Structural Determinants of Arrestin Functions - Progress in Molecular Biology and Translational Science - page 62

interaction. In contrast, the neutralization or reversal of the charge of the
putative phosphate sensor was expected to turn it on, enhancing the binding
to unphosphorylated Rh*.
Since high-affinity arrestin binding was shown to require multiple phos-
phates on rhodopsin, 25 one would expect the multiphosphorylated and
therefore highly negatively charged rhodopsin C-terminus to interact with
several closely spaced positively charged residues in arrestin. The first scan-
ning mutagenesis performed targeted a 14-amino acid stretch of the primary
sequence carrying six positive charges (residues 163-176). 27 Considering
that it was done long before crystal structure became available, this attempt
proved amazingly well targeted: five out of six mutations reduced P-Rh and
P-Rh* binding. The sixth, Arg175Asn, showed even more interesting phe-
notype: somewhat enhanced binding to P-Rh* and a dramatic

fivefold
increase in Rh* binding. 27 This is consistent with the idea that Arg175
serves as a phosphate sensor, which was artificially activated by charge neu-
tralization, “fooling” arrestin-1 into perceiving any active form of rhodopsin
as phosphorylated. However, the data with full-length protein did not prove
one key point, that Arg175 actually interacts with phosphates, because the
binding of Arg175Asn mutant to inactive P-Rh and P-Rh* was also
increased. 27 Luckily, the N-terminal half of arrestin-1, residues 1-191 con-
taining Arg175, was previously shown to be functional and act as an unso-
phisticated version of arrestin; it interacts P-Rh and Rh*, and its binding to
P-Rh* is essentially the sum of the two. 24,25 In the context of this mini-
arrestin, the Arg175Asn mutation reduced the binding to P-Rh and
P-Rh* just like the other mutations, proving that Arg175 actually engages
rhodopsin-attached phosphates. 27 Replacement of Arg175 with all 19 alter-
native residues showed that positive charge is the key: Arg175Lys retained
high selectivity for P-Rh*, whereas all other substitutions showed enhanced
binding to Rh*, with the charge reversal mutation Arg175Glu having the
most potent effect. 28 The simplest interpretation of these data was that
Arg175 interacts with a negatively charged partner within arrestin, and
the breaking of this salt bridge by negatively charged phosphates turns
the sensor on, allowing arrestin transition into high-affinity receptor-
binding state. 27,28 Later, the crystal structure revealed that Arg175 is part
of solvent-excluded arrangement of five charged residues between the
two arrestin domains ( Fig. 3.1 ), identifying three possible negatively charged
partners. 17,18 Further mutagenesis proved that the salt bridge between
Arg175 and Asp296 is the key phosphate sensor: charge reversal mutations
of either greatly increase Rh* binding, whereas simultaneous reversal of