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

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.

Figure 1. 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.

Figure 2. Effects of RACK1 overexpression on mAChR internalization.

HEK cells were cotransfected with either M₁ (A), M₂ (B), M₃ (C), or M₄ (D) and either PCDNA3.1 (open circles) or RACK1 (black squares). Cell surface receptors were measured by [³H]NMS binding following stimulation for 5, 15, 30 and 60 minutes with 1 mM carbachol. Internalization is expressed as percent of receptor remaining on the cell surface compared to control unstimulated cells. Inset: data from panel B were fit to an exponential decay curve. The rate-constants are M₂ + PCDNA3.1, 3.2×10⁻² min⁻¹; M₂ + RACK1, 2.1×10⁻² min⁻¹. *indicates p≤0.04.

Figure 3. Effects of RACK1 overexpression on mAChR down regulation.

HEK cells were cotransfected with either M₁ (A) or M₂ (B) and either PCDNA3.1 (black bars) or RACK1 (white bars). Total receptors were measured by [³H]QNB binding following 8 hours of stimulation with 1 mM carbachol. Down regulation is expressed as percent of control cells. * indicates p<0.001.

Figure 4. RACK1 in lysates of HEK cells stably expressing M₂.

Lysates from four clonal HEK cell lines stably transfected with either PCDNA3.1 (PC-1 and PC-2) or Flag-M₂ (M2-1 and M2-2) were run on SDS-PAGE. Anti-RACK1 antibodies were used to blot. Blot shown are representative of ≥3 experiments.

Figure 5. 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.

Figure 6. Effects of decreased RACK1 expression on M₂ mAChR down regulation.

HEK cells stably expressing Flag-M₂ with either normal levels of RACK1 (black bar) or with low levels of RACK1 (white bar) expression. Total receptors were measured by [³H]QNB binding following 8 hours of stimulation with 1 mM carbachol. Down regulation is expressed as percent of receptor down regulated compared to unstimulated cells. * indicates p<0.001.