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domain for binding of the sole SUMO-E2 enzyme, UBC9. In bovine
b -arrestin2, SUMOylation predominantly occurs at a specific lysine residue
and is important for b -arrestin interaction with the endocytic adaptor pro-
tein b -adaptin2. 54 The SUMO-E3 ligase that mediates this modification
remains to be identified.
2.2. Biology: Kinetics and stability
7TMR-stimulated b -arrestin2 ubiquitination can be categorized into two
patterns: (1) transient ubiquitination which occurs rapidly (e.g., acute stim-
ulation of the b 2 AR by the agonist isoproterenol leads to transient b -arrestin
ubiquitination signals which are rapidly reduced to baseline levels within a
fewminutes) and (2) sustained ubiquitination in which the signals evoked by
agonist treatment do not deteriorate for up to 1 h or more (e.g., stimulation
of AT 1a R by angiotensin II leads to sustained b -arrestin ubiquitination;
Fig. 7.3 A and B; Refs. 55,56 ). Apart from routinely used Western blot tech-
niques mentioned above, both transient and sustained ubiquitination of
b -arrestin as induced by the b 2 AR and the V 2 vasopressin receptor (V 2 R)
are detectable as dynamic modifications in live cells by monitoring BRET
(bioluminescence resonance energy transfer) between b -arrestin2 fused with
luciferase and GFP 2 -tagged ubiquitin. 57 The two patterns of b -arrestin
ubiquitination parallel previously shown trafficking profiles of green fluores-
cent protein (GFP)-tagged b -arrestins, in which transient recruitment to
activated b 2 ARs at the plasma membrane defines “class A” and stable
recruitment to activated AT 1a Rs, first to the plasma membrane followed
by cointernalization of receptor- b -arrestin complexes, defines “class B”
receptor. 58 In general, class A receptors recycle and resensitize at the plasma
membrane more rapidly than the class B receptors, and their affinities for the
two b -arrestin isoforms are also different. Class A receptors preferentially
recruit b -arrestin2, whereas class B receptors bind both b -arrestins with
equal affinity. These internalization, recycling, b -arrestin binding, and
ERK activation patterns are attributed to particular serine-threonine-rich
phosphorylation motifs found only in the class B receptors. 58,59 Interest-
ingly, when the C-terminal region of a class A receptor is replaced with that
of a class B receptor, trafficking, ERK activation, and ubiquitination patterns
are transformed to the class B type. 55,58,59 Translational fusion of Ub to
b -arrestin2 promotes its sustained binding to even a class A receptor,
whereas removal of all ubiquitination sites results in transient recruitment
to even class B receptors ( Fig. 7.3 C and D; Refs. 51,55,56 ). These findings
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