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internalization of either
b
2
AR or V
2
R, confirming that ubiquitination of
b
-arrestin is crucial for its role in promoting receptor endocytosis
(
Fig. 7.3
D). In contrast, a
b
-arrestin-Ub chimera that remains ubiquitinated
can enhance
b
2
AR internalization and is stably recruited to endosomal com-
partments with the
b
2
AR (
Fig. 7.3
C; Refs.
51,56,60
). Correlating with
these effects on internalization, these two forms of
b
-arrestins show recip-
rocal binding affinities with clathrin, suggesting that ubiquitin moieties on
b
-arrestin2 are crucial “tags” for its efficient binding to clathrin and entry
into the endocytic pathway.
51
In cellular coimmunoprecipitation assays,
b
-arrestin2-0K displays similar
binding with overexpressed c-Raf and GFP-ERK2 as the wild type, whereas
b
-arrestin2-Ub exhibits enhanced interaction.
51
However, pERK con-
tained in isolated
b
2
AR complexes is significantly decreased when
b
-arrestin2-0K is coexpressed when compared with
b
-arrestin2 wild type.
In contrast,
b
-arrestin2-Ub coexpression markedly increases pERK levels in
receptor complexes and further stabilizes the kinase activity on endosomes
(
Fig. 7.3
C). Thus, although both ubiquitinated and nonubiquitinated forms
of
b
-arrestin can form complexes with pERK, only the ubiquitinated form is
capable of this function in a receptor complex.
51
Subcellular fractionation
experiments indicate that ubiquitination of
b
-arrestin2 favors its membrane
association, suggesting that although ubiquitination is dispensable for
b
-arrestin's interactions with cytososlic partners, it may be necessary to facil-
itate the formation of functional 7TMR-
b
-arrestin endocytic and signaling
complexes in a membrane environment.
51
3. DEUBIQUITINATION OF ARRESTINS
The DUB, ubiquitin-specific protease 33 (USP33, also known as von-
Hippel Lindau interacting deubiquitinase1 or VDU1; Ref.
61
), was identi-
fied as a
b
-arrestin-interacting protein in a yeast two-hybrid screen and its
binding promotes deubiquitination of
b
-arrestin2.
49
Upon USP33 over-
expression, V
2
R-stimulated effects, namely, stable
b
-arrestin binding,
sustained
b
-arrestin2 ubiquitination, and persistent ERK activation, are all
inhibited. On the other hand, targeted gene silencing of USP33 promotes
stable interaction and cointernalization of
b
-arrestin2 with the
b
2
AR,
sustained ubiquitination of
b
-arrestin2, and prolonged activation of ERK.
Accordingly, the kinetics of
b
-arrestin ubiquitination and deubiquitination
are tightly regulated by USP33 ensuring the appropriate duration and mag-
nitude of
b
-arrestin-biased signaling.
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