Highlights d Quantified the proteomes of 375 cell lines from diverse lineages in the CCLE d Correlated expression of proteins across many pathways d Downregulation of multiple protein complexes in microsatellite instability d Protein complexes associated with sensitivity to gene knockdown and mutation

DNA double-strand breaks (DSBs) are acutely hazardous for cells, as they can cause genome instability. DSB repair involves the sequential recruitment of repair factors to the DSBs, followed by Rad51-mediated homology probing, DNA synthesis, and ligation. However, little is known about how cells react if no homology is found and DSBs persist. Here, by monitoring a single persistent DNA break, we show that, following DNA resection and RPA recruitment, Rad51 spreads chromosome-wide bidirectionally from the DSB but selectively only on the broken chromosome. Remarkably, the persistent DSB is later fixed to the nuclear periphery in a process that requires Rad51, the histone variant H2A.Z, its SUMO modification, and the DNA-damage checkpoint. Indeed, H2A.Z is deposited close to the break early but transiently and directs DNA resection, single DSB-induced checkpoint activation, and DSB anchoring. Thus, a persistent DSB induces a multifaceted response, which is linked to a specific chromatin mark.

Increased cardiac contractility during fight-or-flight response is caused by β-adrenergic augmentation of Ca V 1.2 channels 1-4. In transgenic murine hearts expressing fully PKA phosphorylation-site-deficient mutant Ca V 1.2 α 1C and β subunits, this regulation persists, implying involvement of extra-channel factors. Here, we identify the mechanism by which β-adrenergic agonists stimulate voltage-gated Ca 2+ channels. We expressed α 1C or β 2B subunits conjugated to ascorbate-peroxidase 5 in mouse hearts and used multiplexed, quantitative proteomics 6,7 to track hundreds of proteins in proximity of Ca V 1.2. We observed that the Ca 2+ channel inhibitor Rad 8,9 , a monomeric G-protein, is enriched in the Ca V 1.2 micro-environment but is depleted during β-adrenergic stimulation. PKA-catalyzed phosphorylation of specific Ser residues on Rad decreases its affinity for auxiliary β-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

SUMMARY G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans, and they regulate processes ranging from neurotransmission to cardiovascular biology. Although GPCRs have been studied for decades, current methods for tracking GPCR signaling often suffer from low throughput, modification or overexpression of effector proteins, and low temporal resolution. Here, we introduce a new approach using peroxidase-catalyzed proximity labeling to track GPCR signaling and internalization in living cells. Combination of this technique with isobaric labeling and triple-stage mass spectrometry enables precise, quantitative, and time-resolved measurement of thousands of receptor-proximal proteins at their native levels to comprehensively track GPCR agonist response. Using this technique, we examine the response of the angiotensin II type 1 receptor to both balanced and biased agonists. In addition, we extend the approach to the β2 adrenergic receptor, underscoring the generalizability of this technology.