Structural Determinants of Arrestin Functions - Progress in Molecular Biology and Translational Science - page 73

module has multipartite site, interacting with both arrestin domains, 162 this
approach identified the C-domain as the key element in JNK3 activation. 158
Interestingly, this region included residues 182-376, encompassing the
Arg196-Ser197 sequence in bovine arrestin-3 that is replaced with
Lys195-Pro196 in arrestin-2. To further dissect the key arrestin-3 elements
necessary for JNK3 activation, the residues unique for this subtype were rep-
laced with their arrestin-2 homologues. 158 Quite a few mutations, including
RS KP, had no detectable effect, ruling out the role of those residues in

JNK3 activation. Multiple other substitutions reduced arrestin-3 potency
in JNK3 activation assay. 158 This study identified Val343 as the most impor-
tant residue, as a Val343Thr point mutation reduced the ability of arrestin-3
to activate JNK3 by two-thirds. Residues Leu278, Ser280, His350, Asp351,
His352, and Ile353 were found to play supporting roles, as their substitution
with arrestin-2 homologues also reduced the ability of arrestin-3 to facilitate
JNK3 phosphorylation. 158 Interestingly, virtually all arrestin-3 mutants with
reduced or even abolished ability to activate JNK3 demonstrated essentially
normal binding to all three kinases in this module, supporting the idea that
the interaction with these kinases and the ability to promote JNK3 activation
are different arrestin-3 functions that can be separated by targeted mutagen-
esis. Evolution actually separated these functions in arrestin-2, which binds
the same kinases but does not facilitate JNK3 phosphorylation. 162

!

5. DESIGNING SIGNALING-BIASED ARRESTIN MUTANTS
To a certain extent, the natural structural organization of arrestins
makes targeting different aspects of their function easier. The concave sides
of both domains contain allknown receptor-binding residues ( Section 3.3 ),
most other partners interact with elements on the other side of the molecule
that remain accessible in the arrestin-receptor complex, whereas clathrin
and AP2 engage distinct sites on the arrestin C-tail ( Section 4.1 ) that do
not appear to overlap with the binding sites of any other known partner.
These sites were the first to be eliminated by targeted mutagenesis. 145,147,153
Predictably, the disruption of either site precluded arrestin interactions with
its respective partners without affecting GPCR binding. Moreover, arrestin
mutants with disabled binding sites for clathrin, AP2, and particularly both,
acted as dominant-negative, selectively suppressing arrestin-dependent
GPCR internalization via coated pits. 145,147,153 These findings firmly
established that arrestin functions can be manipulated independently of each
other. In addition, these studies showed that an arrestin can act as adominant