Transmembrane Notch Research Articles

Multifaceted regulation of Notch signaling by glycosylation.

To build a complex body composed of various cell types and tissues and to maintain tissue homeostasis in the postembryonic period, animals use a small number of highly conserved intercellular communication pathways. Among these is the Notch signaling pathway, which is mediated via the interaction of transmembrane Notch receptors and ligands usually expressed by neighboring cells. Maintaining optimal Notch pathway activity is essential for normal development, as evidenced by various human diseases caused by decreased and increased Notch signaling. It is therefore not surprising that multiple mechanisms are used to control the activation of this pathway in time and space. Over the last 20years, protein glycosylation has been recognized as a major regulatory mechanism for Notch signaling. In this review, we will provide a summary of the various types of glycan that have been shown to modulate Notch signaling. Building on recent advances in the biochemistry, structural biology, cell biology and genetics of Notch receptors and the glycosyltransferases that modify them, we will provide a detailed discussion on how various steps during Notch activation are regulated by glycans. Our hope is that the current review article will stimulate additional research in the field of Notch glycobiology and will potentially be of benefit to investigators examining the contribution of glycosylation to other developmental processes.

What Determines Fringe Specificity for a Notch EGF?

There are four transmembrane Notch (N) signaling receptors in humans involved in developmental, cell differentiation, and cell regulatory events, and dysregulation of N signaling leads to pathologies such as developmental disorders or cancer. The extracellular domain (ECD) of the N receptors are composed of 29 to 36 tandem Epidermal Growth Factor‐like repeats (EGFs) and N signaling occurs when EGFs of the ECD interact with a membrane tethered N ligand on an adjacent cell. These EGFs are heavily modified with O‐glycans that directly modulate interaction with, and activation by, the N ligands. PROTEIN O‐FUCOSYLTRANSFERASE 1 is an ER localized enzyme that adds an O‐linked fucose to Serines or Threonines within a properly folded EGF containing the C2‐X‐X‐X‐X‐ S/T‐C3 consensus sequence. The O‐fucose affects binding affinity for ligand, as well as N signaling levels. In the Golgi, O‐fucosylated EGFs are exposed to a family of β3‐N‐acetylglucosaminyltransferases named Fringes which can elongate the O‐fucose to a GlcNAc‐β1‐3Fuc‐O disaccharide. Mirroring effects seen after addition of fucose, there are additional effects on ligand binding affinity and signaling after Fringe elongation. There are three Fringe enzymes termed LUNATIC (Lfng), MANIC (Mfng), and RADICAL FRINGE (Rfng). We have mapped the extent of Fringe elongation across N1 and N2 EGFs and we find that generally speaking, Lfng modifies 50% or more of fucosylated EGFs, Mfng modifies a sub‐set of the Lfng sites, and Rfng modifies an even smaller subset of the Lfng sites. Additionally, despite high sequence identity over the entirety of the tandem EGFs in the ECD, the pattern of Fringe‐modified EGFs is different between N1 and N2. Prior work suggested that while primary amino acid sequence of a particular EGF can determine the level of elongation by Fringe enzymes, it may not be sufficient to explain all specificity differences. Our mapping data has shown that some corresponding EGFs in N1 and N2, such as EGF5 and EGF12, show different levels of Fringe modification, despite high sequence identity. These data suggested that the position of the EGF in the linear sequence of tandem EGFs could be another determinant of Fringe enzyme specificity. To test the degree to which linear position in a N receptor or EGF amino acid sequence determines specificity, we swapped corresponding EGFs between N1 and N2 and determined whether the propensity for elongation lies within a given EGF sequence. We now have evidence that EGF amino acid sequence is the primary driver of Fringe elongation potential. We are now testing whether in vitro specificity correlates with in vivo specificity for a given EGF in well‐established Fringe enzyme assays. We will take what we learn from this work, and test whether we can eliminate Fringe elongation at specific EGFs without eliminating the O‐fucose glycan. This will allow a highly refined version of current experiments testing the importance of Fringe elongation at individual EGFs, where glycosylation is completely eliminated by mutating the glycan modified amino acid.Support or Funding InformationThis work is supported by NIH grant GM061126.

CNS myelin protein 36K regulates oligodendrocyte differentiation through Notch.

