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

Kovoor A, Celver J, Abdryashitov RI, Chavkin C, Gurevich VV
J Biol Chem. 1999 274 (11): 6831-4

PMID: 10066734 · DOI:10.1074/jbc.274.11.6831

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 beta-arrestin that were expected to disrupt two crucial elements that make beta-arrestin binding to receptors phosphorylation-dependent. We found that two beta-arrestin mutants (Arg169 --> Glu and Asp383 --> Ter) (Ter, stop codon) are indeed "constitutively active." In vitro these mutants bind to the agonist-activated beta2-adrenergic receptor (beta2AR) regardless of its phosphorylation status. When expressed in Xenopus oocytes these beta-arrestin mutants effectively desensitize beta2AR in a phosphorylation-independent manner. Constitutively active beta-arrestin mutants also effectively desensitize delta 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 beta-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.

MeSH Terms (8)

Animals Arrestins beta-Arrestins GTP-Binding Proteins Mutagenesis, Site-Directed Phosphorylation Receptors, Adrenergic, beta Xenopus

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