Biology Reference
In-Depth Information
particles. This cellular response is known to require the Ras/Raf/MEK/
ERK pathway, suggesting that in at least some cases Ras-dependent ERK
activation by
b
-arrestins is physiologically relevant.
6
The possibility of direct effects of
b
-arrestins on Ras activation had not
been addressed until very recently. Using BRET-based biosensors, angio-
tensin II (Ang II) stimulation was shown to promote Ras activation in
the Golgi and endoplasmic reticulum, but not in early endosomes, of
HEK293 cells stably expressing the Ang II type 1 receptor (AT
1
R). Plasma
membrane Ras activation was also detected in this study, but at a more subtle
level. In cells expressing an AT
1
R mutant impaired in its ability to internal-
ize (AT
1A
R
D
319
), Ang II stimulation enhanced total Ras activation to a
greater extent than in cells expressing wild-type AT
1
Rs, suggesting that
b
-arrestin predominantly played a negative regulatory role in Ras activation.
Instead, expression of a mutant receptor defective in G
a
q
coupling
(AT
1
R
DRY/AAY
) inhibited Ras activation, suggesting that this process is pre-
dominantly G
q
-mediated.
7
Such results underscore the multiplicity of differ-
ent means, including heterotrimeric G protein activation, receptor tyrosine
kinase transactivation, and
b
-arrestin scaffolding, that 7TM receptors use to
signal to this key pathway, and it is clear that the role of
b
-arrestins in mod-
ulating Ras activity will need to be defined for a broad variety of receptors in
different cellular models. BRET-based Ras probes, as well as more classical
approaches such as pull down of activated Ras by Raf1-Ras-binding
domain fusion proteins, should provide a means to directly test whether
b
-arrestins contribute to receptor-dependent Ras activation. Knockdown
of
b
-arrestin is likely to impair Ras activity if a specific receptor utilizes this
scaffold protein to promote MAPK activation and enhance it if
b
-arrestin-
mediated desensitization is providing negative regulation.
2.2. Ral
A multifunctional GTPase
The Ras-like GTPases RalA and RalB were identified based on sequence
similarity to H-, K-, and N-Ras.
36
The two Ral isoforms, therefore, con-
stitute a distinct subfamily within the Ras family. Among all GTPases, Rals
are the most closely related small G proteins to Ras with more than 50%
homology. RalA and RalB are ubiquitously expressed and highly homo-
logous sharing close to 85% homology among themselves. They both
participate in the signaling events leading to endocytosis and exocytosis,
actin remodeling, and transcription.
37
However, they also display distinct
functions. For example, RalA is important for anchorage-independent
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