Targeted construction of phosphorylation-independent β-arrestin mutants with constitutive activity in cells

Abstract

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 β-arrestin that were expected to disrupt two crucial elements that make β-arrestin binding to receptors phosphorylation-dependent. We found that two β-arrestin mutants (Arg169 → Glu and Asp383 → Ter) (Ter, stop codon) are indeed 'constitutively active.' In vitro these mutants bind to the agonist-activated β2-adrenergic receptor (β2AR) regardless of its phosphorylation status. When expressed in Xenopus oocytes these β- arrestin mutants effectively desensitize β2AR in a phosphorylation- independent manner. Constitutively active β-arrestin mutants also effectively desensitize δ 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 β-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.