Arrestin binding to the M-2 muscarinic acetylcholine receptor is precludedby an inhibitory element in the third intracellular loop of the receptor

Desensitization of G protein-coupled receptors (GPCRs) involves the binding of members of the family of arrestins to the receptors, In the model syste m involving the visual GPCR rhodopsin, activation and phosphorylation of rh odopsin is thought to convert arrestin from a low to high affinity binding state. Phosphorylation of the M-2 muscarinic acetylcholine receptor (mAChR) has been shown to be required for binding of arrestins 2 and 3 in vitro an d for arrestin-enhanced internalization in intact cells (Pals-Rylaarsdam, R ., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158), For the M, mAC hR, arrestin binding requires phosphorylation at multiple serine and threon ine residues at amino acids 307-311 in the third intracellular (i3) loop. H ere, we have investigated the molecular basis for the requirement of recept or phosphorylation for arrestin binding. Constructs of arrestin 2 that can bind to other GPCRs in a phosphorylation independent manner were unable to interact with a mutant M, mAChR in which the Ser/Thr residues at 307-311 we re mutated to alanines. However, although phosphorylation-deficient mutants of the M, mAChR that lacked 50-157 amino acids from the i3 loop were unabl e to undergo agonist-dependent internalization when expressed alone in tsA2 01 cells, co-expression of arrestin 2 or 3 restored agonist-dependent inter nalization. Furthermore, a deletion of only 15 amino acids (amino acids 304 -319) was sufficient to allow for phosphorylation-independent arrestin-rece ptor interaction. These results indicate that phosphorylation at residues 3 07-311 does not appear to be required to activate arrestin into a high affi nity binding state. Instead, phosphorylation at residues 307-311 appears to facilitate the removal of an inhibitory constraint that precludes receptor -arrestin association in the absence of receptor phosphorylation.