Biology Reference
In-Depth Information
receptors.
80,138,139
Thus, evolution created only one receptor-specific sub-
type, arrestin-1, which demonstrates clear preference for rhodopsin over
other receptors.
80,140
Arrestin-1 binds rhodopsin efficiently and demonstrates fairly low binding
to M2 muscarinic receptor, whereas arrestin-2 has the opposite prefer-
ence.
80,140
Therefore, the first attempt to identify arrestin elements that deter-
mine its receptor specificity involved the construction of a series of arrestin-1/2
chimeras and testing their ability to bind these two receptors.
79
The premise of
these experiments was that if an element is important for receptor prefer-
ence, the introduction of that part from arrestin-2 into arrestin-1 would
decrease rhodopsin and increase M2 binding, whereas the introduction of
the corresponding arrestin-1 part into arrestin-2 would decrease M2 and
increase rhodopsin binding. N-domain residues 49-90 (
b
-strands V and VI
with adjacent loops) and C-domain residues 237-268 (
b
-strands XV and
XVI) of arrestin-1 and homologous parts of arrestin-2 were found to play
key role in receptor preference. The exchange of these two elements between
arrestin-1 and -2 completely reversed receptor specificity of both, creating
one chimera that was
>
90% arrestin-1, yet bound M2 muscarinic receptor
much better than rhodopsin, and a symmetrical chimera with
>
90% of
arrestin-2 residues that clearly preferred rhodopsin to M2.
79
Fewer than 25 residues in these two elements are nonconservative sub-
stitutions. Their individual contributions to receptor preference were tested
by introducing point mutations.
71
This approach led to the identification of
10 exposed side chains that collectively determine which receptor an arrestin
protein prefers. Interestingly, the replacement of all 10 with alanines in
arrestins-1, -2, and -3 yielded mutants that virtually lost the ability to bind
any GPCRs.
71
One of the nonexposed residues in the N-domain element,
Val90, was found to play an unexpectedly important role; its substitution
with serine (Ser86 is the homologous arrestin-2 residue) increased
arrestin-1 binding to M2 muscarinic receptor more than any other point
mutation.
19
Arrestin-3, with similarly broad receptor specificity, has
Ala87 in the same position,
20
which suggests that a small side chain is impor-
tant. Crystal structure shows that arrestin-1 Val90, localized between the
two
b
-sheets, makes contacts with several other hydrophobic residues,
apparently stabilizing the core of the N-domain.
18
All potential part-
ners are present in arrestin-2
19
and -3,
20
but they do not contact the
smaller side chain of Ser86 or Ala 87. This analysis indicates that the
presence of the bulky Val90 increases the rigidity of the N-domain, ap-
parently enhancing receptor specificity of arrestin-1, whereas the smaller
Search WWH ::
Custom Search