In contrast to humans and other mammals, zebrafish can successfully regenerate and remyelinate central nervous system (CNS) axons following injury. In addition to common myelin proteins found in mammalian myelin, 36K protein is a major component of teleost fish CNS myelin. Although 36K is one of the most abundant proteins in zebrafish brain, its function remains unknown. Here we investigate the function of 36K using translation-blocking Morpholinos. Morphant larvae showed fewer dorsally migrated oligodendrocyte precursor cells as well as upregulation of Notch ligand. A gamma secretase inhibitor, which prevents activation of Notch, could rescue oligodendrocyte precursor cell numbers in 36K morphants, suggesting that 36K regulates initial myelination through inhibition of Notch signaling. Since 36K like other short chain dehydrogenases might act on lipids, we performed thin layer chromatography and mass spectrometry of lipids and found changes in lipid composition in 36K morphant larvae. Altogether, we suggest that during early development 36K regulates membrane lipid composition, thereby altering the amount of transmembrane Notch ligands and the efficiency of intramembrane gamma secretase processing of Notch and thereby influencing oligodendrocyte precursor cell differentiation and further myelination. Further studies on the role of 36K short chain dehydrogenase in oligodendrocyte precursor cell differentiation during remyelination might open up new strategies for remyelination therapies in human patients.

Notch receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The canonical Notch signalling pathway has four type I transmembrane Notch receptors (Notch1-4) and five ligands (DLL1, 2 and 3, and Jagged 1-2). Each member of this highly conserved receptor family plays a unique role in cell-fate determination during embryogenesis, differentiation, tissue patterning, proliferation and cell death [2]. As the Notch ligands are also membrane bound, cells have to be in close proximity for receptor-ligand interactions to occur. Cleavage of the intracellular domain (ICD) of activated Notch receptors by γ-secretase is required for downstream signalling and Notch-induced transcriptional modulation [15, 3, 11, 22]. This is why γ-secretase inhibitors can be used to downregulate Notch signalling and explains their anti-cancer action. One such small molecule is RO4929097 [8], although development of this compound has been terminated following an unsuccessful Phase II single agent clinical trial in metastatic colorectal cancer [19].Aberrant Notch signalling is implicated in a number of human cancers [12, 20, 6, 16], with demcizumab and tarextumab identified as antibody inhibitors of ligand:receptor binding [13].

Abstract 2591: miR-193b is a novel regulator of Inhibition of Notch signaling by for targeting cancer stem cell and tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive human cancers with extremely poor survival rates with 6 months of median patient survival, representing an unmet therapeutic challenge. The exact mechanisms by which PDAC progresses and still remain poorly understood. Strategies targeting only tumor cells have proven to be unsuccessful in eliminating PDAC and prevent metastasis, drug resistance and patient deaths. Accumulating evidence suggests that cancer stem cells (CSC) are also responsible for relapses and resistance to therapies as well as promoting invasive and metastatic growth of the tumor. Notch signaling has been implicated in the survival of CSC, suggesting that NOTCH pathway can be targeted for treatment of PDAC. In four known transmembrane Notch receptors, antibodies targeting Notch 2/3 have shown efficacy in solid tumors and PDAC in phase I clinical trials, suggesting that these are viable targets. Targeting Notch 2 expression has been shown to induce apoptosis in PDAC but its role and regulation of in PDAC CSC is not well understood. We found that Notch2 significantly upregulated in pancreatic cancer stem cells and its expression is associated with shorter overall patient survival in pancreatic cancer. Recent discoveries revealed that small non-coding RNAs such as microRNAs (miRNAs) play significant role in the pathogenesis of human cancers. Therefore, microRNAs have attracted great interest as a tool for cancer treatment and entered clinical trials in the US. Using in silico prediction algorithms we found that miR-193b-3p has binding sites on the 3’-untranslated region (3’-UTR) of NOTCH2 gene and demonstrated that transfection of PDAC and CD133+/CD44+/EpCam+ pancreatic CSC cells with miR193b-3p mimic or NOTCH 2 siRNA suppressed NOTCH2 expression by Western blot and qPCR analysis and proliferation, migration and metastasis of PDAC (Panc-1 and MiaPaCa-2) and the tumorosphere forming ability of CSC cells in Mammocult Human Tumorosphere Culture. CD133+/CD44+/EpCam+ pancreatic cancer stem cells transfected with miR-193b inhibited Notch 2 and colony forming capabilities in soft agar. Currently we are performing a gene reporter assay to show miR193b directly binds to 3’-UTR of NOTCH2 mRNA and inhibits its expression. We demonstrated that injection of nanoliposome incorporated miR193b-3p into mice from tail vein (0.15 mg/kg once a week) significantly inhibited PDAC tumor growth. However, animal studies transplanted with CD133+/CD44+/EpCam+ pancreatic cancer stem cells after miR193b-3p transfection are underway to detect in vivo effects of targeting miR193 in CSC and tumor growth. Overall, our findings provide new insights into the tumor suppressor role of miR-193b by targeting of Notch 2 signaling and suggest that miR-193b-based therapy may be a potential therapeutic strategy to targeted pancreatic cancer stem cells. Citation Format: Ummu Guven, Hamada Ahmed Mokhlis, Nermin Kahraman, Cristina Ivan, Fahriye Duzagac, Gabriel Lopez-Berestein, Gulperi Oktem, Bulent Ozpolat. miR-193b is a novel regulator of Inhibition of Notch signaling by for targeting cancer stem cell and tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2591.

