The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed. We present a list of 1,580 human core fitness genes and describe their general properties. Moreover, we demonstrate that context-dependent fitness genes accurately recapitulate pathway-specific genetic vulnerabilities induced by known oncogenes and reveal cell-type-specific dependencies for specific receptor tyrosine kinases, even in oncogenic KRAS backgrounds. Thus, rigorous identification of human cell line fitness genes using a high-complexity CRISPR-Cas9 library affords a high-resolution view of the genetic vulnerabilities of a cell.

The Wnt family of secreted ligands act through many receptors to stimulate distinct intracellular signalling pathways in embryonic development, in adults and in disease processes. Binding of Wnt to the Frizzled family of receptors and to low density lipoprotein receptor-related protein 5 (LRP5) or LRP6 co-receptors stimulates the intracellular Wnt-beta-catenin signalling pathway, which regulates beta-cateninstability and context-dependent transcription. This signalling pathway controls many processes, such as cell fate determination, cell proliferation and self-renewal of stem and progenitor cells. Intriguingly, the transmembrane receptor Tyr kinases Ror2 and Ryk, as well as Frizzledreceptors that act independently of LRP5 or LRP6, function as receptors for Wnt and activate beta-catenin-independent pathways. This leads to changes in cell movement and polarity and to the antagonism of the beta-catenin pathway.

Protein ubiquitination is a common form of post-translational modification that regulates a broad spectrum of protein substrates in diverse cellular pathways. Through a three-enzyme (E1-E2-E3) cascade, the attachment of ubiquitin to proteins is catalysed by the E3 ubiquitin ligase, which is best represented by the superfamily of the cullin-RING complexes. Conserved from yeast to human, the DDB1-CUL4-ROC1 complex is a recently identified cullin-RING ubiquitin ligase, which regulates DNA repair, DNA replication and transcription, and can also be subverted by pathogenic viruses to benefit viral infection. Lacking a canonical SKP1-like cullin adaptor and a defined substrate recruitment module, how the DDB1-CUL4-ROC1 E3 apparatus is assembled for ubiquitinating various substrates remains unclear. Here we present crystallographic analyses of the virally hijacked form of the human DDB1-CUL4A-ROC1 machinery, which show that DDB1 uses one beta-propeller domain for cullin scaffold binding and a variably attached separate double-beta-propeller fold for substrate presentation. Through tandem-affinity purification of human DDB1 and CUL4A complexes followed by mass spectrometry analysis, we then identify a novel family of WD40-repeat proteins, which directly bind to the double-propeller fold of DDB1 and serve as the substrate-recruiting module of the E3. Together, our structural and proteomic results reveal the structural mechanisms and molecular logic underlying the assembly and versatility of a new family of cullin-RING E3 complexes.

mRNA processing, transport, translation, and ultimately degradation involve a series of dedicated protein complexes that often assemble into large membraneless structures such as stress granules (SGs) and processing bodies (PBs). Here, systematic in vivo proximity-dependent biotinylation (BioID) analysis of 119 human proteins associated with different aspects of mRNA biology uncovers 7424 unique proximity interactions with 1,792 proteins. Classical bait-prey analysis reveals connections of hundreds of proteins to distinct mRNA-associated processes or complexes, including the splicing and transcriptional elongation machineries (protein phosphatase 4) and the CCR4-NOT deadenylase complex (CEP85, RNF219, and KIAA0355). Analysis of correlated patterns between endogenous preys uncovers the spatial organization of RNA regulatory structures and enables the definition of 144 core components of SGs and PBs. We report preexisting contacts between most core SG proteins under normal growth conditions and demonstrate that several core SG proteins (UBAP2L, CSDE1, and PRRC2C) are critical for the formation of microscopically visible SGs.

Gli zinc-finger proteins are transcription factors involved in the intracellular signal transduction controlled by the Hedgehog family of secreted molecules. They are frequently mutated in human congenital malformations, and their abnormal regulation leads to tumorigenesis. Genetic studies in several model systems indicate that their activity is tightly regulated by Hedgehog signaling through various posttranslational modifications, including phosphorylation, ubiquitin-mediated degradation, and proteolytic processing, as well as through nucleocytoplasmic shuttling. In vertebrate cells, primary cilia are required for the sensing of Hedgehog pathway activity and involved in the processing and activation of Gli proteins. Two evolutionarily conserved Hedgehog pathway components, Suppressor of fused and Kif7, are core intracellular regulators of mammalian Gli proteins. Recent studies revealed that Gli proteins are also regulated transcriptionally and posttranslationally through noncanonical mechanisms independent of Hedgehog signaling. In this review, we describe the regulation of Gli proteins during development and discuss possible mechanisms for their abnormal activation during tumorigenesis.

