Acidic amino acids flanking phosphorylation sites in the M-2 muscarinic receptor regulate receptor phosphorylation, internalization, and interaction with arrestins

The studies reported here address the molecular events underlying the inter actions of arrestins with the M-2 muscarinic acetylcholine receptor (mAChR) , In particular, we focused on the role of receptor phosphorylation in this process. Agonist-dependent phosphorylation of the M-2 mAChR can occur at c lusters of serines and threonines at positions 286-290 (site pi) or 307-311 (site P2) in the third intracellular loop (Pals-Rylaarsdam, R,, and Hosey, M, M. (1997) J, Biol, Chem, 272, 14152-14158). Phosphorylation at either P 1 or P2 can support agonist-dependent internalization. However, phosphoryla tion at P2 is required for receptor interaction with arrestins (Pals-Rylaar sdam, R,, Gurevich, V, V,, Lee, K. B,, Ptasienski, J, A., Benovic, J, L,, a nd Hosey, M, M, (1997) J, Biol, Chem. 272, 23682-26389), The present study investigated the role of acidic amino acids between P1 and P2 in regulating receptor phosphorylation, internalization, and receptor/arrestin interacti ons. Mutation of the acidic amino acids at positions 298-300 (site Al) and/ or 304-305 (site A2) to alanines had significant effects on agonist-depende nt phosphorylation, P2 was identified as the preferred site of agonist-depe ndent phosphorylation, and full phosphorylation at P2 required the acidic a mino acids at Al or their neutral counterparts. In contrast, phosphorylatio n at site P1 was dependent on site A2, In addition, sites Al and A2 signifi cantly affected the ability of the wild type and P1 and P2 mutant receptors to internalization and to interact with arrestina. Substitution of asparag ine and glutamine for the aspartates and glutamates at sites Al or A2 did n ot influence receptor phosphorylation but did influence arrestin interactio n with the receptor. We propose that the amino acids at sites Al and A2 pla y important roles in agonist-dependent phosphorylation at sites P2 and Fl, respectively, and also play an important role in arrestin interactions with the M-2 mAChR.