Biology Reference
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While the effect of receptor-attached phosphates on its conformational state
is completely unknown, activation-induced changes were well character-
ized, first by a series of site-directed spin-labeling studies of rhodopsin
55-59
then by the solution of
several crystal
structures of active forms of
rhodopsin,
60-63
b
2AR,
42
adenosine A2A receptor,
49,64
agonist-activated
b
2AR stabilized by nanobody,
65
and
b
2AR in a physiologically relevant
complex with the G protein.
66,67
The common theme in GPCR activation
is the outward movement of helices 5 and 6, and less dramatic
rearrangements of other elements, which collectively open the cavity in
the middle of the cytoplasmic side of the receptor, where the C-terminus
of the
a
-subunit of their cognate G protein binds.
68-70
These movements
increase the diameter of the receptor,makingitlargerthantheshortaxis
of arrestin, although still much smaller than its long axis (
Fig. 3.2
). To even
begin meaningful fitting of an arrestin to a receptor, one needs to iden-
tify interacting residues in both proteins. While the arrestin-binding
parts of the receptor have been identified imprecisely and only in a few
GPCRs,
74-76
the receptor “footprint” on different arrestins was mapped
fairlycomprehensivelybyseveralgroupsusingavarietyofmethods
(
Fig. 3.2
).
3.1. Receptor-binding surface of arrestins
Receptor-binding residues in arrestins were identified using
mutagenesis,
22,25-28,71,73,77
H/D exchange,
78
element swapping,
79,80
pep-
tide competition,
81
epitope insertion,
82
solution NMR,
83-85
and site-
directed spin-labeling EPR.
73,86
All these experiments yielded essentially
the same answer: an extensive surface on the concave sides of both arrestin
domains is involved (reviewed in Ref.
14
)(
Fig. 3.2
). The distance between
the extreme positions of arrestin residues implicated in receptor binding
appears to be larger than even the expanded active receptor, at least the parts
visible in crystal structures. However, we should not forget that spatial local-
ization of the phosphates necessary for high-affinity arrestin binding
in vitro,
25,80,87,88
in cells,
89
and in living animals
90,91
remains completely
unknown. This is true for rhodopsin and the
b
2AR, where phosphorylation
sites are localized in the C-terminus,
92-100
which was not resolved in any of
the structures. This is equally true for M2 muscarinic cholinergic receptor,
where phosphorylation sites are localized on the large third cytoplasmic
loop,
101-103
which was deleted to obtain well-diffracting crystals.
45
Thus,
we do not really know the size of the arrestin-binding surface of any GPCR.
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