Abstract
Receptor internalization from the cell surface occurs through several mechanisms. Some of these mechanisms, such as clathrin coated pits, are well understood. The M2 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 M2-immunoprecipitates from M2-expressing cells over those of non-M2 expressing cells. Treatment of cells with the agonist carbachol disrupted the interaction of RACK1 with M2. We further found that RACK1 overexpression inhibits the internalization and subsequent down regulation of the M2 receptor in a receptor subtype-specific manner. Decreased RACK1 expression increases the rate of agonist internalization of the M2 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 M2 receptors to the degradative pathway.
Highlights
Endocytosis of cell surface proteins has several functions, e.g., delivery of nutrients and viruses into the cell
To identify polypeptides associated with the M2 receptor, we used isotope-coded affinity tagging (ICAT), a technique in which the protein content of two samples is compared by covalently labeling peptides from the sample and control preparations with a heavy or light isotope [13]
We report that RACK1 interacts with M2 and regulates trafficking of the receptor following agonist stimulation
Summary
Endocytosis of cell surface proteins has several functions, e.g., delivery of nutrients and viruses into the cell. Internalization of cell surface receptors allows for coupling to additional signal transduction pathways [1], resensitization and recycling to the cell surface for further signaling [2], or degradation (down regulation) [3]. Endocytosis can occur both constitutively, as in the case of the transferrin receptor, or in response to agonist stimulation, as in the case of most G-protein coupled receptors (GPCRs). One clathrin independent pathway is caveolar internalization, which involves caveolin and is associated with lipid rafts [5,6]. The M2 muscarinic acetylcholine receptor (mAChR) does not associate with clathrin or caveolin in JEG-3 and HEK cells [7,8,9,10,11,12]
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