Notch-mediated crosstalk of antitumor CD8+T and NK cells

Abstract A large majority of immune-oncology research efforts focus on T-cell immunotherapy. However, limited clinical efficacy has been achieved with adoptive T-cell effectors alone. In addition, research has shown that the sole use of adoptive T-cells leads to the development of tumor escape variants and “occult cancer”. Conversely, tumor-infiltrating natural killer (NK) cells have been shown to cause elimination of these escape variants. These observations indicate that the interaction between the innate and adaptive immune effectors is indispensable for effective tumor immunity. Notch is a major signaling system important for communication between neighboring cells. Our previous studies show that it plays a crucial role in the differentiation and antitumor function of CD8+T cells. However, its role in NK cell activation and antitumor function is not clear. Thus, we analyzed Notch receptor-ligand system on NK cells and in the context of their crosstalk with CD8+ T cells. Our data indicate that inhibiting Notch signaling in NK cells decreases NK cell activation. In addition, Notch intracellular domain (NICD) expression is increased upon NK activation, suggesting that the Notch signaling pathway may be involved in NK activation. Our data also show that activated CD8+T cells activate naïve NK cells through direct physical contact. Transmembrane notch receptor expression is decreased during this interaction suggesting that the Notch receptors may be involved in CD8+T cell – NK cell crosstalk. Experiments are underway to further understand the relevance of Notch signaling pathway in innate-adaptive immune crosstalk. The findings will help develop novel strategies for adoptive cell immunotherapies, specifically against solid tumors.

Extracellular vesicle-carried Jagged-1 inhibits HUVEC sprouting in a 3D microenvironment.

NOTCH signalling is an evolutionarily conserved juxtacrine signalling pathway that is essential in development. Jagged1 (JAG1) and Delta-like ligand 4 (DLL4) are transmembrane NOTCH ligands that regulate angiogenesis by controlling endothelial cell (EC) differentiation, vascular development and maturation. In addition, DLL4 could bypass its canonical cell-cell contact-dependent signalling to influence NOTCH signalling and angiogenesis at a distance when it is packaged into extracellular vesicles (EVs). However, it is not clear whether JAG1 could also be packaged into EVs to influence NOTCH signalling and angiogenesis. In this work, we demonstrate that JAG1 is also packaged into EVs. We present evidence that JAG1-EVs inhibit NOTCH signalling and regulate EC behaviour and function. JAG1-EVs inhibited VEGF-induced HUVEC proliferation and migration in 2D culture condition and suppressed sprouting in a 3D microfluidic microenvironment. JAG1-EV treatment of HUVECs leads to a reduction of Notch1 intracellular domain(N1-ICD), and the proteasome and the intracellular domain of JAG1 (JAG1-ICD) are both required for this reduction to occur. These findings reveal a novel mechanism of JAG1 function in NOTCH signalling and ECs through EVs.

A novel reporter allele for monitoring Dll4 expression within the embryonic and adult mouse.

ABSTRACTCanonical Notch signaling requires the presence of a membrane bound ligand and a corresponding transmembrane Notch receptor. Receptor engagement induces multiple proteolytic cleavage events culminating in the nuclear accumulation of the Notch intracellular domain and its binding to a transcriptional co-factor to mediate gene expression. Notch signaling networks are essential regulators of vascular patterning and angiogenesis, as well as myriad other biological processes. Delta-like 4 (Dll4) encodes the earliest Notch ligand detected in arterial cells, and is enriched in sprouting endothelial tip cells. Dll4 expression has often been inferred by proxy using a lacZ knockin reporter allele. This is problematic, as a single copy of Dll4 is haploinsufficient. Additionally, Notch activity regulates Dll4 transcription, making it unclear whether these reporter lines accurately reflect Dll4 expression. Accordingly, precisely defining Dll4 expression is essential for determining its role in development and disease. To address these limitations, we generated a novel BAC transgenic allele with a nuclear-localized β-galactosidase reporter (Dll4-BAC-nlacZ). Through a comparative analysis, we show the BAC line overcomes previous issues of haploinsufficiency, it recapitulates Dll4 expression in vivo, and allows superior visualization and imaging. As such, this novel Dll4 reporter is an important addition to the growing Notch toolkit.

