Phosphorylation-dephosphorylation of muscarinic acetylcholine receptors: evidence for the in vivo and in vitro release of receptors from rat brain plasma membrane.
The effects of phosphorylation on muscarinic acetylcholine receptors (mAChRs) were studied in vitro and in vivo using rat brain plasma membrane and receptors partially purified at least 2500-fold. Purified mAChRs were phosphorylated in vitro by cAMP-dependent protein kinase and dephosphorylated by calcineurin. Phosphorylation of purified mAChRs was enhanced by carbachol and blocked by atropine. The filtrate which passed through glass fiber filters and high speed supernates were assayed for mAChRs by an ammonium sulfate precipitation method. Following incubation of the plasma membrane under phosphorylating conditions and ultracentrifugation at 300,000 g, the mAChRs appeared in the high speed supernate. This release was stimulated by adding carbachol to the incubation medium. In rats treated with carbachol, brain mAChRs redistributed from the heavy into the light membrane fractions. Ultrastructural examination of the light membrane fractions and the 300,000 g supernatant fractions after in vivo and in vitro carbachol treatment calcineurin increased the reincorporation of added partially purified receptors into the plasma membrane. The release and reincorporation of mAChRs strongly imply that there is a translocation and recycling of mAChRs between plasma membrane and cytosol in vivo. The significance and the function of the translocation of mAChRs remain to be investigated.
Ho
A K
AK
Department of Basic Science, University of Illinois, College of Medicine, Peoria 61605.
Zhang
Y J
YJ
Duffield
R
R
Zheng
G M
GM
D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000648:ultrastructure; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008297:Male; D008854:Microscopy, Electron; D010749:Phosphoprotein Phosphatases / Q000494:pharmacology; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000378:metabolism*
D002148:Calmodulin-Binding Proteins; D011976:Receptors, Muscarinic; D002217:Carbachol; D011494:Protein Kinases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases