Biology Reference
In-Depth Information
and activate transcription of target genes. Yet they are not redundant.
Not only have the three copies been kept over 400 million years of ver-
tebrate evolution, but knock-out (KO) experiments show paralog-
specific phenotypes, mostly affecting development. 50 Like other nuclear
receptors, RARs have a DNA-binding domain and a ligand-binding
domain. The latter contains about 270 amino acids in RARs, and is com-
posed of 12
-helices. Of these, 25 amino acids in the hydrophobic
ligand-binding pocket make direct contact with the ligand. The binding
pockets of the human RAR paralogs differ in three of these 25 positions.
They also differ in their in vitro binding to different synthetic retinoids,
and in the resulting transactivation of target genes.
To gain further insight into the evolution of these differences, we
compared all chordate RARs, including vertebrates, amphioxus, and
tunicates. 51 The phylogeny confirms the dating of the duplications at the
origin of vertebrates, with single-copy orthologs in amphioxus and tuni-
cates. The amino acid sequence of the ligand-binding domain of the
RAR immediately predating the duplications was predicted using maxi-
mum likelihood, and synthesized. As expected, all homologs and the pre-
dicted ancestral protein transactivate with all-trans retinoic acid, with
similar EC50 values. On the other hand, the ancestral, amphioxus, and
tunicate RARs do not transactivate in the presence of retinoids that are
specific to human RAR
α
-specific
retinoid, with strong transactivation in amphioxus RAR and with weaker
transactivation in tunicate RAR and predicted ancestral RAR. Targeted
mutations of the amphioxus RAR show that the three positions identi-
fied in human are indeed key to the differences in specificity. These
results suggest that RAR
α
or RAR
γ;
they do bind the RAR
β
is closest to the ancestral function, with
changes evolving by point mutations in the ligand-binding pocket of
RAR
β
. This is confirmed by limited proteolytic experiments,
which show that all RARs do bind the
α
and RAR
γ
-specific retinoid, even when it is
not sufficient for transactivation, but do not bind the other specific
retinoids. Interestingly, in situ hybridization shows that the expression
pattern of amphioxus RAR is most similar to that of RAR
β
. Thus, it
appears that, after whole-genome duplications, one copy remained simi-
lar to the ancestral function in terms of both sequence and expression
pattern, while the other two acquired derived characteristics. 51
β
Search WWH ::




Custom Search