Quantitative bioluminescence resonance energy transfer (BRET) analysis was applied to the study of ␤ 1 -and ␤ 2 -adrenergic receptor homo-and heterodimerization. To assess the relative affinity between each of the protomers, BRET saturation experiments were carried out in HEK-293T cells. ␤ 1 -and ␤ 2 -adrenergic receptors were found to have similar propensity to engage in homo-and heterotropic interactions suggesting that, at equivalent expression levels of the two receptor subtypes, an equal proportion of homo-and heterodimers would form. Analysis of the data also revealed that, at equimolar expression levels of energy donor and acceptor, more than 80% of the receptor molecules exist as dimers and that this high incidence of receptor dimerization is insensitive to receptor density for expression levels varying between 1.4 and 26.9 pmol of receptor/mg of membrane protein. Taken together, these results indicate that most of the receptors expressed in cells exist as constitutive dimers and that, at least in undifferentiated fibroblasts, the proportion of homo-and heterodimers between the closely related ␤ 1 -and ␤ 2 -adrenergic receptors is determined by their relative levels of expression. G protein-coupled receptors (GPCRs)1 represent the largest family of transmembrane receptors involved in cell signaling. In the past few years, many studies indicated that GPCR dimerization can occur between two identical receptors (homodimerization), between two different receptor subtypes of the same family, or even between receptors that are only distantly related (heterodimerization) (for a review, see Refs. 1 and 2). In most instances, co-immunoprecipitation was used as the primary experimental evidence supporting the existence of such dimers. More recently, however, light resonance energy transfer techniques such as fluorescence and bioluminescence resonance energy transfer (FRET and BRET) were also used. These "non-invasive" proximity-based assays confirmed that GPCR dimerization does not represent biochemical artifacts due to receptor solubilization and can occur in living cells. They have been used to demonstrate homodimerization of the ␤ 2 -adrenergic (3), the yeast alpha mating factor (4), the SST5 somatostatin (5), the gonadotropin releasing hormone (6), the luteinizing hormone (7), the ␦-opioid (8), the thyrotropin-releasing hormone (9), the cholecystokinin (10), and the melatonin (11) receptors as well as heterodimerization between somatostatin receptor subtypes (5), somatostatin and dopamine receptors (12), melatonin receptor subtypes (11), and opioid receptor subtypes (13).An advantage of BRET and FRET over co-immunoprecipitation approaches lies in the more quantitative nature of the assay. However, relatively few studies exploited this quantitative potential for the study of GPCR dimerization. For the melatonin receptors, Ayoub et al. (11) recently used BRET competition assays to determine that the transfer of energy resulted from the formation of dimers and not of higher order oligomers. They also showed that...

Heptahelical receptors that interact with heterotrimeric G proteins represent the largest family of proteins involved in signal transduction across biological membranes. Although these receptors generally were believed to be monomeric entities, a growing body of evidence suggests that they may form functionally relevant dimers. However, a definitive demonstration of the existence of G protein-coupled receptor (GPCR) dimers at the surface of living cells is still lacking. Here, using bioluminescence resonance energy transfer (BRET), as a protein-protein interaction assay in whole cells, we unambiguously demonstrate that the human ␤2-adrenergic receptor (␤ 2AR) forms constitutive homodimers when expressed in HEK-293 cells. Receptor stimulation with the hydrophilic agonist isoproterenol led to an increase in the transfer of energy between ␤2AR molecules genetically fused to the BRET donor (Renilla luciferase) and acceptor (green fluorescent protein), respectively, indicating that the agonist interacts with receptor dimers at the cell surface. Inhibition of receptor internalization did not prevent agonist-promoted BRET, demonstrating that it did not result from clustering of receptors within endosomes. The notion that receptor dimers exist at the cell surface was confirmed further by the observation that BS3, a cell-impermeable cross-linking agent, increased BRET between ␤2AR molecules. The selectivity of the constitutive interaction was documented by demonstrating that no BRET occurred between the ␤2AR and two other unrelated GPCR. In contrast, the well characterized agonist-dependent interaction between the ␤2AR and the regulatory protein ␤-arrestin could be monitored by BRET. Taken together, the data demonstrate that GPCR exist as functional dimers in vivo and that BRET-based assays can be used to study both constitutive and hormone-promoted selective protein-protein interactions. G protein-coupled receptors (GPCR) represent the single largest family of transmembrane receptors involved in cell signaling. Until recently, they were believed, unlike most other membrane receptors, to function as monomeric entities that interact with G proteins once stabilized in their active conformation by agonist binding. However, a growing body of functional and biochemical evidence suggests that they may exist as homo-or heterodimers. The functional evidence is based largely on positive and negative effects that dominant receptor mutants have on wild-type receptor function and on the observation that coexpression of two defective receptors can restore activity (1-6). More recently, coexpression of the type-2b ␥-aminobutyric acid receptor GABAb-R2 was found to be essential for the cell surface expression and the function of the GABAb-R1 subtype (7-9), suggesting that heterodimerization between the two receptor molecules is required for function. Biochemically, coimmunoprecipitation of receptors bearing different epitope tags was used to support the notion that GPCR homo-(10 -13) and heterodimers (9, 14) can form. However, the cons...

In the last four to five years, the view that G protein-coupled receptors (GPCRs) function as monomeric proteins has been challenged by numerous studies, which suggests that GPCRs exist as dimers or even higher-structure oligomers. Recently, biophysical methods based on luminescence and fluorescence energy transfer have confirmed the existence of such oligomeric complexes in living cells. Although no consensus exists on the role of receptor dimerization, converging evidence suggests potential roles in various aspects of receptor biogenesis and function. In several cases, receptors appear to fold as constitutive dimers early after biosynthesis, whereas ligand-promoted dimerization at the cell surface has been proposed for others. The reports of heterodimerization between receptor subtypes suggest a potential level of receptor complexity that could account for previously unexpected pharmacological diversities. In addition to fundamentally changing our views on the structure and activation processes of GPCRs, the concept of homo- and heterodimerization could have dramatic impacts on drug development and screening.