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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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Effects of decreased RACK1 expression on M2 mAChR internalization.HEK cells stably expressing Flag-M2 with normal levels of RACK1 (cell line M2-1; open circles) or low levels of RACK1 (cell line M2-2; black squares) expression. Cell surface receptors were measured by [3H]NMS binding following stimulation for 15 or 30 minutes with 1 mM carbachol. Internalization is expressed as percent of receptors remaining on the cell surface compared to unstimulated cells. * indicates p<0.02.
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pone-0013517-g005: Effects of decreased RACK1 expression on M2 mAChR internalization.HEK cells stably expressing Flag-M2 with normal levels of RACK1 (cell line M2-1; open circles) or low levels of RACK1 (cell line M2-2; black squares) expression. Cell surface receptors were measured by [3H]NMS binding following stimulation for 15 or 30 minutes with 1 mM carbachol. Internalization is expressed as percent of receptors remaining on the cell surface compared to unstimulated cells. * indicates p<0.02.

Mentions: During the course of isolating stably transfected M2 expressing cells, we found that one M2 expressing clonal cell line (M2-2) expressed significantly lower levels of RACK1 compared both to several clonal lines transfected with pCDNA3.1 alone (PC-1, PC-2) and to another M2 expressing clonal cell line (M2-1), with M2-2 expressing approximately 1/5 the level of the M2-1 cell line as determined by densitometry analysis (Fig. 4). While RACK1 could be co-immunoprecipitated with M2 in M2-1 cells (Fig. 1), we were not able to detect RACK1 co-immunoprecipitation from the M2-2 cells (data not shown). We took advantage of this to test the effects of the decreased levels of RACK1 expression on M2 trafficking. Receptor expression in both cell lines was found to be mainly (≥80%) on the cell surface in unstimulated conditions. We found that in cells with decreased RACK1 expression levels there is an increase in the extent of M2 internalization with over 60% of receptors internalized after 30 minutes compared to almost 45% of receptors internalized in cells with normal levels of RACK1 expression (Fig. 5). When we tested M2 receptor down regulation in the stable cell lines relatively lacking in RACK1, we found that down regulation was again inhibited with only about 20% of receptors down regulated compared to almost 60% down regulated in cells with normal levels of RACK1 expression (Fig. 6).


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)

Effects of decreased RACK1 expression on M2 mAChR internalization.HEK cells stably expressing Flag-M2 with normal levels of RACK1 (cell line M2-1; open circles) or low levels of RACK1 (cell line M2-2; black squares) expression. Cell surface receptors were measured by [3H]NMS binding following stimulation for 15 or 30 minutes with 1 mM carbachol. Internalization is expressed as percent of receptors remaining on the cell surface compared to unstimulated cells. * indicates p<0.02.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0013517-g005: Effects of decreased RACK1 expression on M2 mAChR internalization.HEK cells stably expressing Flag-M2 with normal levels of RACK1 (cell line M2-1; open circles) or low levels of RACK1 (cell line M2-2; black squares) expression. Cell surface receptors were measured by [3H]NMS binding following stimulation for 15 or 30 minutes with 1 mM carbachol. Internalization is expressed as percent of receptors remaining on the cell surface compared to unstimulated cells. * indicates p<0.02.
Mentions: During the course of isolating stably transfected M2 expressing cells, we found that one M2 expressing clonal cell line (M2-2) expressed significantly lower levels of RACK1 compared both to several clonal lines transfected with pCDNA3.1 alone (PC-1, PC-2) and to another M2 expressing clonal cell line (M2-1), with M2-2 expressing approximately 1/5 the level of the M2-1 cell line as determined by densitometry analysis (Fig. 4). While RACK1 could be co-immunoprecipitated with M2 in M2-1 cells (Fig. 1), we were not able to detect RACK1 co-immunoprecipitation from the M2-2 cells (data not shown). We took advantage of this to test the effects of the decreased levels of RACK1 expression on M2 trafficking. Receptor expression in both cell lines was found to be mainly (≥80%) on the cell surface in unstimulated conditions. We found that in cells with decreased RACK1 expression levels there is an increase in the extent of M2 internalization with over 60% of receptors internalized after 30 minutes compared to almost 45% of receptors internalized in cells with normal levels of RACK1 expression (Fig. 5). When we tested M2 receptor down regulation in the stable cell lines relatively lacking in RACK1, we found that down regulation was again inhibited with only about 20% of receptors down regulated compared to almost 60% down regulated in cells with normal levels of RACK1 expression (Fig. 6).

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