Download Evolutionary Conservation in Retinoid Signalling and Metabolism

AMER. ZOOL., 39:783-795 (1999)
Evolutionary Conservation in Retinoid Signalling and Metabolism1
BARBARA R. BECKETT AND MARTIN PETKOVICH 2 *
Cancer Research Labs, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada
•Tel: 613 545 6791, Fax 6830—to whom correspondence should be addressed
SYNOPSIS. In accord with the notion that retinoid signalling is of central importance in vertebrate evolution, a number of its components are evolutionarily conserved. Retinoid X nuclear receptors (RXRs), which interact directly with a number of signalling pathways, are highly conserved among mammals, Xenopus, and
chick. We have studied RXRs in zebrafish and find that they are also very well
conserved with respect to amino acid sequence and function, compared to mammalian RXRs. However, zebrafish has additional subtypes (RXRS and RXRe)
which are altered in structure and function. New information which has come to
light since these were first described suggests ways in which these unique subtypes
could fine-tune retinoid signalling in zebrafish. We have performed phylogenetic
analysis with the zebrafish RXRs and RXRs from other species to try to understand
the evolutionary relationships among them. In addition, we have found a retinoic
acid (RA)-inducible, RA-metabolizing cytochrome P450 (P450RAI/CYP26) which
is evolutionarily conserved among vertebrates and has an important role in controlling retinoid signalling by regulating the level of biologically available RA.
INTRODUCTION
The retinoid signalling system
Retinoid signalling involves a complex
system of interacting proteins and small
molecules which play critical roles in the
development of a diverse range of organisms. The components of this system include two families of nuclear receptors, interacting nuclear proteins which modulate
the transcriptional activity of the receptors,
a number of high affinity cellular retinoid
binding proteins, an array of naturally occurring ligands, and enzymes which synthesize and metabolize these ligands. We discuss the evolutionary conservation of two
of these components that we have studied
in zebrafish (Danio rerid), the retinoid X
receptors and cytochrome P450RAI
(CYP26), and their possible roles in retinoic
acid signalling.
Biological effects of vitamin A
Vitamin A (retinol) is essential for a
number of biological processes, including
' From the Symposium Evolution of the Steroid/Thyroid/Retinoic Acid Receptors presented at the Annual
Meeting of the Society for Integrative and Comparative Biology, 3-7 January 1998, at Boston, Massachusetts.
2
E-mail: petkovic@post.queensu.ca
growth, vision, reproduction, hematopoiesis, immune function, and maintenance of
epithelial tissues. Retinoic acid (RA), the
principal active metabolite of vitamin A, affects the growth and differentiation of many
different cultured mammalian cell lines in
vitro and has been found to affect the expression of a wide variety of genes (see Gudas et al., 1994). RA also has spectacular
effects on vertebrate development, influencing the establishment of the embryonic axes
and morphogenesis of the nervous system
and limbs (for review see Hofmann and Eichele, 1994). The effects of vitamin A have
been observed in conditions of vitamin A
excess and vitamin A deficiency (for references see Chambon, 1994). During embryonic development vitamin A deficiency
results in congenital malformations of many
structures including the eye, heart, and genito-urinary tract. Administration of teratogenic doses of RA during mammalian embryonic development results in craniofacial,
cardiac, thymic, limb, and central nervous
system malformations (Means and Gudas,
1995). Recently, RA has been found to be
effective in the prevention and treatment of
certain cancers (Hong et al, 1990; Meyskens and Manetta, 1995; Warrell, 1996).
