains (e.g. AB or DE regions) of RXR[gamma] to the GAL4 DNA binding domain and examined the ability of these
chimeras to regulate expression of the G5E1b-CAT reporter in COS-1 cells. The GAL-RXR[gamma] chimera containing the full open reading frame (ORF)
of RXR[gamma] in the presence of 9-
cis
-RA activated transcription ~4-fold above the control, pGALO (GAL4 DBD), and GAL-RXR[gamma] in the absence of 9-
cis
-RA. This demonstrated that the GAL-RXR[gamma] chimera conferred appropriate 9-
cis
-RA-dependent
trans
-activation via RXR[gamma] to the GAL4 DBD (Fig.
1
). The GAL-RXR[gamma]AB plasmid, which contains only the AB regions of RXR[gamma], with the DBD and LBD deleted, increased transcription of
the reporter construct ~14-fold over the GAL DBD alone, independent of 9-
cis
-RA treatment (Fig.
1
). This indicated that the AB region of RXR[gamma] contained a ligand-independent transactivation function. GAL-RXR[gamma]DE, which contains the LBD of RXR[gamma] with the AB and C regions deleted, activated
transcription of the G5E1b-CAT reporter ~8-fold in a 9-
cis
-RA-dependent manner. The GAL-RXR[gamma]ABDE plasmid (which lacks the C region of RXR[gamma], which contains the DBD), increased
transcription ~10-fold in the presence of 9-
cis
-RA. Deletion of only the C region of RXR[gamma] increased 9-
cis
-RA-mediated activation by 2- to 4-fold in comparison with the full-length RXR[gamma] linked to the GAL DBD (GAL-RXR[gamma]). The lower activity of full-length RXR[gamma] may be
attributed to the presence of two DNA binding domains in the GAL-RXR[gamma] chimeric protein, causing possible steric hindrance in either the
DNA binding or transactivation function. Possible repression of chimeric
constructs containing two DBDs with the GAL system have been previously
reported (
14
). These experiments indicate that there are two domains involved in
transactivation by RXR[gamma]; AF-1 in the AB domain, which is ligand-independent, and the ligand-dependant AF-2 in the DE domain, which activates transcription
in response to the ligand 9-
cis
-RA.
Recent reports have indicated that transcriptional activation by RXRs can be
regulated by phosphorylation (
23
,
24
). We therefore investigated the affect of 8-Br-cAMP (a stimulator of cAMP-dependent protein kinases) and OA (an inhibitor of serine-threonine protein phosphatases) on the AF-1 and AF-2 functions of RXR[gamma].
In control studies (to examine non-specific effects of 8-Br-cAMP on the GAL4 hybrid system) when COS-1 cells were transfected with the reporter plasmid G5E1b-CAT the presence of 0.5 mM 8-Br-cAMP increased CAT expression by 1.4 +- 0.25-fold (data not shown).
Furthermore, the presence of 0.5 mM 8-Br-cAMP increased CAT expression from the reporter in the presence of
the GAL4 DBD (pGALO) by 2.4-fold (Fig.
2
A). The ability of GAL-RXR[gamma]AB to
trans
-activate gene expression (in a ligand-independent manner) was increased ~4.0-fold by 8-Br-cAMP treatment (Fig.
2
A). GAL-RXR[gamma]DE increased expression of the reporter by 10-fold in the presence of 8-Br-cAMP (and more importantly, in the absence of the
ligand 9-
cis
-RA). Activation of GAL-RXR[gamma]DE in the presence of 8-Br-cAMP was 1.2- to 2-fold greater than that seen in GAL-RXR[gamma]DE-transfected cells
treated only with 9-
cis
-RA. Co-treatment of GAL-RXR[gamma]DE with 9-
cis
-RA and 8-Br-cAMP resulted in a similar activation of gene expression
compared with treatment with 8-Br-cAMP. For GAL-RXR[gamma]ABDE results similar to GAL-RXR[gamma]DE were observed when treated with either
8-Br-cAMP and/or 9-
cis
-RA.
Activation of the AF-2 domain by 8-Br-cAMP (independent of ligand) was higher than activation
mediated by 9-
cis
-RA and indicates that activation of AF-2 by phosphorylation is not dependant upon hormone binding.
Therefore, this set of controlled experiments clearly illustrates that both the
AF-1 and AF-2 functions of RXR[gamma] are regulated either directly or indirectly by cAMP-dependent protein kinase cascades in a ligand-independent manner.
The ability of GAL-RXR[gamma]AB to
trans
-activate gene expression (in a ligand-independent manner) was increased ~5.0-fold by OA treatment (Fig.
2
B). However, in control studies (to examine non-specific effects of OA on the GAL4 hybrid system) when COS-1 cells were transfected with the reporter plasmid G5E1b-CAT the presence of 50 nM OA increased CAT expression by 1.3 +- 0.14-fold (data not shown). Furthermore, 50 nM OA or
OA + 9-
cis
-RA increased CAT expression from the reporter in the presence of the GAL4
DBD by 4.6- and 4-fold respectively. This suggested that the effects of OA on GAL-RXR[gamma]AB were not specific to RXR. GAL-RXR[gamma]DE increased expression of the reporter by
5-fold in the presence of OA (and more importantly in the absence of the
ligand, 9-
cis
-RA). However, this was 2-fold less than that seen in cells treated only with 9-
cis
-RA and similar to the increased activity of the GAL 4 DBD after OA
treatment. Co-treatment of GAL-RXR[gamma]DE with 9-
cis
-RA and OA resulted in a 4.5-fold greater activation of gene expression compared with treatment
with either 9-
cis
-RA or OA. For GAL-RXR[gamma]ABDE results similar to GAL-RXR[gamma]DE were seen when cells were treated with either
OA and/or 9-
cis
-RA. Our controls demonstrated that it was difficult to interpret the
specific effects of OA on RXR[gamma] in the GAL4 hybrid system, because of the generalized effect of OA on
the reporter construct and the GAL4 DBD. Therefore, we cannot determine or make
firm statements about the role of serine-threonine phosphorylation in the activity of RXR[gamma], however, we note that OA can activate the LBD in a ligand-independent manner.
We have shown that the N-terminus or AB domain of RXR[gamma] contains a ligand-independent activation function (AF-1) located between amino acids 1 and 138 (Fig.
1
). In order to further characterize the AF-1 region of RXR[gamma] we have constructed various deletions of the AB region and fused
these sub-domains to the GAL DBD (Fig.
3
A). These constructs were transfected into COS-1 cells in the absence of ligand and assayed with respect to the ability
to
trans
-activate the reporter (Fig.
3
B). A construct (GAL-RXR[gamma]1-103) containing the first 103 amino acids of the 138 amino
acid AB region of RXR[gamma] increased activation 10.3-fold above the control, pGALO (GAL-DBD) alone, and had similar activity to the entire AB domain
of RXR[gamma] (GAL-RXR[gamma]AB). The plasmid GAL-RXR[gamma]104-138 did not
trans
-activate gene expression in this assay system. This and the previous
experiment demonstrate that amino acids 104-138 are not essential for activity of the AB region and do not contain an
activation domain.
We went on to examine the role of the AB region of RXR[gamma] and the effect of phosphorylating agents in RXR-mediated transactivation from an optimal RXRE (G18) cloned into the
heterologous herpes simplex virus thymidine kinase (tk) promoter (
27
) linked to the
CAT
gene in non-muscle and myogenic cells (pG18
2
-tkCAT). We transfected both cell types because RXR[gamma] is selectively expressed in skeletal and cardiac muscle. The G18
RXRE utilized was derived from a RXR[gamma] binding site selection experiment and was the optimal sequence with
respect to binding of RXR[gamma] homodimers and RXR-dependent
trans
-activation
in vivo
by 9-
cis
-RA (
28
).
We investigated the ability of full-length RXR[gamma], RXR[gamma] lacking the AB region (the RXR[gamma][Delta]AB construct contains amino acids 129-463) and RXR[gamma] lacking the D/E region (the RXR[gamma][Delta]LBD construct
contains amino acids 1-229) to transactivate an RXRE in non-muscle and muscle cells in the presence and absence of
phosphorylating agents. We utilized the RXR[gamma][Delta]LBD construct as a control construct, since removal of the LBD of
RXRs has been shown by Zhang
et al.
(
10
) to abolish receptor function (i.e. homodimerization, ligand binding and
transactivation). Therefore, the use of plasmid RXR[gamma][Delta]LBD acts as a proper negative vector control and serves to
highlight the contribution of endogenously expressed RXRs (
35
).
In control studies when COS-1 cells were transfected with the reporter plasmid ptkCAT relative CAT
activities in the presence of 0.5 mM 8-Br-cAMP or 50 nM OA compared with untreated cells were 2.1 +- 0.2 and 2.9 +- 0.18 respectively (data not shown). COS-1 cells (Fig.
