Arrestin proteins play a key role in the desensitization of G protein-coupled receptors (GPCRs). Recently we proposed a molecular mechanism whereby arrestin preferentially binds to the activated and phosphorylated form of its cognate GPCR. To test the model, we introduced two different types of mutations into -arrestin that were expected to disrupt two crucial elements that make -arrestin binding to receptors phosphorylation-dependent. We found that two -arrestin mutants (Arg 169 3 Glu and Asp 383 3 Ter) (Ter, stop codon) are indeed "constitutively active." In vitro these mutants bind to the agonist-activated  2 -adrenergic receptor ( 2 AR) regardless of its phosphorylation status. When expressed in Xenopus oocytes these -arrestin mutants effectively desensitize  2 AR in a phosphorylationindependent manner. Constitutively active -arrestin mutants also effectively desensitize ␦ opioid receptor (DOR) and restore the agonist-induced desensitization of a truncated DOR lacking the critical G protein-coupled receptor kinase (GRK) phosphorylation sites. The kinetics of the desensitization induced by phosphorylation-independent mutants in the absence of receptor phosphorylation appears identical to that induced by wild type -arrestin ؉ GRK3. Either of the mutations could have occurred naturally and made receptor kinases redundant, raising the question of why a more complex two-step mechanism (receptor phosphorylation followed by arrestin binding) is universally used.
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