Recent Insights into Notch Signaling in Embryonal Rhabdomyosarcoma.

The Notch signaling pathway is an evolutionarily conserved developmental network critical for embryonic and postnatal regulation of tissue growth, homeostasis, and repair. Signaling is initiated when transmembrane Notch ligands bind to transmembrane Notch receptors on nearby cells. Sequential proteolytic steps generate an activated Notch fragment that translocates to the nucleus, where it drives activation of canonical Notch target genes. In skeletal muscle, Notch signaling governs myogenic cell fate and stem cell maintenance. In the human soft tissue sarcoma rhabdomyosarcoma, which bears markers of skeletal muscle commitment and so is thought to be related to the skeletal muscle lineage, Notch signaling is also found to be upregulated and dysregulated. This review provides an overview of Notch signaling during normal embryonic and postnatal myogenesis, information on the recently discovered aberrant Notch signaling occurring in muscular dystrophies, the upregulation and mechanism of Notch signaling in the embryonal variant of rhabdomyosarcoma and related soft tissue sarcomas, and Notch cross-talk with other metazoan developmental pathways including Hippo, Hedgehog, Wnt, and TGF-β. The review concludes with updates on current promising efforts to target and inhibit Notch signaling in human sarcomas including rhabdomyosarcoma.

An interaction that guides cell fate

Structural Biology Notch signaling is important in cell fate determination in mammals. Signaling is initiated when the extracellular domain of the transmembrane Notch protein on one cell binds to a surface ligand on another cell. Luca et al. report the crystal structure of the interacting regions of

Notch signaling and new therapeutic options in liver disease

Notch signaling is a crucial determinant of cell fate decision during development and disease in several organs. Notch effects are strictly dependent on the cellular context in which it is activated. In the liver, Notch signaling is involved in biliary tree development and tubulogenesis. Recent advances have shed light on Notch as a critical player in liver regeneration and repair, as well as in liver metabolism and inflammation and cancer. Notch signaling is finely regulated at several levels. The complexity of the pathway provides several possible targets for development of therapeutic agents able to inhibit Notch. Recent reports have shown that persistent activation of Notch signaling is associated with liver malignancies, particularly hepatocellular with stem cell features and cholangiocarcinoma. These novel findings suggest that interfering with the aberrant activation of the Notch pathway may have therapeutic relevance. However, further studies are needed to clarify the mechanisms regulating physiologic and pathologic Notch activation in the adult liver, to better understand the mechanistic role(s) of Notch in liver diseases and to develop safe and specific therapeutic agents.

The Ubiquitin-specific Protease 12 (USP12) Is a Negative Regulator of Notch Signaling Acting on Notch Receptor Trafficking toward Degradation

Notch signaling is critical for development and adult tissue physiology, controlling cell fate in a context-dependent manner. Upon ligand binding, the transmembrane Notch receptor undergoes two ordered proteolytic cleavages releasing Notch intracellular domain, which regulates the transcription of Notch target genes. The strength of Notch signaling is of crucial importance and depends notably on the quantity of Notch receptor at the cell surface. Using an shRNA library screen monitoring Notch trafficking and degradation in the absence of ligand, we identified mammalian USP12 and its Drosophila melanogaster homolog as novel negative regulators of Notch signaling. USP12 silencing specifically interrupts Notch trafficking to the lysosomes and, as a consequence, leads to an increased amount of receptor at the cell surface and to a higher Notch activity. At the biochemical level, USP12 with its activator UAF1 deubiquitinate the nonactivated form of Notch in cell culture and in vitro. These results characterize a new level of conserved regulation of Notch signaling by the ubiquitin system.

ALK1 Signaling Inhibits Angiogenesis by Cooperating with the Notch Pathway

Activin receptor-like kinase 1 (ALK1) is an endothelial-specific member of the TGF-β/BMP receptor family that is inactivated in patients with hereditary hemorrhagic telangiectasia (HHT). How ALK1 signaling regulates angiogenesis remains incompletely understood. Here we show that ALK1 inhibits angiogenesis by cooperating with the Notch pathway. Blocking Alk1 signaling during postnatal development in mice leads to retinal hypervascularization and the appearance of arteriovenous malformations (AVMs). Combined blockade of Alk1 and Notch signaling further exacerbates hypervascularization, whereas activation of Alk1 by its high-affinity ligand BMP9 rescues hypersprouting induced by Notch inhibition. Mechanistically, ALK1-dependent SMAD signaling synergizes with activated Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2, thereby repressing VEGF signaling, tip cell formation, and endothelial sprouting. Taken together, these results uncover a direct link between ALK1 and Notch signaling during vascular morphogenesis that may be relevant to the pathogenesis of HHT vascular lesions.

The intracellular domain of Jagged-1 interacts with Notch1 intracellular domain and promotes its degradation through Fbw7 E3 ligase

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. Jagged-1 also undergoes proteolytic cleavage by gamma-secretase and releases an intracellular fragment. In this study, we have demonstrated that the Jagged-1 intracellular domain (JICD) inhibits Notch1 signaling via a reduction in the protein stability of the Notch1 intracellular domain (Notch1-IC). The formation of the Notch1-IC-RBP-Jk-Mastermind complex is prevented in the presence of JICD, via a physical interaction. Furthermore, JICD accelerates the protein degradation of Notch1-IC via Fbw7-dependent proteasomal pathway. These results indicate that JICD functions as a negative regulator in Notch1 signaling via the promotion of Notch1-IC degradation.

Regulation of Notch1 signaling by Delta-like ligand 1 intracellular domain through physical interaction.

Notch signaling involves the proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands. The Delta-like ligand 1 also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. In this study, we have demonstrated that the Delta-like 1 intracellular domain (Dll1-IC) specifically binds to Notch1-IC in the nucleus, thereby disrupting the association of the Notch1-IC-RBP-Jk-MAM transcription activator complex. Additionally, the Notch1-mediated blockage of the induction of MyoD is abolished by the co-expression of Dll1-IC. Collectively, our results show that Dll1-IC functions as a negative regulator in Notch signaling via the disruption of the Notch1-IC-RBP-Jk complex.

Polarized targeting of DNER into dendritic plasma membrane in hippocampal neurons depends on endocytosis

The targeting of membrane proteins into axons and dendrites is of critical importance for directional signal transmission within specific neural circuits. Many dendritic proteins have been shown to reach the somatodendritic membrane based on selective sorting and transport of carrier vesicles. Using rat hippocampal neurons in culture, we investigated the trafficking pathways of Delta/Notch-like EGF-related receptor (DNER), a transmembrane Notch ligand which is specifically expressed in CNS dendrites. Mutations in the cytoplasmic domain of DNER that abolished somatodendritic localization also increased its surface expression. Furthermore, inhibition of endocytosis resulted in disruption of the somatodendritic localization of DNER, indicating that the somatodendritic targeting of DNER is dependent on endocytosis. The DNER cytoplasmic domain binds to a clathrin adaptor protein complex-2 via a proximal tyrosine motif and a 40 amino acid stretch in the mid-domain, but not by the C-terminal tail. Molecular and pharmacological inhibition revealed that the surface expression of DNER is regulated by clathrin-dependent and -independent endocytosis. In contrast, the somatodendritic targeting of DNER is predominantly regulated by clathrin- and adaptor protein complex-2-independent endocytosis via the C-terminal tail of DNER. Our data suggest that clathrin-independent endocytosis is critical for the polarized targeting of somatodendritic proteins.

Notch signaling is not essential in sonic hedgehog-activated medulloblastoma

Dysregulated signal transduction through the notch pathway has been noted in human and mouse medulloblastoma studies. Gamma secretase inhibitors (GSIs) impair notch signaling by preventing the cleavage of transmembrane notch proteins into their active intracellular domain fragments. Previous studies have shown that GSI treatment caused apoptosis and impaired medulloblastoma cell engraftment in xenograft systems. In this study, we used in vivo genetic and pharmacologic approaches to quantify the contribution of notch signaling to sonic hedgehog (shh)-activated mouse medulloblastoma models. In contrast to prior in vitro studies, pharmacologic inhibition of notch pathways did not reduce the efficiency of medulloblastoma xenotransplantation nor did systemic therapy impact tumor size, proliferation, or apoptosis in genetically engineered mouse medulloblastoma models. The incidence and pathology of medulloblastomas driven by the SmoA1 transgene was unchanged by the bi-allelic absence of Notch1, Notch2, or Hes5 genes. These data show that notch signaling is not essential for the initiation, engraftment, or maintenance of sonic hedgehog pathway-driven medulloblastomas.

