title: The interaction of a constitutively active arrestin with the arrestin-insensitive 5-HT(2A) receptor induces agonist-independent internalization.
abstract: 5-HT(2A) 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-HT(2A) receptors is unknown but is probably caused, in part, by the apparent lack of agonist-induced 5-HT(2A) receptor phosphorylation. Because the arrestin-insensitivity of 5-HT(2A) 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-HT(2A) receptors to be regulated by arrestins. Cotransfection of 5-HT(2A) receptors with Arr2-R169E induced agonist-independent 5-HT(2A) 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-HT(2A) receptor (R*). Intriguingly, the agonist-independent interaction of Arr2-R169E with 5-HT(2A) receptors was inhibited by inverse agonist treatment and is thus probably caused by the high level of 5-HT(2A) receptor 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.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4935, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Gray
authors.forename: Anushree
authors.initials: A
authors.lastname: Bhatnagar
authors.forename: Vsevolod V
authors.initials: VV
authors.lastname: Gurevich
authors.forename: Bryan L
authors.initials: BL
authors.lastname: Roth
mesh_terms: D019943:Amino Acid Substitution; D019390:Arrestins / Q000378:metabolism*; D002478:Cells, Cultured; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D006868:Hydrolysis; D009474:Neurons / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000235:genetics
chemical_list: D019390:Arrestins; D010750:Phosphoproteins; D044402:Receptor, Serotonin, 5-HT2A; D011985:Receptors, Serotonin; D018698:Glutamic Acid; D014443:Tyrosine