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predominates, as some data suggest.
156
It is even harder to explain mecha-
nistically how ERK1/2 can prefer both receptor-bound and D7-like con-
formation over basal; existing data suggest that the arrestin domains shift in
opposite directions in these two states.
34,35
It is becoming increasingly clear
that without direct structural information obtained by cocrystallization of
arrestins with different partners these questions will remain unanswered.
4.4. Binding and activation do not go hand-in-hand
The original explanation for why arrestin-3 promotes JNK3 phosphoryla-
tion while the closely related arrestin-2 does not was beautifully simple; it
was suggested that arrestin-2 does not bind this MAP kinase,
148
and this idea
appeared to be supported by the identification of putative JNK3-binding
sequence RRS present in the C-domain of arrestin-3, whereas arrestin-2
has KP in homologous position.
159
However, RRS is only found in rodent
arrestin-3, while the prevalent sequence in mammalian species is RS.
12
Sub-
sequent studies showed that arrestin-3 from other species also binds
JNK3
134,163
and effectively facilitates its activation.
136,158,162
In fact, all four
mammalian arrestins were found to bind JNK3 comparably and remove it
from the nucleus, where it spontaneously localizes, to the cytoplasm.
134,161
This was the first indication that JNK3 binding and its activation are two
distinct functions of arrestins. The idea that arrestin-2 might not bind the
upstream kinases ASK1 and MKK4, and therefore fails to assemble the com-
plete MAPK module necessary for JNK3 activation, also was not supported
by the evidence; both nonvisual arrestins appeared to interact with all three
kinases comparably in cells,
162
although subsequent more precise direct
binding experiments with purified proteins suggested that the affinity of
arestin-3 for JNK3 and MKK4 is higher than that of arrestin-2.
160
Since
to assemble a productive complex arrestin must hold all three kinases simul-
taneously, even a subtle reduction in affinity for two of them could make a
qualitative difference in the cellular environment, where the concentrations
of all proteins involved are fairly low.
Arrestins-2 and -3 are 78% identical (and 88% similar),
8
both bind ASK1,
MKK4, and JNK3,
134,162
yet only arrestin-3 facilitates JNK3 phosphoryla-
tion in cells.
148,158,162
Thus, arrestin-3-specific residues must be responsible
for its unique ability to promote JNK3 activation. This idea was tested by the
construction of arrestin-2/3 chimeras.
158
It turned out that virtually every
chimera was less efficient than WT arrestin-3,
158
indicating that it is much
easier to destroy a function than to build it. Although these data were con-
sistent with earlier conclusion that each kinase in the ASK1-MKK4-JNK3
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