Abstract
Arrestin binding to agonist-occupied phosphorylated G protein-coupled receptors typically increases the affinity of agonist binding, increases resistance of receptor-bound agonist to removal with high acid/salt buffer, and leads to receptor desensitization and internalization. We tested whether thyrotropin-releasing hormone (TRH) receptors lacking phosphosites in the C-terminal tail could form stable and functional complexes with arrestin. Fibroblasts from mice lacking arrestins 2 and 3 were used to distinguish between arrestin-dependent and -independent effects. Arrestin did not promote internalization or desensitization of a receptor that had key Ser/Thr phosphosites mutated to Ala (4Ala receptor). Nevertheless, arrestin greatly increased acid/salt resistance and the affinity of 4Ala receptor for TRH. Truncation of 4Ala receptor just distal to the key phosphosites (4AlaStop receptor) abolished arrestin-dependent acid/salt resistance but not the effect of arrestin on agonist affinity. Arrestin formed stable complexes with activated wild-type and 4Ala receptors but not with 4AlaStop receptor, as measured by translocation of arrestin-green fluorescent protein to the plasma membrane or chemical cross-linking. An arrestin mutant that does not interact with clathrin and AP2 did not internalize receptor but still promoted high affinity TRH binding, acid/salt resistance, and desensitization. A sterically restricted arrestin mutant did not cause receptor internalization or desensitization but did promote acid/salt resistance and high agonist affinity. The results demonstrate that arrestin binds to proximal or distal phosphosites in the receptor tail. Arrestin binding at either site causes increased agonist affinity and acid/salt resistance, but only the proximal phosphosites evoke the necessary conformational changes in arrestin for receptor desensitization and internalization.
Footnotes
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This work was supported by National Institutes of Health grant DK19974 (to P.M.H.), a National Institutes of Health Cardiovascular Research Training grant, and a Pharmaceutical Manufacturers' Association Predoctoral Fellowship (to B.W.J.).
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ABBREVIATIONS: GPCR, G protein-coupled receptor; TRH, thyrotropin-releasing hormone; Arr2/3KO MEF, mouse embryo fibroblast from arrestin 2 and 3 knock-out animals; HEK, human embryonic kidney; GFP, green fluorescent protein; HA, hemagglutinin; InsP, inositol phosphate; PAGE, polyacrylamide gel electrophoresis; ANOVA, analysis of variance.
- Received January 31, 2008.
- Accepted April 15, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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