448
Antitumor Steroids
from unstimulated cells may derive from
its nuclear release at the time of homoge-
nization.
Conveyance of ER into the cell nucleus is
governed by speciFc amino acid sequences
and nuclear localization signals (NLSs)
situated between its DNA and hormone
binding domains (HBDs) (hinge region).
The existence of nuclear export signals
(NESs) has also been reported; both NESs
and NLSs play an important role in nuclear
export of the steroid receptors. Nuclear
uptake of receptors requires ATP, while
nuclear export apparently occurs in the
absence of energy production.
Investigations
performed
on
ER-
transfected cells revealed an additional
subnuclear
movement
of
the
receptor
under ligand stimulations. E
2
changes
the
receptor
pattern
from
a
diffuse
nucleoplasmic aspect to a hyper-speckled
distribution, suggesting that the ligand-
dependent organization of the receptors
within
the
nuclear
matrix
involves
more
complex
events
than
a
simple
recognition of
DNA-binding sites
and
coregulators. Interestingly, such a hyper-
speckled distribution
also
occurs
with
the partial antiestrogen OH-Tam, while
a
substantial
effect
is
solely
detected
with the pure antagonist ICI 182,780.
In this context, it should be stressed
that several other investigations localize
the receptor within the cytoplasm under
exposure to pure antiestrogens. Hence,
the cellular localization of the receptor
under
pure
antiestrogens
is
still
not
established, although the tendency for a
major distribution within the cytoplasm
dominates (when downregulation does not
occur, see Sect. 3.4).
Concepts outlined here suggest that
linkage of cytotoxic residues aimed at
altering
DNA
and
associated
proteins
should be restricted to estrogens (and
SERM), pure antiestrogen having only
a chance to strongly associate to the
nuclear matrix.
3.3
Ligand-induced Regulation of Estrogen
Binding Capacity
Shuttling of ER between the cytoplasm
and the nucleus influences its ligand-
binding ability. The nuclear localization
of a receptor is associated with a loss of
ligand-binding ability, which is restored
by its return into the cytoplasm (dephos-
phorylation/phosphorylation
cycle).
Af-
ter several cycles, ER is degraded by
the
proteasomal
pathway,
most
prob-
ably
into
the
cytoplasm
(half-life
3
4 h); ubiquitination of the receptor fa-
vors
its
shipment
to
the
proteasome.
Inhibitors of protein synthesis (cyclohex-
imide,
puromycin)
interfere
with
this
mechanism
leading
to
a
stabilization
of the receptor within the cell nucleus
in
a
form
devoid
of
strong
ligand-
binding ability.
As stated above (Sect. 2.2), E
2
is stably
incorporated within the HBD, decreasing
the chance of exchange with unbound
ligands (closing of the binding pocket).
Such a property Fxes the majority of the
receptor molecules into a conformation ap-
propriate for a speciFc anchorage within
the cell nucleus (hyper-specked pattern) to
accomplish functions imposed by the hor-
mone. A progressive decrease of estrogen
binding capacity subsequently occurs. As
expected, this decrease is closely related
to the binding afFnity of the estrogen for
the receptor, which is a major determi-
nant of its nuclear stabilization. One may
anticipate that this receptor form, unable
to bind the hormone is subjected to a
return into the cytoplasm to be recycled
or degraded.
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