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Targeted construction of phosphorylation-independent beta-arrestin mutants with constitutive activity in cells
A Kovoor1, J Celver, R I Abdryashitov
1Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280, USA.
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 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.
Insights
Mutant arrestin proteins bind activated G protein-coupled receptors (GPCRs) independently of phosphorylation, demonstrating a constitutive activity that bypasses normal receptor desensitization pathways.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Arrestin proteins are crucial for desensitizing G protein-coupled receptors (GPCRs).
- A proposed mechanism suggests arrestin binds preferentially to activated, phosphorylated GPCRs.
Purpose of the Study:
- To investigate the phosphorylation-dependent binding mechanism of arrestin to GPCRs.
- To create and test beta-arrestin mutants with altered phosphorylation-dependent binding properties.
Main Methods:
- Introduced specific mutations (Arg169 --> Glu, Asp383 --> Ter) into beta-arrestin.
- Assessed in vitro binding of mutant beta-arrestin to activated beta2-adrenergic receptor (beta2AR).
- Evaluated desensitization of beta2AR and delta opioid receptor (DOR) in Xenopus oocytes using mutant beta-arrestins.
Main Results:
- Two beta-arrestin mutants exhibited "constitutive activity," binding activated beta2AR irrespective of its phosphorylation status.
- These mutants induced phosphorylation-independent desensitization of beta2AR in oocytes.
- Mutants also desensitized DOR and restored desensitization in a GRK-phosphorylation-site-deficient DOR mutant.
Conclusions:
- Beta-arrestin mutations can confer phosphorylation-independent GPCR desensitization.
- The findings challenge the necessity of the canonical two-step GPCR desensitization mechanism.
- Raises questions about the evolutionary advantage of the complex phosphorylation-dependent pathway.