Clearly, it is of interest to understand at a
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B. R. BECKETT AND MARTIN PETKOVICH
molecular level the effects of a substance forming heterodimers with other nuclear rewhich has such wide-ranging and important ceptors (Leid et al, 1992; Mangelsdorf et
effects on adult and developing organisms. al, 1992a). Unlike RARs, which bind both
aU-trans and 9-cis RA, RXRs are selective
Retinoic acid receptors
for the 9-cis RA isomer. In vivo, the role of
The discovery of nuclear receptors for 9-cis RA in the activation of RXRs has not
retinoic acid was a crucial leap forward in been fully established, although synthetic
our understanding of how RA affects gene retinoids with RXR selectivity appear to act
transcription (Giguere et al, 1987; Petkov- through these receptors in vivo (Jones and
ich et al, 1987). These RA receptors Petkovich, 1996; Lala et al, 1996). In ad(RAR) are members of the large steroid/ dition to its actions as a heterodimer partner
thyroid receptor superfamily and are acti- for other nuclear receptors, RXR is also cavated by the binding of RA. There are three pable of homodimerization (Lala et al,
forms of RARs; a, (3, and 7 subtypes, each 1996; Mangelsdorf et al, 1992a; Rottman
of which has been found to have distinct et al, 1991; Zhang et al, \992b), although
tissue distribution and developmental pat- the biological significance of this homoditerns of expression (Ang and Duester, 1997; merization is unclear (Leblanc and StunDolle et al, 1990; Mendelsohn et al., 1992; nenberg, 1995). The result of this promisRuberte et al, 1990, 1991, 1993). The a, cuity in its interactions with other nuclear
(3, and 7 subtypes in turn consist of a num- receptors is that the RXR signalling pathber of isoforms generated by alternate splic- ways intersect with, and is able to influence,
ing and promoter usage (Leid et al., 1993). many other signalling systems. Among
The RARs are classified as type II nuclear these are vitamin D signalling (through inreceptors, along with retinoid X receptors teraction with VDR); thyroid hormone sig(RXRs, see below), vitamin D receptors nalling (through TR); adipogenesis, fatty
(VDRs), thyroid receptors (TRs), peroxi- acid metabolism, and glucose homeostasis
some proliferator-activated receptors (through PPAR; Mangelsdorf and Evans,
(PPARs), and many orphan receptors for 1995; Mukherjee et al, 1997); growth facwhich ligands have not been identified tor signalling pathways (NGFI-B and
(Mangelsdorf and Evans, 1995; Meier, NURR1; Perlmann and Jansson, 1995); and
1997). Type II receptors recognize a se- cholesterol homeostasis (LXR; Janowski et
quence on the target DNA (response ele- al, 1996; Lehmann et al, 1997). Like
ment) consisting of two half sites with the RARs, RXRs are also expressed in distinct
consensus AG(G/T)TCA, arranged as direct tissue and developmental patterns (Manor inverted repeats separated by a variable gelsdorf et al., 1992a).
but characteristic number of nucleotides
(Naar et al, 1991; Umesono et al., 1991). Ligand Modulation in RA signalling: RA
These receptors bind DNA in the absence Metabolism and P450RAI
of ligand and form heterodimers with RXRs
There is also diversity in naturally oc(Mangelsdorf and Evans, 1995).
curring retinoids, ligands for retinoid receptors. Among those which have been idenRetinoid X receptors
tified are ail-trans and 9-cis RA, 3,4-dideRXRs, first identified in 1990 (Mangels- hydroretinoic acid (Thaller and Eichele,
dorf et al., 1990), are necessary for high- 1990); 14-hydroxy-refro-retinol and anhyefficiency DNA binding and transcriptional droretinol (Eppinger et al, 1993); 13,14-diactivation of a large number of nuclear re- dydroxy-retinol (Derguini et al, 1995); 4ceptors in the type II category (see above; oxoretinaldehyde (Blumberg et al, 1996);
Bugge et al., 1992; Kliewer et al., 1992; 4-oxoretinol (Achkar et al, 1996); and 4Kliewer et al., 1992; Mangelsdorf and oxo-retinoic acid (Pijnappel et al, 1993). A
Evans, 1995; Marks et al, 1992; Yu et al, large number of synthetic retinoids specific
1991; Zhang et al, 1992a). Like RARs, to RAR and RXR subtypes have been synthey exist in a number of different subtypes, thesized, and among these are compounds
designated a, (3, and 7, each capable of whose function depends on the receptor
RETINOID SIGNALLING AND METABOLISM
context. For example, the synthetic retinoid
LG100754 is an antagonist in the context
of RXR/RXR homodimers, but it activates
RXR/PPAR heterodimers ("mixed function
retinoid"; Lala et al, 1996). Other synthetic retinoids can antagonize transactivation
by RAR, but retain the ability to block the
activity of the transcription factor API,
characteristic of RA ("dissociating retinoids"; Chen et al, 1995). Naturally occurring retinoids may also prove discriminating. For example, 4-oxo-RA, although a
weak agonist, may have selectivity for
RAR3 (Pijnappel et al, 1993). The relevance of this selectivity in vivo is not clear
at present and will ultimately depend on the
intracellular concentrations of RA metabolites.
The pathways responsible for synthesis
and metabolism of RA and other naturally
occurring retinoids have been the subject of
investigation for many years. It is known
that RA is synthesized from retinal and retinol precursors and that the process may involve cellular retinol-binding protein
(CRBP) (Napoli, 1996). Some candidate
enzymes have been identified (Ang and
Duester, 1997), but the processes have not
been well characterized at the molecular
level. The oxidative pathways responsible
for metabolizing RA have been well-characterized biochemically and are known to
involve RA-inducible cytochrome P450
(Roberts et al., 1979a; Roberts et al,
1979b) and intermediates whic include 4OH-RA, 4-oxo-RA, 18-OH-RA, and possibly 5,6-epoxy RA (Fiorella and Napoli,
1994; Frolik et al, 1979; Kurlandsky et al.,
1994). Recently a RA-inducible, developmentally-regulated cytochrome P450
(P450RAI or Cyp26) has been cloned and
been shown to metabolize RA to 4-OH-RA,
4-oxo-RA, and other metabolites in vitro
(Abu-Abed et al., 1998; Fujii et al., 1997;
Ray et al., 1997; White et al., 1997; White
et al., 1996). Its expression in mammalian
cells (Stuart, Chithalen, White, Jones, and
Petkovich, personal communication) and in
developing mouse embryos (Fujii et al.,
1997; B.B. and MR, unpublished data)
suggests that P450RAI is responsible for
RA metabolism occurring in RA target tissues.
785
Interacting proteins
In addition to the complexity resulting
from multiple receptors and multiple ligands, retinoid signalling is complicated by
a number of other interactions which are
shared with other nuclear receptors. The interaction between nuclear receptors and the
transcriptional apparatus recently has been
found to be mediated by adaptor proteins.
These include co-activator and co-repressors and co-integrator proteins which serve
as a platform for assembly of these positive
and negative effectors (Glass et al., 1997;
Korzus et al, 1998; Kurokawa et al, 1998;
Shibata et al, 1997; see references in Wolffe, 1997). There is evidence that some coactivators and co-repressors may have tissue-specific expression or be developmentally regulated. In the case of ACTR and
N-CoR this has been directly observed
(Chen et al, 1997; Soderstrom et al, 1997).
Tissue-specific expression of co-regulators
has been inferred from the cell-dependent
agonistic and antagonistic properties of the
antiestrogen tamoxifen.
The interactions between nuclear receptors and these modulatory proteins may be
allosterically affected by both ligand and
DNA sequence. In the case of retinoid receptors, different ligands induce distinct interactions with co-repressors (Lala et al,
1996), suggesting that specificity in the ligand-induced conformational changes that
results in differences in the complement of
interacting proteins. In addition, the nature
and affinity of binding of co-repressors to
receptors is influenced allosterically by the
sequence of the DNA to which the receptor
is bound (Kurokawa et al, 1995). On DR5
response elements, RAR/RXR heterodimers
dissociate from the co-repressor N-CoR in
response to ligand, whereas on DR1 elements this dissociation does not occur.
