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RACK1 associates with muscarinic receptors and regulates M(2) receptor trafficking.

Reiner CL, McCullar JS, Kow RL, Le JH, Goodlett DR, Nathanson NM - PLoS ONE (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
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.

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Agonist-sensitive interaction of RACK1 with M2.HEK cells stably expressing either PCDNA3.1 or Flag-M2 were treated with 1 mM carbachol (CCH) for 30 minutes as indicated. Receptors were immunoprecipitated with anti-Flag antibodies and run on SDS-PAGE. Anti-RACK1 antibodies were used to blot. Blot shown is representative of ≥3 experiments.
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pone-0013517-g001: Agonist-sensitive interaction of RACK1 with M2.HEK cells stably expressing either PCDNA3.1 or Flag-M2 were treated with 1 mM carbachol (CCH) for 30 minutes as indicated. Receptors were immunoprecipitated with anti-Flag antibodies and run on SDS-PAGE. Anti-RACK1 antibodies were used to blot. Blot shown is representative of ≥3 experiments.

Mentions: In order to confirm an interaction between M2 and RACK1, we used immunoprecipitation of Flag-M2 from stably transfected HEK cells with anti-Flag antibodies, followed by Western blot analysis with anti-RACK1 antibodies. We also tested the effects of carbachol stimulation on the interaction by immunoprecipitation from unstimulated and carbachol-stimulated cells, and compared the amount of RACK1 immunoprecipitated by anti-Flag antibody from non-M2 expressing cells as a test for the specificity of co-immunoprecipitation (Fig. 1). We found that RACK1 and M2 specifically co-immunoprecipitate and that this interaction is disrupted by carbachol treatment, despite having used agonist stimulated cells in the original proteomics experiments. We also found that RACK1 could be co-immunoprecipitated with M2 in an agonist-sensitive manner from transiently transfected JEG-3 cells (data not shown.) Interestingly, the crosslinker used in the proteomics experiment was not necessary to observe the interaction between RACK1 and M2 in either cell type.


RACK1 associates with muscarinic receptors and regulates M(2) receptor trafficking.

Reiner CL, McCullar JS, Kow RL, Le JH, Goodlett DR, Nathanson NM - PLoS ONE (2010)

Agonist-sensitive interaction of RACK1 with M2.HEK cells stably expressing either PCDNA3.1 or Flag-M2 were treated with 1 mM carbachol (CCH) for 30 minutes as indicated. Receptors were immunoprecipitated with anti-Flag antibodies and run on SDS-PAGE. Anti-RACK1 antibodies were used to blot. Blot shown is representative of ≥3 experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958127&req=5

pone-0013517-g001: Agonist-sensitive interaction of RACK1 with M2.HEK cells stably expressing either PCDNA3.1 or Flag-M2 were treated with 1 mM carbachol (CCH) for 30 minutes as indicated. Receptors were immunoprecipitated with anti-Flag antibodies and run on SDS-PAGE. Anti-RACK1 antibodies were used to blot. Blot shown is representative of ≥3 experiments.
Mentions: In order to confirm an interaction between M2 and RACK1, we used immunoprecipitation of Flag-M2 from stably transfected HEK cells with anti-Flag antibodies, followed by Western blot analysis with anti-RACK1 antibodies. We also tested the effects of carbachol stimulation on the interaction by immunoprecipitation from unstimulated and carbachol-stimulated cells, and compared the amount of RACK1 immunoprecipitated by anti-Flag antibody from non-M2 expressing cells as a test for the specificity of co-immunoprecipitation (Fig. 1). We found that RACK1 and M2 specifically co-immunoprecipitate and that this interaction is disrupted by carbachol treatment, despite having used agonist stimulated cells in the original proteomics experiments. We also found that RACK1 could be co-immunoprecipitated with M2 in an agonist-sensitive manner from transiently transfected JEG-3 cells (data not shown.) Interestingly, the crosslinker used in the proteomics experiment was not necessary to observe the interaction between RACK1 and M2 in either cell type.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, University of Washington, Seattle, Washington, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus