Biology Reference
In-Depth Information
androgen receptor (AR, (NR3C4)) bind essentially as homodimers to response
elements containing palindromic repeats of the hexamerix half-site sequence
5
0
-AGAACA-3
0
(
Khorasanizadeh & Rastinejad, 2001
).
Dimerization is a general mechanism to increase binding site affinity, specificity,
and diversity due to (i) cooperative DNA binding, (ii) the lower frequency of two
hexamer-binding motifs separated by a defined spacer compared to that of single
hexamers, and (iii) heterodimers that may have recognition sites distinct from those
of homodimers. In this regard, RXRs play a central role in various signal transduction
pathways since they can both homodimerize and act as promiscuous heterodimeriza-
tion partner for almost fifteen NRs. Crystal structures of DBD and LBD homo- and
heterodimers have defined the surfaces involved in dimerization. It is important to
point out that the response element repertoire for receptor homo- and heterodimers
is dictated by the DBD while LBDs stabilize the dimers, but do not contribute to
response element selection. Two types of dimerization functions mediate homo-
and heterodimerization. One involves several surface residues in the DBD that
establish weak response element-specific interfaces with corresponding surfaces
in the partner DBD (
Fig. 2.1
). The second is a strong dimerization function in the
LBD
s of both partners that differs between homo- and heterodimers and to some
extent between the partners of RXR. We present here different procedures, which
comprise both cellular and biochemical approaches, to investigate the interactions
that occur between the NRs, that is, the formation of homodimers or heterodimers.
2.1
METHODS
2.1.1
Studies of NR-NR interactions through protein crystallization
One of the very effective structural methods to study protein-protein interactions is
based on the preparation of crystals of the complexes followed by their analysis
using X-ray beams. The determination of the high-resolution three-dimensional
(3D) structures that derive from this technique provides atomic-level information
on the protein-protein interface. The vast majority of NR-NR interactions are medi-
ated by the dimerization surface located in the LBD of receptors. In this regard, several
structures of homo- and heterodimers of NRs have been reported, thereby identifying
the structural organization of NR dimers. In particular, crystal structures of several
NRs in their homodimeric form (
Bourguet, Ruff, Chambon, Gronemeyer, & Moras,
1995; Brzozowski et al., 1997; Greschik et al., 2002; Nolte et al., 1998
) or containing
RXR LBD in complex with various partner LBDs, including RAR (
Bourguet, Vivat,
et al., 2000; Pogenberg et al., 2005; Sato et al., 2010
), PPAR (
Gampe et al., 2000
),
TR (
Putcha, Wright, Brunzelle, & Fernandez, 2012
), LXR (
Svensson et al., 2003
),
or CAR (
Suino et al., 2004; Xu et al., 2004
), have been reported. All these structures
demonstrated a topologically conserved dimerization surface with identical structural
elements generating the interface in homo- and heterodimers. However, amino acid
variations at the surface of the various NRs determine their specific dimerization
characteristics. An extensive analysis of the dimerization interfaces of NR LBDs,