5
) and C
2
C
12
muscle cells (Fig.
6
) were co-transfected with the expression vector RXR[gamma][Delta]LBD, RXR[gamma][Delta]AB or RXR[gamma] and the reporter plasmid pG18
2
-tkCAT. Transfection of reporter plasmid pG18
2
-tkCAT with an expression vector containing the RXR[gamma][Delta]LBD construct in the presence of 9-
cis
-RA and/or 8-Br-cAMP or OA (in COS-1 cells)
trans
-activated gene expression 1.6- to 2.3-fold and negligibly in COS-1 and C
2
C
12
cells respectively (Figs
5
and
6
). These experiments in COS-1 cells and C
2
C
12
cells verified the inability of RXR lacking the LBD to
trans
-activate gene expression. When cells were co-transfected with full-length RXR[gamma] and the reporter pG18
2
-tkCAT addition of 9-
cis
-RA induced a 7.2-fold increase in CAT expression in COS-1 cells (Fig.
5
). After treatment with either 8-Br-cAMP or OA CAT expression was increased only 2.8- and 1.8-fold respectively. However, these increases in
activation were not significant, as 8-Br-cAMP and OA stimulated CAT expression mediated by RXR[gamma][Delta]LBD ~2-fold (Fig.
5
). Simultaneous 9-
cis
-RA + 8-Br-cAMP or 9-
cis
-RA + OA treatment stimulated CAT expression mediated by RXR[gamma] 13.7- and 13.2-fold respectively (Fig.
5
). Whether this truly reflects a synergistic activation or simply a
generalized/indirect increase mediated by the non-specific effects of 8-Br-cAMP and OA on transcription in COS-1 cells is unclear. Co-transfection with RXR[gamma][Delta]AB and the reporter pG18
2
-tkCAT and addition of 9-
cis
-RA induced a 5.9-fold increase in CAT expression. Co-treatment with 9-
cis
-RA and 8-Br-cAMP or OA resulted in 14.2- and 8.8-fold increases respectively (Fig.
5
). These studies in COS-1 cells indicate that RXR[gamma][Delta]AB and RXR[gamma]
trans
-activate gene expression in a similar manner. These results are in
agreement with studies by Nagpal
et al.
(
13
), which showed that co-transfection of RXR[gamma] with the AB domain removed (RXR[gamma][Delta]AB) did not affect activation of an RXRE (DR-1) placed upstream of the tk promoter in COS-1 cells.
We have shown in these studies utilizing the GAL4 hybrid system that RXR[gamma] contains two transactivation functions, AF-1 and AF-2. The first of these is located in the N-terminal AB region (AF-1) and is constitutively active, independent of 9-
cis
-RA, while the second is located in the DE (LBD) region (AF-2) and functions in a ligand-dependant manner. These experiments are in agreement with
previous studies by Nagpal
et al.
(
8
,
13
), which identified two separate domains involved in transactivation by RXR[gamma], by linkage to the estrogen receptor DBD. Interestingly, the GAL-RXR[gamma]ABCDE and GAL-RXR[gamma]ABDE constructs were non-functional in the absence of the ligand,
9-
cis
-RA. This indicates that in the presence of the ligand-dependent AF-2 domain the function of AF-1 (AB region) is repressed in COS-1 cells.
We examined the effect of 8-Br-cAMP and OA on GAL-RXR[gamma] chimeras to assess the ability of phosphorylation to
modulate
trans
-activation of the modular AF-1 and AF-2 domains of RXR[gamma] in the GAL4 hybrid system. Although several studies
have reported that protein kinase C- and cAMP-dependant protein kinase pathways are involved in retinoid-mediated transcription (
19
-
21
), our studies are the first to address specific activation of the AF-1 and AF-2 domains of RXR[gamma] by phosphorylation. Our study has demonstrated that both the
AF-1 and AF-2 domains of RXR[gamma] are regulated either directly or indirectly by cAMP-dependent protein kinase cascades in a ligand-independent manner. By examination of the amino
acid sequence of mRXR[gamma] with respect to PKA consensus phosphorylation sites (RXS, RRXS or RXXS)
we identified some putative PKA targets (e.g. RTLS, RVIT, RQRS, RAES and RSVS)
in the AB and DE domains of the receptor, which we are evaluating by
mutagenesis and transfection studies. Although OA activated the AF-1 (AB) and AF-2 (LBD) regions in the absence of ligand, the generalized effects of
OA in the GAL hybrid system obscured the effects of this agent on receptor
function. There are various mechanisms by which 8-Br-cAMP and OA could influence transcriptional activation mediated by
the AF-1 and AF-2 domains of RXR[gamma]. The AF-1 and AF-2 domains of RXR[gamma] may be phosphorylated, with a consequent
change in receptor conformation/activity, whereby the ability to interact with
the transcriptional machinery and/or other accessory proteins is enhanced. The
use of phosphorylation enhancing agents like 8-Br-cAMP and OA may also phosphorylate proteins in the transcriptional
machinery (e.g. TF II A-J and/or TAFs), other accessory proteins and/or other protein kinase
pathways, resulting in a change in their activities.
