Received for publication January 31, 2008.
Revised April 14, 2008.
Accepted for publication April 15, 2008.

ARRESTIN BINDS TO DIFFERENT PHOSPHORYLATED REGIONS OF THE TRH RECEPTOR WITH DISTINCT FUNCTIONAL CONSEQUENCES

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 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 wildtype and 4Ala receptors, but not with 4AlaStop receptor, as measured by translocation of arrestin-GFP 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.

Key words: Thyrotropin/TRH, Gq/11 family, Receptor synthesis/trafficking, Desensitization/uncoupling, Sequestration/Internalization, GRKs, barrestins, Phosphorylation/Dephosphorylation

This article has been cited by other articles:

				V. V. Gurevich and E. V. Gurevich Rich Tapestry of G Protein-Coupled Receptor Signaling and Regulatory Mechanisms Mol. Pharmacol., August 1, 2008; 74(2): 312 - 316. [Abstract] [Full Text] [PDF]