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negative in a particular regard only when a single function is destroyed in a
way that preserves all other functional capabilities. Similarly, to generate
signaling-biased arrestins, a single function should be enhanced or disabled
by the mutations that do not significantly affect others. In reality, it turned
out to be extremely difficult to change anything within the arrestin molecule
without affecting more than one function. However, as long as a single
aspect of arrestin functions is affected to a much greater extent than the
others, the mutant can serve a particular purpose.
5.1. Enhanced phosphorylation-independent mutants
The first arrestin that binds with high affinity to any active form of its cog-
nate receptor regardless of phosphorylation, arrestin-1-Arg175Asn, was cre-
ated in the process of elucidation of the mechanism arrestins use to respond
to the receptor-attached phosphates in 1995.
27
This happened when
arrestins were still believed to interact only with GPCRs, before arrestin
binding to the first nonreceptor partner, clathrin, was discovered in
1996.
145
Arg175 was later substituted by every residue in the topic, and
charge reversal mutation Arg175Glu was found to be the most potent.
28
Later, a whole family of structurally distinct phosphorylation-independent
mutants of arrestin-1 was constructed.
77,165
The Arg175Glu mutant was
shown to effectively suppress transducin activation by unphosphorylated
light-activated rhodopsin (Rh*).
166
Several mutations homologous to
those that enable arrestin-1 binding to unphosphorylated Rh* were
shown to yield similar phosphorylation-independent binding of both
nonvisual arrestins to their cognate receptors,
125-128
and to block the
coupling of unphosphorylated WT and mutant GPCRs to their cognate
G proteins.
125,126,129
One of the phosphorylation-independent arrestin-1 mutants was
recently tested for its ability to compensate for defects of rhodopsin
phosphorylation
in vivo.
91
This enhanced arrestin-1 was shown to signifi-
cantly improve survival and functional performance of rod photoreceptors
lacking rhodopsin kinase, and to facilitate the rate of rod recovery
threefold, as compared to parental WT arrestin-1.
91
This proof-
of-concept study showed that one mutant protein can be used to compen-
sate for a molecular defect in another.
141
However, photoresponse recovery
in “compensated” rods was much slower than in WT photoreceptors,
suggesting that a significant further redesign of arrestin-1 is necessary to
achieve a perfect fit and high-affinity binding to unphosphorylated
Rh*.
91
These results clearly showed that, even though we know about
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