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its activation can be stimulated by agonist treatment of the cells. Evidence
demonstrating regulation of its function by
b
-arrestins is described below.
5.1. ARF6 and receptor endocytosis
The first evidence that ARF6 could be a modulator of 7TM receptor func-
tion came from the early studies describing the function of GIT, a new ARF
GAP and interacting partner of the G protein-coupled receptor kinases
(GRKs).
98
The observation that an ARF GAP would modulate internaliza-
tion of a receptor sparked some interest in addressing the role of ARFs them-
selves in this process. Expression of dominant negative and constitutively
active mutants of ARF6 inhibited sequestration of the
b
2
-adrenergic recep-
tor.
13
Later, it was shown that inhibition of ARF6 expression by RNA inter-
ference blocked internalization of this particular receptor, as well as the
AT
1
R, the V2R, the ET
B
R, and the M2MR.
99
The proposed mechanisms
by which ARF6 regulated receptor internalization involve
b
-arrestins.
It was shown that both
b
-arrestin isoforms could be found in complex with
the GTPase upon agonist stimulation of the
b
2
-adrenergic receptor in
HEK293 cells. According to
in vitro
data, the interaction appeared to be
direct and modulated by the nature of the nucleotide bound to the ARF,
in that ARF6-GDP preferentially bound the C-terminal region of
b
-arrestin.
13,14
In this same context,
b
-arrestin was shown to be constitu-
tively associated with ARNO, an ARF GEF that promoted loading of
GTP on ARF6.
13
In addition, a second exchange factor was also reported
to interact with
b
-arrestin, the protein EFA6.
14
Activated ARF6 was shown
to mediate the recruitment of AP-2 and clathrin to the receptor/
b
-arrestin
complex, a step essential for the formation of endocytic vesicles.
100,101
A role
of
b
-arrestin in the activation process of ARF6 had been suggested after
addition of purified recombinant
b
-arrestin proteins improved the ability
of ARNO to promote loading of GTP on the small G in an
in vitro
setting.
13
The direct demonstration that
b
-arrestin is a key protein controlling
b
2
-
adrenergic receptor-mediated ARF6 activation was recently reported in
HEK293 cells. Depletion of
b
-arrestin by siRNA markedly inhibited
isoproterenol-induced GTP loading of ARF6.
14
In addition to mediating
the initial step of receptor endocytosis,
99
activation of ARF6 controlled
recycling. In these conditions, the pool of EFA6-activated ARF6 inhibited
fast recycling through the Rab4 pathway and relocalized receptors to late
endosomes and lysosomes.
14
A role for the
b
-arrestin/ARF signaling axis
has also been described in
Dictyostelium discoideum
. In this model organism,
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