RACK1 Associates with Muscarinic Receptors and Regulates M2 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 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

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Agonist-sensitive interaction of RACK1 with M₂.

<p>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.</p

Effects of decreased RACK1 expression on M₂ mAChR down regulation.

<p>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.</p

RACK1 in lysates of HEK cells stably expressing M₂.

<p>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.</p

Effects of RACK1 overexpression on mAChR internalization.

<p>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. <i>Inset</i>: 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.</p

Effects of decreased RACK1 expression on M₂ mAChR internalization.

<p>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.</p

Sex differences in oncogenic mutational processes

Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research.Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research.Peer reviewe

Sex differences in oncogenic mutational processes

Funder: Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada (NSERC Canadian Network for Research and Innovation in Machining Technology); doi: https://doi.org/10.13039/501100002790Funder: Genome Canada (Génome Canada); doi: https://doi.org/10.13039/100008762Funder: Canada Foundation for Innovation (Fondation canadienne pour l'innovation); doi: https://doi.org/10.13039/501100000196Funder: Terry Fox Research Institute (Institut de Recherche Terry Fox); doi: https://doi.org/10.13039/501100004376Abstract: Sex differences have been observed in multiple facets of cancer epidemiology, treatment and biology, and in most cancers outside the sex organs. Efforts to link these clinical differences to specific molecular features have focused on somatic mutations within the coding regions of the genome. Here we report a pan-cancer analysis of sex differences in whole genomes of 1983 tumours of 28 subtypes as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium. We both confirm the results of exome studies, and also uncover previously undescribed sex differences. These include sex-biases in coding and non-coding cancer drivers, mutation prevalence and strikingly, in mutational signatures related to underlying mutational processes. These results underline the pervasiveness of molecular sex differences and strengthen the call for increased consideration of sex in molecular cancer research