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side chains in arrestin-2 and -3, as well as in the arrestin-1-V90S mutant,
make the N-domain more flexible, which appears to correlate with broad
receptor specificity. These results suggest that to construct a nonvisual
arrestin with narrow receptor preference, one needs to place valine in this
position.
The fact that the same 10 exposed residues determine receptor specificity
and largely drive the arrestin-receptor interaction
71,89
suggests that the
targeted manipulation of these side chains has the potential to yield nonvisual
arrestin mutants with much narrower receptor specificity than their parental
proteins. Since arrestins that can selectively target particular GPCRs would
be very useful research tools and have clear therapeutic potential,
141,142
this
idea was recently tested experimentally.
143
Ten different amino acid substi-
tutions affecting 8 out of 10 identified receptor discriminator residues were
introduced on the background of an arrestin-3-A87V base mutant. The
binding of WT arrestin-3, A87V, and other mutant arrestins to
b
2AR,
M2 muscarinic, as well as D1 and D2 dopamine receptors was tested in cells
using a BRET-based assay.
143
Seven out of 10 mutations resulted in differ-
ential changes in the binding to these receptors, yielding up to fourfold
increase in GPCR selectivity over WT protein.
143
This unexpectedly high
success rate clearly indicates that correct targets were chosen to change the
receptor preference of arrestin-3. The combination of two mutations that
individually reduced
b
2AR binding, but did not affect the interactions with
M2 or D2 receptors, yielded a variant with more than 50-fold preference for
these receptors over
b
2AR. Similarly, two other mutations were shown to
act additively; their combination yielded a version of arestin-3 with
>
fivefold preference for D1 over D2 receptor.
143
Numerous GPCR mutations have been shown to underlie a variety of
human disorders (reviewed in Ref.
144
). Currently, there are no viable
approaches to counteract the effects of gain-of-function mutations that
result in excessive receptor signaling. Arrestins with enhanced ability to
quench this signaling, such as the phosphorylation-independent mutants
described above, have a potential to become a solution to this problem.
However, WT nonvisual arrestins have very broad receptor specificity, so
the introduction of an enhanced arrestin-2 or -3 will likely simultaneously
blunt the signaling by the “bad” mutant receptor and numerous other per-
fectly normal GPCRs expressed in the same cell. Therefore, arrestin variants
selectively targeting the overactive receptor are needed for therapeutic use in
combination with other mutations changing its functional capabilities. This
study was the first attempt to create something that evolution did not; a
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