%0 Journal Article %1 Lee2000 %A Lee, K. B. %A Ptasienski, J. A. %A Bunemann, M. %A Hosey, M. M. %D 2000 %J J Biol Chem %K *Endocytosis 50 Acid Acids/*metabolism Agonists/metabolism/pharmacology Amino Animals Arrestins/chemistry/genetics/*metabolism Binding Carbachol/metabolism/pharmacology Cell Cholinergic Concentration Data Humans Inhibitory Line M2 Molecular Muscarinic Muscarinic/*chemistry/genetics/*metabolism Mutagenesis/genetics Phosphorylation/drug Precipitin Protein Sequence Substitution/genetics Tests Transfection effects Receptor %N 46 %P 35767-77 %T Acidic amino acids flanking phosphorylation sites in the M2 muscarinic receptor regulate receptor phosphorylation, internalization, and interaction with arrestins %U http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10952973 %V 275 %X The studies reported here address the molecular events underlying the interactions 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 clusters of serines and threonines at positions 286-290 (site P1) 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 P1 or P2 can support agonist-dependent internalization. However, phosphorylation at P2 is required for receptor interaction with arrestins (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and 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 interactions. Mutation of the acidic amino acids at positions 298-300 (site A1) and/or 304-305 (site A2) to alanines had significant effects on agonist-dependent phosphorylation. P2 was identified as the preferred site of agonist-dependent phosphorylation, and full phosphorylation at P2 required the acidic amino acids at A1 or their neutral counterparts. In contrast, phosphorylation at site P1 was dependent on site A2. In addition, sites A1 and A2 significantly affected the ability of the wild type and P1 and P2 mutant receptors to internalization and to interact with arrestin2. Substitution of asparagine and glutamine for the aspartates and glutamates at sites A1 or A2 did not influence receptor phosphorylation but did influence arrestin interaction with the receptor. We propose that the amino acids at sites A1 and A2 play important roles in agonist-dependent phosphorylation at sites P2 and P1, respectively, and also play an important role in arrestin interactions with the M(2) mAChR.

@article{Lee2000, abstract = {The studies reported here address the molecular events underlying the interactions 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 clusters of serines and threonines at positions 286-290 (site P1) 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 P1 or P2 can support agonist-dependent internalization. However, phosphorylation at P2 is required for receptor interaction with arrestins (Pals-Rylaarsdam, R., Gurevich, V. V., Lee, K. B., Ptasienski, J. A., Benovic, J. L., and 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 interactions. Mutation of the acidic amino acids at positions 298-300 (site A1) and/or 304-305 (site A2) to alanines had significant effects on agonist-dependent phosphorylation. P2 was identified as the preferred site of agonist-dependent phosphorylation, and full phosphorylation at P2 required the acidic amino acids at A1 or their neutral counterparts. In contrast, phosphorylation at site P1 was dependent on site A2. In addition, sites A1 and A2 significantly affected the ability of the wild type and P1 and P2 mutant receptors to internalization and to interact with arrestin2. Substitution of asparagine and glutamine for the aspartates and glutamates at sites A1 or A2 did not influence receptor phosphorylation but did influence arrestin interaction with the receptor. We propose that the amino acids at sites A1 and A2 play important roles in agonist-dependent phosphorylation at sites P2 and P1, respectively, and also play an important role in arrestin interactions with the M(2) mAChR.}, added-at = {2010-12-14T18:12:02.000+0100}, author = {Lee, K. B. and Ptasienski, J. A. and Bunemann, M. and Hosey, M. M.}, biburl = {https://www.bibsonomy.org/bibtex/2196816a25cce581bae145e41b8c03390/pharmawuerz}, endnotereftype = {Journal Article}, interhash = {ac4cae8d4cc37233fc732915c6a354d8}, intrahash = {196816a25cce581bae145e41b8c03390}, issn = {0021-9258 (Print) 0021-9258 (Linking)}, journal = {J Biol Chem}, keywords = {*Endocytosis 50 Acid Acids/*metabolism Agonists/metabolism/pharmacology Amino Animals Arrestins/chemistry/genetics/*metabolism Binding Carbachol/metabolism/pharmacology Cell Cholinergic Concentration Data Humans Inhibitory Line M2 Molecular Muscarinic Muscarinic/*chemistry/genetics/*metabolism Mutagenesis/genetics Phosphorylation/drug Precipitin Protein Sequence Substitution/genetics Tests Transfection effects Receptor}, month = {Nov 17}, note = {Lee, K B Ptasienski, J A Bunemann, M Hosey, M M HL50121/HL/NHLBI NIH HHS/United States T32GM08061/GM/NIGMS NIH HHS/United States Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. United states The Journal of biological chemistry J Biol Chem. 2000 Nov 17;275(46):35767-77.}, number = 46, pages = {35767-77}, shorttitle = {Acidic amino acids flanking phosphorylation sites in the M2 muscarinic receptor regulate receptor phosphorylation, internalization, and interaction with arrestins}, timestamp = {2010-12-14T18:22:02.000+0100}, title = {Acidic amino acids flanking phosphorylation sites in the M2 muscarinic receptor regulate receptor phosphorylation, internalization, and interaction with arrestins}, url = {http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=10952973}, volume = 275, year = 2000 }