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of supporting evidence. Unfortunately, it also did not survive experimental
testing. The model predicts that the saturation point would be achieved
upon binding of one arrestin molecule to two molecules of rhodopsin. This
prediction was tested in mice
in vivo.
110
In rod photoreceptors in the dark,
arrestin-1 is largely localized away from the outer segment (OS), where all
rhodopsin resides. In contrast, in bright light, the bulk of arrestin-1 moves to
this compartment,
32,111-114
where it remains due to high-affinity binding to
rhodopsin.
32
Considering that the expression ratio of arrestin-1 to rhodopsin
in mouse rods is
0.8:1,
110,115-117
if this model were true, one would never
expect virtually quantitative translocation of arrestin-1 to the OS, which was
reproducibly observed by many labs.
32,111-114
Genetic manipulation of
arrestin-1 and rhodopsin expression levels in mouse rods revealed that the
amount of arrestin-1 that can move to the OS in the light is, indeed, limited
by the amount of rhodopsin there, but that saturation is achieved at the ratio
of translocated arrestin-1 to rhodopsin that is greater than 0.8:1, which is
consistent only with 1:1 binding model.
110
Obviously, in the photorecep-
tors of live mice, one cannot exclude the role of other proteins in arrestin-1
translocation. However, a variety of arrestin-1:rhodopsin ratios were tested
in vitro
using two carefully quantified pure proteins, which again yielded sat-
uration at
1:1.
110
Since arrestin-1 readily self-associates, forming dimers
and tetramers,
118-121
this binding ratio could have been explained by an
interaction of an arrestin-1 dimer with a rhodopsin dimer. However, it
was shown that only monomeric arrestin-1 binds rhodopsin
120
because its
receptor-binding surface is shielded by sister subunits in the arrestin-1
tetramer and both possible dimers.
122
Finally, monomeric rhodopsin was
reconstituted into nanodiscs (HDL particles containing membrane-like lipid
bilayer) and shown to bind arestin-1 not just efficiently,
123
but with phys-
iologically relevant high affinity (
K
D
3-4 nM) and 1:1 stoichiometry.
124
Thus, while the problem exists, neither model proposed so far has with-
stood the rigors of experimental testing. It appears very likely that in real life
a single molecule of activated phosphorylated receptor fits arrestin in its
active conformation well enough, but we do not know how exactly this
fit is achieved. Two new ideas, which are not mutually exclusive, are
suggested by the available evidence. On the arrestin side, unexpectedly large
movement of the “139 loop” in the central crest, apparently out of the way
of the incoming receptor,
72
likely allows the “finger loop” to insert itself
fairly deeply into the cavity opening in the middle of the active receptor
69
(
Fig. 3.2
). This would result in extensive contacts between the cytoplasmic
loops and extended helices of the receptor and the cavities of both arrestin
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