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both, restoring the salt bridge, also restores normal arrestin-1 selectivity for
P-Rh*.
29
Interestingly, these five charges are conserved in arrestin evolution
from
C. elegans
to mammals,
12
attesting to their important role in arrestin
function. Virtually identical positions of all five side chains in different sub-
types of mammalian arrestins (
Fig. 3.1
) support this notion. Charge reversals
of homologous arginines in arrestin-2 and -3 also yield enhanced
phosphorylation-independent mutants,
125-130
demonstrating that this resi-
due plays the same role in all arrestins.
Further mutagenesis identified additional phosphate-binding elements in
arrestins. Arg18 in the loop between
b
-strands I and II is unique for
arrestin-1,
22
likely making is much more dependent on receptor-attached
phosphates than the other subtypes.
89
In contrast, two lysines in
b
-strand
I are present in all arrestins.
12
These charged residues appear to be critical
for the “delivery” of phosphates to the shielded polar core.
77
Importantly,
they are adjacent to the bulky hydrophobic residues in
b
-strand I that par-
ticipate in its interactions with the arrestin C-tail and
a
-helix I (
Fig. 3.1
).
18
This suggested that their interaction with phosphates likely disrupts this
three-element interaction, which would destabilize the basal arrestin con-
formation, similar to the effect of the disruption of the polar core.
77
These
data support the main premise of the sequential multisite binding model of
the arrestin-receptor interaction
25
that receptor binding is accompanied by a
global conformational change in arrestin.
The action of the phosphate sensor is based on pure electrostatics; all that
receptor-attached phosphates need to do to activate the sensor is to break the
key salt bridge.
30
This makes it essentially insensitive to the sequence context
of the phosphorylated residues. This mechanism explains how just two non-
visual arrestins in mammals and only one in
Drosophila
can interact with hun-
dreds of different GPCRs, in which serines and threonines phosphorylated
by GRKs are found within diverse sequences that can be localized in the
receptor C-terminus, or any of the intracellular loops (reviewed in Ref.
14
).
3.4. The conformation of the receptor-bound arrestin
Several lines of indirect evidence suggested that the conformation of receptor-
bound arrestins is likely quite different from their basal state revealed by
crystal structures. The first indication that this must be the case was unusually
high-energy barrier of arrestin-1 binding to rhodopsin.
104
Receptor-
binding-induced release of the arrestin C-tail has been well documented
for more than 20 years.
73,86,109
In addition, the movement and/or structural
rearrangement of the “finger loop” in the central crest of the receptor-
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