RACK1 associates with muscarinic receptors and regulates M(2) receptor trafficking.
Receptor internalization from the cell surface occurs through several mechanisms. Some of these mechanisms, such as clathrin coated pits, are well understood. The M(2) muscarinic acetylcholine receptor undergoes internalization via a poorly-defined clathrin-independent mechanism. We used isotope coded affinity tagging and mass spectrometry to identify the scaffolding protein, receptor for activated C kinase (RACK1) as a protein enriched in M(2)-immunoprecipitates from M(2)-expressing cells over those of non-M(2) expressing cells. Treatment of cells with the agonist carbachol disrupted the interaction of RACK1 with M(2). We further found that RACK1 overexpression inhibits the internalization and subsequent down regulation of the M(2) receptor in a receptor subtype-specific manner. Decreased RACK1 expression increases the rate of agonist internalization of the M(2) receptor, but decreases the extent of subsequent down-regulation. These results suggest that RACK1 may both interfere with agonist-induced sequestration and be required for subsequent targeting of internalized M(2) receptors to the degradative pathway.
Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.
Cindy L
CL
Reiner
Jennifer S
JS
McCullar
Rebecca L
RL
Kow
Joshua H
JH
Le
David R
DR
Goodlett
Neil M
NM
Nathanson
D000345:Affinity Labels; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D009363:Neoplasm Proteins / Q000378:metabolism*; D021381:Protein Transport; D043585:Receptor, Muscarinic M2 / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000378:metabolism*
D000345:Affinity Labels; D009363:Neoplasm Proteins; C419368:RACK1 protein, human; D043585:Receptor, Muscarinic M2; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D019204:GTP-Binding Proteins