In addition to these interacting nuclear
proteins, there are high-affinity retinol(CRBP) and RA-binding proteins (CRABP)
in the cytoplasm which are developmentally
regulated (Ruberte et al, 1993) and may
have some role in RA synthesis and/or metabolism (Napoli, 1996). However, their biological role is poorly understood; mice in
which both CRABP forms have been de-
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B. R. BECKETT AND MARTIN PETKOVICH
leted by homologous recombination have
an essentially normal phenotype (Lampron
et al, 1995).
RESULTS AND DISCUSSION
H7 REGION OF LBD:
283
RXKS
RXRE
RXRa
RXRy
309
PKESTHNLGVEAFFDRESSHSAEVGALFDRVLTEL
HRNSAHSAGVGAIFDRESAHNAEVGAIFDRVLTEL
HRSSAHSAGVGSIFDR
VLTEL
HRNSAHTAGVGAIFDR
VLTEL
Evolutionary conservation of zebrafish
C-TERMINDS:
RXRs
413
422
The preceding discussion gives some inFLMEMLESPH
dication of the complexity of the retinoid RXR5
RXRE
FLMEMLEAPHOLT
signalling system with regard to receptors, RXRa
FLMEMLEAPHOIT
EiHEiaEAPHOIT
ligands, and interacting proteins. RXRs RXRy
play a unique role in linking several sig- FIG. 1. Amino acid sequence of zebrafish RXRs (see
nalling pathways together, including that for Jones et al., 1995). The sequence from the regions
RA. If RXRs in vivo are indeed activated where RXRS and/or RXRe differ significantly from
of mammalian RXRs is shown: 1) the region of
by 9-cis RA, as much of the in vitro data that
the ligand binding domain (LBD) in conserved helical
suggest, RA will play a central role in in- domain H7 (Bourguet et al., 1995) in which RXR8 and
tegrating these pathways.
RXRe have 14 additional amino acids, and 2) the CIn our lab we have cloned RXRs from terminus. The underlined region is the conserved AFzebrafish and we find that the subtypes are 2 core motif (Durand et al, 1994).
somewhat different from those which have
been cloned from other vertebrates. Unlike amino acid truncation at the C-terminus
mammals which have three subtypes of compared to RXRe and the other zebrafish
RXR (Leid et al, 1992; Mangelsdorf et al, and mammalian RXRs. The zebrafish
1992), zebrafish have at least 5 distinct sub- RXRs do not have an obvious correspontypes. These include the a and 7 subtypes dence with mammalian RXRs a, (3, or 7
which we have previously described and based on amino acid sequence, and the deswhich bind 9-cis RA and are transcription- ignation of zebrafish RXRa and RXR7 are
ally active; the 8 and e subtypes which do based on affinity for 9-cis RA and their exnot bind 9-cis RA and are transcriptionally pression levels of expression in developing
inactive; (Jones et al., 1995) and an addi- embryos (Jones et al., 1995) and regenertional subtype which has not been charac- ating caudal fin. The latter is shown in the
terized (Jos Joore, personal communica- northern blot in Figure 2, which shows that
tion). The 8 and e subtypes have a 14 amino (like mammalian RXRa) zebrafish RXRa
acid insert in an alpha-helical region in the mRNA is abundantly expressed, whereas
middle of the ligand-binding domain, in the RXR7 mRNA is expressed at much lower
conserved alpha-helical region designated levels.
H7 in X-ray crystallographic studies (Fig.
In order to understand the phylogenetic
1; Bourguet et al., 1995). At the amino acid relationships among the zebrafish RXRs
level this insert appears to be an approxi- and those which have been isolated from
mate repeat of the preceding region. X-ray other species, we performed neighbor-joincrystallographic studies which have been ing analysis of the RXRs with the complete
done on the ligand-binding domains of amino acid sequences of the receptors using
RXRa and RAR7 show that while the H7 ClustalX and excluding gaps. Figure 3
helical domain in which the 14 amino acid shows the results of this analysis. Zebrafish
insert of RXRS and RXRe is found, does RXR7 appears from the phylogenetic analnot actually form part of the ligand binding ysis to be more closely related to mammapocket, it is very close by, (Wurtz et al, lian RXRa while zebrafish RXRa and the
1996) and it would not be surprising if it uncharacterized zebrafish receptor designatresulted in a subtle change in the confor- ed only "RXR" in Figure 2, group with
mation. This could explain the inability of mammalian RXR7 in this analysis (also in
RXRs 8 and e to bind 9-cis RA in vitro. It parsimony analysis; V. Laudet, personal
should also be noted that RXRS has a 3- communication). Note that the evolutionary
787
RETINOID SIGNALLING AND METABOLISM
RXRa
RXRy
RXR5
RXRe
N R
N R
N R
NR
9 kb
6.5kb-v
3.4 kb
2.2 kb
FIG. 2. Northern blot of zebrafish RXRs expressed in normal (N) and regenerating (5 days post-amputation;
R) zebrafish caudal fin. Northern blots were done using 3 u.g of poly A+ RNA, using the Quik-Hyb (Stratagene)
kit as previously described (Jones et al., 1995). Blots were exposed to Kodak X-Omat film for 24 hr. Expression
of zebrafish RXRs was not upregulated in regenerating caudal fin, as in the zebrafish RARs (White et al., 1994)
but in contrast to the RA-inducible, RA-metabolizing cytochrome P450RAI (White et al., 1996).
ZRXR5
ZRXRY
xRXRa
/ hRXRa
/n686
hRXR0
zRXR
0.1
FIG. 3. Phylogeny of RXRs based on complete coding sequence, showing the relationship between zebrafish
(z), mammalian (h, human), Xenopus (x) and chick (c) RXRs. The zebrafish RXR labelled only "RXR" has
not been characterized (Jos Joore, personal communication). The tree was plotted using neighbor joining analysis
and the ClustalX package, eliminating gaps. The bar represents 10% difference in sequence and the numbers
represent bootstrap values. The Genbank accession numbers for the sequences are: hRXRa (S09592), hRXRfi
(P28702), hRXR-y (P48443), xRXRp (P51128), xRXRp (S73269), xRXR-y (P51129), cRXRP (P28701).
788
B. R. BECKETT AND MARTIN PETKOVICH
distance between the zebrafish receptors
and mammalian RXR7 is much larger than
between zebrafish RXR7 and mammalian
RXRa. The non-ligand-binding receptors
RXR8 and RXRe, although they share a
similar 14 amino acid insert in the same
location in the ligand binding domain, are
actually quite distantly related to each other.
They both group with the mammalian
RXRp by this analysis and by parsimony
analysis (V. Laudet, personal communication). Interestingly, it has been found in
mapping studies of the zebrafish genome
that RXRe and mammalian RXRp map in
regions of the genome which appear to be
syntenic (J. Postlethwaite, personal communication), giving credence to the idea
that RXRe may be evolutionarily related to
mammalian RXR(3. It has been proposed
that the ancestral nuclear receptor did not
have a ligand, and that ligand-binding nuclear receptors have evolved more recently
(Escriva et al, 1997). Perhaps RXR8 and
RXRe are "fossils" in the RXR family, representing an older, non-ligand binding form
of the RXR. However, when neighbor joining analysis is performed using members of
the USP family (the arthropod homolog of
RXR) in addition to RXRs (Fig. 4), it is
clear that the zebrafish "orphan" 8 and e
receptors are no more closely related to the
non-ligand binding USP family than are
other RXRs.