In our study deletion analysis of the AF-1 domain of RXR[gamma] demonstrated that the AB region contained multiple motifs in the
first 103 amino acids that function synergistically to activate transcription
in a ligand-independent manner and are targets either directly or indirectly of cAMP-dependent protein kinase cascades. The AF-1 domain of RAR[beta] has been analysed using the GAL4 hybrid system in P19
embryonal carcinoma cells and has been shown to be located in the first 32
amino acids of the AB region (
14
), which is in contrast to our findings with AF-1 of RXR[gamma]. This indicates that the AF-1 domains of RXR[gamma] and RAR[beta] may function by different mechanisms.
Transfection experiments in COS-1 cells revealed that simultaneous treatment with 9-
cis
-RA and OA or 8-Br-cAMP produced a synergistic activation of an optimal RXRE
linked to tkCAT in an RXR[gamma]-dependent manner. However, whether the effects of these agents were
direct or indirect was masked by the effects of these phosphorylating agents on
the basal reporter in the absence of functional receptor. During the course of
this study two recent reports have indicated that OA is able to regulate the
DNA binding activities and/or function (independent of ligand) of RXR and RAR (
23
,
24
). Differences between those studies and ours probably reflect the use of
different response elements and/or cell lines, which have been demonstrated to
influence receptor-mediated
trans
-activation.
Important observations of these studies were the demonstration that: (i) RXR[gamma]
trans
-activated more efficiently in a myogenic background; (ii) the AB region of
RXR[gamma] functions in a cell-specific manner and is required for optimal ligand-dependant transactivation of an RXRE in muscle cells.
Specifically, full-length RXR[gamma] produced a 7.2- and 19-fold induction of G18 RXRE linked to tkCAT in COS-1 and myogenic C
2
C
12
cells respectively. Furthermore, deletion of the AB region reduced
trans
-activation from 19- to 11-fold in myogenic cells, whereas in non-muscle cells the impact of this deletion on
trans
-activation was minimal (7- versus 5.9-fold). Nagpal
et al.
(
13
), using COS-1 cells and RXREs, demonstrated that deletion of the AB domain of RXR[gamma] did not have any significant affect on the ability of the receptor
to activate transcription. Our results correlate with the preferential
expression of this isoform in skeletal and cardiac muscle. In COS-1 cells activity of GAL-RXR[Delta]AB is contradictory to the similar ability of native RXR[gamma] and RXR[gamma][Delta]AB to
trans
-activate gene expression (in accordance with Nagpal
et al.
;
13
). This discrepancy is probably a result of the different assay systems
involved; the GAL4 hybrid sytem examines the ability of a modular domain to
independently complement the GAL4 DBD and
trans
-activate gene expression, whereas, the other assay examines the function
of the entire receptor.
The results obtained with our RXR[gamma] deletion constructs in COS-1 and C
2
C
12
muscle cells indicate that the two AFs of RXR[gamma] function by different mechanisms, which could be explained by the
presence or absence of cell-specific auxiliary/accessory factors needed for
trans
-activation in the cell lines tested. The presence of an AF-1 in RXR[gamma] which preferentially activates transcription in a cell-specific manner suggests that RXR[gamma] may function in a programmed spatio-temporal manner.
We sincerely thank Drs David J.Mangelsdorf and Ronald M.Evans for the murine RXR[gamma]1 cDNA, Dr Pierre Chambon for the pSG5 expression vector containing
murine RXR[gamma]1 and Dr M.Klaus of Hoffmann-LaRoche Ltd for the 9-
cis
retinoic acid. This work was supported by the National Health and Medical
Research Council (NHMRC) of Australia. We also wish to thank Michael Downes and
Dr Amanda Carozzi for excellent technical assistance and helpful discussions.
REFERENCES
Return