TY - JOUR T1 - The Interaction of a Constitutively Active Arrestin with the Arrestin-Insensitive 5-HT<sub>2A</sub> Receptor Induces Agonist-Independent Internalization JF - Molecular Pharmacology JO - Mol Pharmacol SP - 961 LP - 972 DO - 10.1124/mol.63.5.961 VL - 63 IS - 5 AU - John A. Gray AU - Anushree Bhatnagar AU - Vsevolod V. Gurevich AU - Bryan L. Roth Y1 - 2003/05/01 UR - http://molpharm.aspetjournals.org/content/63/5/961.abstract N2 - 5-HT2A serotonin receptors are unusual among G-protein coupled receptors in that they can be internalized and desensitized, in some cell types, in an arrestin-independent manner. The molecular basis of the arrestin-insensitivity of 5-HT2A receptors is unknown but is probably caused, in part, by the apparent lack of agonist-induced 5-HT2A receptor phosphorylation. Because the arrestin-insensitivity of 5-HT2A receptors is cell-type selective, we used a “constitutively active” arrestin mutant that can interact with agonist-activated but nonphosphorylated receptors. We show here that this “constitutively active” arrestin mutant (Arr2-R169E) can force 5-HT2A receptors to be regulated by arrestins. Cotransfection of 5-HT2A receptors with Arr2-R169E induced agonist-independent 5-HT2A receptor internalization, and a constitutive translocation of the Arr2-R169E mutant to the plasma membrane, whereas wild-type Arrestin-2 had no effect. Additionally, Arr2-R169E, unlike wild-type arrestin-2, induced a significant decrease in efficacy of agonist-induced phosphoinositide hydrolysis with an unexpected increase in agonist potency. Radioligand binding assays demonstrated that the fraction of receptors in the high-affinity agonist binding-state increased with expression of Arr2-R169E, indicating that Arr2-R169E stabilizes the agonist-high affinity state of the 5-HT2A receptor (R*). Intriguingly, the agonist-independent interaction of Arr2-R169E with 5-HT2A receptors was inhibited by inverse agonist treatment and is thus probably caused by the high level of 5-HT2Areceptor constitutive activity. This is the first demonstration that a constitutively active arrestin mutant can both induce agonist-independent internalization and stabilize the agonist-high affinity state of an arrestin-insensitive G protein coupled receptor. The American Society for Pharmacology and Experimental Therapeutics ER -