The organization of the introns and exons
comprising a gene also give some indication of the evolutionary relatedness of
genes. In the case of the mouse RXR7 and
RXRa genes, the genomic structure has
been found to differ significantly from most
other nuclear receptors, for example RAR
(Lehmann et al, 1991; van der Leede et al,
1992), androgen receptor (Lubahn et al.,
1989), and estrogen receptor (Ponglikitmongkol et al., 1988), in two ways: 1) the
intron within the DNA binding (C) domain
is located within the first zinc finger, instead
of between the two zinc fingers, and 2)
there are five introns in the RXR genes instead of the usual four (Liu and Linney,
1993). Examination of a RXRS genomic
clone reveals that the intron within the first
zinc finger is conserved between zebrafish
and mouse, suggesting that the appearance
of RXRs 8 and e occurred after the divergence of the RXRs from other nuclear receptors.
The fact that there are more RXR genes
in zebrafish than in other vertebrates is consistent with what has been found in other
gene families isolated from zebrafish. For
example, there are more members of the
homeodomain protein families Dll (distalless), Msx (muscle segment homeobox),
and En (engrailed) in zebrafish than in
mammals, and more Hox clusters (M. Ekker, personal communication). In addition,
in mapping studies of the zebrafish genome,
blocks of genes are found to be repeated (J.
Postlethwaite, M. Ekker, etc., manuscript in
preparation), indicative of an ancient genome duplication in zebrafish sometime after the divergence of the teleost and mammalian lines.
Possible importance of zebrafish RXRS
and RXRe
Since RXRS and RXRe were cloned, Xray crystallographic studies (Bourguet et
al., 1995; Renaud et al., 1995; Wurtz et al,
1996) have localized ligand binding pockets and other studies have indicated that
there may be a more important role for the
C-terminal domain of RXR in a heterodimer pair than was previously realized. In
light of this new information, it becomes
easier to appreciate that variant RXRs such
as 8 and e may have an important biological
role.
For some time it was thought that as a
member of a heterodimer pair, RXR was a
"silent partner," with RXR allosterically
blocked by its partner from interacting with
its ligand and activation of the heterodimer
pair occurring solely as a result of the partner's ligand (Forman et al, 1995; Leblanc
and Stunnenberg, 1995). This has been
shown very elegantly with direct assays of
the binding of receptor-specific ligands and
RAR/RXR heterodimers (Kurokawa et al,
1994). However it has been known for several years that under certain circumstances
RXR can be an active partner in the RAR/
RXR heterodimer, for example in the presence of RXR ligand (Forman et al, 1995)
or on certain retinoic acid responsive elements (RAREs) (Durand et al, 1994). Also
789
RETINOID SIGNALLING AND METABOLISM
hRXRy
cRXRoc.
xRXRy.
zRXRy
hRXRa
xRXRa
zRXRa
zRXRS
V^xRXRp
hRXRp
mUSP
bUSP
dUSP
FIG. 4. Phylogenies of RXRs and USPs (see Fig. 3 for details). The Genbank accession numbers for the USP
sequences are: bUSP (Bombyx mori. P49700), mUSP {Manduca sexla, P54779), dUSP (Drosoplula, P20153).
with certain heterodimer partners such as
LXR, PPAR, and NGFI-B (Forman et al,
1995; Kliewer et al, 1992; Perlmann and
Jansson, 1995; Willy et al, 1995) RXRspecific ligands can in fact bind to and activate the heterodimer. There has also been
considerable circumstantial evidence for an
active partner role for RXR in biological
responses in which RAR and RXR ligands
act synergistically, for example in the
growth and differentiation of cell lines (Apfel et al, 1995; Lotan et al., 1995; Roy et
al., 1995) and in the activation of RA genes
(Roy et al., 1995). Recently there has been
direct evidence using receptor-specific synthetic ligands and receptors mutated in their
C-terminal activation domains, that RXR
can be active in RAR/RXR heterodimers
(Schulman et al., 1996) and that the AF2
domains of both receptors contribute to activation (Botling et al., 1997). Additionally,
fluorescence-based, in vivo footprinting,
and proteolytic analyses of RAR/RXR heterodimers have shown physical changes
following treatment with RXR ligands, indicative of ligand binding to the RXR part-
790
B. R. BECKETT AND MARTIN PETKOVICH
ner in the heterodimer (Kersten et al, 1996;
Minucci et al, 1997). It seems that the ability of RXR in a heterodimer partner to respond to ligand can depend on the relative
orientation of the receptors, the activation
status of the heterodimer partner, and the
nature of the response element (La VistaPicard et al., 1996).
