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receptor-bound arrestins. Yet arrestin ubiquitination by Mdm2 is clearly
stimulated by receptor binding,
135,150,164
suggesting that receptor-bound
form is a better substrate for Mdm2. Collectively, these data suggest that
Mdm2 binds free arrestins and is recruited to the receptor, where it has lim-
ited time to ubiquitinate arrestin before falling off due to reduced binding
affinity. This model explains limited and fairly selective ubiquitination of
arrestins by Mdm2 in response to receptor activation.
164
Another E3
ubiquitin ligase, parkin, also shows strict preference for the basal and D7
forms of arrestins over 3A mutants partially mimicking receptor-bound
state.
135
Interestingly, in contrast to Mdm2, parkin appears to be able to shift
the conformational equilibrium of arrestin to the form it prefers.
135
Parkin
greatly increases the binding of Mdm2 to WT arrestins, but not to D7
mutants that show enhanced Mdm2 interaction, suggesting that parkin acts
by stabilizing the D7-like conformation.
135
We found that parkin suppresses
Mdm2-dependent arrestin ubiquitination.
135
This is consistent with the idea
that parkin stabilizes a basal-like arrestin conformation, whereas receptor-
bound arrestins are better Mdm2 substrates.
Even though they appear almost inseparable in evolution (see
Chapter 2
),
12
the two arrestin domains are independent folding units that
can be expressed separately and retain certain functions.
24-26,80
Therefore,
the expression of separated domains has been repeatedly used to determine
which part of arrestin contains the binding sites for different partners. Inter-
estingly, whenever the localization of binding sites was tested by this method
or using peptide arrays, it was found that the protein of interest binds both
arrestin domains. This was shown for microtubules,
35
the MAP kinases
JNK3,
161
ASK1,
162
MKK4,
162
c-Raf1,
162
MEK1,
162
ERK2,
162
the cAMP
phosphodiesterase PDE4D5,
155
as well as the ubiquitin ligases Mdm2
135,161
and parkin.
135
Thus, with very few possible exceptions, the interaction
with both arrestin domains appears to be a general rule. Even though re-
ceptor binding does not induce a large movement of the two arrestin
domains, the domains actually shift relative to each other.
72
Therefore, this
rearrangement can explain conformational preference of certain partners;
the relative positions of the two parts of the binding site localized on differ-
ent domains actually change. The distance between these two parts and/or
their relative orientation can become favorable or unfavorable for the inter-
action in one of the conformations.
31
It is harder to reconcile the bipartite binding site with the observations
that some proteins, such as MEK1, appear to bind arrestins in all confor-
mations equally well,
133
unless the interaction with one of the domains
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