ADAM proteases: ligand processing and modulation of the Notch pathway.

ADAM metalloproteases play important roles in development and disease. One of the key functions of ADAMs is the proteolytic processing of Notch receptors and their ligands. ADAM-mediated cleavage of Notch represents the first step in regulated intramembrane proteolysis of the receptor, leading to activation of the Notch pathway. Recent reports indicate that the transmembrane Notch ligands also undergo ADAM-mediated processing in cultured cells and in vivo. The proteolytic processing of Notch ligands modulates the strength and duration of Notch signals, leads to generation of soluble intracellular domains of the ligands, and may support a bi-directional signaling between cells.

Notch Signaling Is Required for Exocrine Regeneration After Acute Pancreatitis

The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice. Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays. Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain. Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells.

Size, Mates, and Fates

![Figure][1] CREDIT: C. BICKEL/ SCIENCE The Science Perspectives and associated Connections Maps in the database of Cell Signaling at Science 's Signal Transduction Knowledge Environment ([www.sciencemag.org/sciext/cellsignaling06/][2]) highlight pathways initiated by three different types of receptors: brassinosteroid receptors that control plant size, G protein-coupled receptors that control mating responses in yeast, and Notch receptors that control cell fate in animals. Brassinosteroids are plant hormones that contribute to cell growth and division, differentiation, and reproductive development. As Belkhadir and Chory (p. [1410][3]) describe, the brassinosteroid receptor BR1 is a plasma membrane-localized leucine-rich-repeat receptor kinase that initiates a kinase cascade, ultimately controlling gene expression. Experiments in Arabidopsis thaliana have revealed that the active receptor complex is a serine-threonine kinase that initiates a process that inactivates BIN2, the plant homolog of glycogen synthase kinase 3 (GSK-3). Arabidopsis BIN2 is localized to the nucleus, whereas in animals, GSK-3 is cytosolic. BIM, which resembles the animal transcription factor Myc, interacts with the brassinosteroid response factor BES to enhance its activity. Thus, at each step of the brassinosteroid pathway—from the steroid hormone-like ligands to the transcription factors in the nucleus—there are similarities with animal pathways; however, no known pathway in animals assembles this cast of characters in quite the same way. Even well-characterized pathways continue to reveal new secrets. The yeast mating response is an extensively studied G protein-coupled receptor pathway, in which the G βγ subunits have been in the spotlight as the subunits that activate the mitogen-activated protein kinase cascade. Now, Slessareva and Dohlman (p. [1412][4]) describe how the Gα subunit participates in transmitting the mating signal by interacting with the phosphoinositide 3-kinase (PI3K) at the endosome to stimulate the production of phosphoinositide 3-phosphate. Not only was this a previously unknown function and location for Gγ in the yeast mating response, but the regulatory subunit of PI3K appears to serve as a noncanonical Gβ, subunit for the endosomally located Gα, a discovery that will undoubtedly stimulate a search for similar types of interactions in other systems. The Notch signaling pathway is crucial to animal development, and aberrant activity of this pathway is associated with certain types of leukemia. Ehebauer et al . (p. [1414][5]) explain how the transmembrane Notch receptor interacts with the transmembrane ligand on adjacent cells, which leads to cleavage and release of the Notch intracellular domain (NICD) that translocates to the nucleus and regulates gene expression. The cleavage of Notch remains an open area of research, with various candidates for the protease that performs the first cleavage in the extracellular domain and questions surrounding whether the second cleavage event occurs at the plasma membrane or after internalization. Notch acts in concert with other morphogenic signals to control cell fate; thus, understanding Notch signaling within the more complex signaling network remains a critical avenue of investigation. This knowledge may not only yield insight into animal development but also open the doors to new therapeutic opportunities in cases where the loss of differentiation contributes to disease. [1]: pending:yes [2]: http://www.sciencemag.org/sciext/cellsignaling06/ [3]: /lookup/doi/10.1126/science.1134040 [4]: /lookup/doi/10.1126/science.1134041 [5]: /lookup/doi/10.1126/science.1134042