The C-terminal activation domain, AF-2,
is required for ligand-induced binding of
co-activators and ligand-induced release of
co-repressors from heterodimers. SRC-1
and other co-activators bind to both RXR
and its heterodimer partners, and this binding occurs in the AF-2 region (Horwitz et
al., 1996; Jeyakumar et al, 1997; Mengus
et al, 1997; Shibata et al, 1997).
Thus it seems that RXR is likely an active partner with the other receptors in the
heterodimer pair, in ligand binding, and in
determining interactions with both co-activators and co-repressors. The zebrafish
RXR8 receptor is truncated at the C-terminus and is lacking three amino acids forming part of the activation domain AF-2,
which are conserved in other zebrafish and
mammalian RXRs. RXR8, like other RXRs
which have small C-terminal deletions, can
have a dominant negative effect on transcription (Durand et al, 1994; Leng et al,
1995; Zhang et al, 1994). If RXR was considered to be a silent partner necessary only
to enhance DNA binding, these observations would be puzzling, but in light of the
importance of AF-2 for release of co-repressor, the dominant negative effect is easily explained.
Another observation for which we can
now offer an explanation based on new information in the literature, is the apparent
increase in the TR-stimulated activity of
TR/RXR8 and TR/RXRe heterodimers by
9-cis RA. This was difficult to explain
when the accepted wisdom was that RXR
was a silent partner, and was also puzzling
because RXR8 and RXRe both failed to
bind 9-cis RA in in vitro assays. RXR and
its heterodimer partners may allosterically
influence each other. The first indication of
this came from a study in the Evans lab
(mentioned above; Forman et al, 1995) in
which it was noted that ligand binding
properties of RXR in a RAR/RXR hetero-
dimer were altered by ligand binding of the
RAR partner. Subsequently it has been
found that binding of a ligand specific to
RXR can conformationally alter its heterodimer partner (RAR, LXR, or OR1), resulting in binding of co-activator, release of
co-repressor, and activation. This has been
called the "phantom ligand" effect (Schulman et al, 1997; Wiebel and Gustafsson,
1997; Willy and Mangelsdorf, 1997). In
view of these conformational changes imposed by receptors on their heterodimer
partners, it is possible that RXRS and RXRe
might bind ligand in vivo as part of a heterodimer pair, even though they are unable
to bind 9-cis RA in vitro.
Clearly more experiments are needed to
determine the biological role of RXRS and
RXRe. They may have altered affinity for
ligand, co-activator, or co-repressor in heterodimers, and may provide an additional
way of fine-tuning the RA signalling pathway in zebrafish. Their high level of expression in zebrafish caudal fin (Fig. 2) suggests they are biologically significant. It
will be interesting to determine whether
other types of fish and other vertebrates also
express related RXRs.
P450RAI: evolutionary conservation of RA
metabolism
The enzymes involved in synthesis and
metabolism of RA and other biologically
active retinoids are an important part of the
RA signalling system, and if the RA signalling system is evolutionarily conserved,
any enzyme which has a critical role in regulating RA levels should also be conserved.
