Arrestins and Protein Ubiquitination - Progress in Molecular Biology and Translational Science - page 184

vesicular pathways. Substrates that have been shown to require a b -arrestin
isoform as an E3 ubiquitin ligase adaptor are described below.
4.1. 7TMRs
Agonist activation of b 2 ARs induces ubiquitination of the receptor protein
itself in addition to b -arrestin ubiquitination described earlier. 48,50,63-65
Receptor ubiquitination is detected within minutes and remains stable for
several hours, diminishing as subsequent lysosomal degradation of the pro-
tein ensues. Although the activated b 2 ARs internalize into endosomes
within minutes, their trafficking to lysosomes is detected only after several
hours. 50,63 In the commonly employed HEK-293 or COS-7 cells, the
receptor protein remains in these compartments for prolonged periods
and is slowly degraded. 50,66,67 When receptor ubiquitination is prevented,
as in a lysine-less mutant, internalization of the b 2 AR proceeds normally,
but lysosomal trafficking and receptor degradation are impaired. 48,50,64
Additional studies with four b 2 AR mutants each with lysines restricted to
a single intracellular domain showed that both the third intracellular loop
and the carboxyl tails of the b 2 AR are ubiquitinated. 64 The lysines targeted
for ubiquitination have been mapped utilizing mass spectrometry
approaches to residues in the third intracellular loop (lys-263, lys-270)
and in the carboxyl tail (lys-348, lys-372, lys-375) of human b 2 AR. 64
b 2 ARubiquitination is not detectable in b -arrestin1/2 null mouse embry-
onic fibroblasts, but robust receptor ubiquitination is detectedwhen b -arrestin2
is expressed or added back into these cells. 48 In contrast, reexpression of
b -arrestin1 does not promote b 2 AR ubiquitination. Subsequent studies
showed that b -arrestin2 binds and recruits the HECT domain-containing
E3 ubiquitin ligaseNedd4 tomediate b 2 ARubiquitination and lysosomal deg-
radation. 50 Although Nedd4 enzymes and their yeast homolog Rsp5 are
known to interact with substrate or adaptor proteins via WW domains (in
Nedd4) and proline-rich motifs (PPXY in substrate or adaptors), elimination
of all theWWdomains in Nedd4 did not affect its interaction with b -arrestin2
suggesting other modes of adaptor binding. 50 In fact, for some substrates, WW
domain-independent interactions of Nedd4 have been reported. 68-71
Ubiquitination of CXCR4, a chemokine receptor, involves modifica-
tion of lysine residues 327, 331, and 333 in the carboxyl tail and requires
phosphorylation of nearby serine residues. 72 Unlike the b 2 ARmodification,
CXCR4 is modified with only one or small number of ubiquitin moieties.
However, similar to the b 2 AR, a CXCR4 lysine mutant defective in