Recently (White et al, 1996; see above) we
have cloned a new member of the cytochrome P450 family (P450RAI) from zebrafish. It has been designated CYP26 by
the official cytochrome P450 nomenclature
committee. P450RAI has features which indicate that it could play an important role
in RA metabolism: 1) It is rapidly induced
by retinoic acid (Abu-Abed et al, 1998;
White et al, 1996) 2) It is developmentally
regulated in zebrafish and mouse (White et
al, 1996; M.P., unpublished data) 3) When
expressed in transfected cells it rapidly metabolizes RA to 4-OH-RA, 4-oxo-RA, 18OH-RA, and water soluble metabolites
791
RETINOID SIGNALLING AND METABOLISM
3374
hP450RAI
E1
mP450RAI '
67%
E2
E3
51%
E4
65%
E5
30%
E6
59%
E7
31%
3446
FIG. 5. Conservation of genomic structure between human and mouse P450RAI. The numbers above and below
the lines represent the first and last nucleotide of the coding region of the human or mouse genes. E1-E7 refer
to exons 1—7, and the lines between the exons represent introns. The numbers below the representation of the
mouse gene refer to the % nucleotide identity between human and mouse introns. Everything is drawn to scale.
(Abu-Abed et al, 1998; White et al, 1997;
White et al, 1996) and 4) Expression of
P450RAI message and RA metabolic activity are correlated in various cultured cell
lines (Abu-Abed et al, 1998; M.P., personal
communication).
When the amino acid sequence of
P450RAI from zebrafish, human, and
mouse is compared (Abu-Abed et al.,
1998), it can be seen that there is a high
level of homology in all three vertebrate
species, with overall amino acid identity of
68% between zebrafish and human, and
93% between mouse and human. Regions
of nonhomology are short and are distributed throughout the length of the cDNA.
Not only is the amino acid sequence of
P450RAI conserved among the three species, but also the metabolic activity appears
to be identical. It is RA-inducible in all
three species, and at least in zebrafish and
mouse, the developmental expression profile is similar (White et al, 1996, #62; D.
Lohnes, personal communication).
We have sequenced genomic clones of
P450RAI from both mouse and human, and
we find that there is remarkable conservation of the genomic structure and sequence.
This is shown diagrammatically in Figure
5, in which it can be seen that the location,
size, and sequence of the introns are conserved. Intron locations are identical, intron
sizes are almost identical, and the nucleotide sequence varies between 30% and 67%
identical. The presence of only 6 introns
(like CYP1A) makes it one of the simplest
mammalian cytochromes (Degtyarenko and
Archakov, 1993). In addition, the sequence
of the proximal promoter is very similar
among zebrafish, mouse, and human, with
a classic DR5 RARE close to the transcription start site (White et al, manuscript in
preparation). The gene is very small, with
the total size of the coding portion of the
gene being 6.5 kilobases.
CONCLUSIONS
The retinoid signalling system is very
complex, with many components including
cytoplasmic and nuclear binding proteins,
interacting nuclear proteins, and enzymes
which synthesize and metabolize retinoids.
Among the nuclear proteins that translate
the retinoid signal into regulation of gene
activity are retinoic acid receptors (RARs)
and retinoid X receptors (RXRs). RXRs
also interact with a wide range of other signalling systems acting through type II nuclear receptors. We have found that the amino acid sequence of RXRs in zebrafish is
highly conserved compared to those of
mammals, although there are differences
among the subtypes, including two novel
subtypes with altered function. In addition,
we have found that a newly discovered cytochrome P450 (P450RAI, CYP26), which
has properties consistent with its being an
important regulator of RA concentration in
vivo, is also conserved among zebrafish,
mouse and human with regard to amino
acid sequence, function, and expression.
ACKNOWLEDGMENTS
We thank Dr. Jim Gerlach for help with
the phylogenetic analysis, and Jay White
for the sequence of the human P450RAI
792
B. R. BECKETT AND MARTIN PETKOVICH
gene. M.P. was supported by grants from
the National Cancer Institute of Canada and
from the Medical Research Council of Canada.
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