Notch receptor activation and function

Lz Notch signaling completely changes the way that cells respond to other signaling pathways Flores et al., Cell 103:75-85 (2000) R7 Photoreceptor D-Pax2 Cone cell

Question: What are Notch receptors normally used for? Answer: Lateral Specification – Required to generate two distinct cell types in the correct ratio and configuration – e.g. spacing of neurons vs epidermal cells. Inductive Signaling – Induces differentiation of a new cell fate in response to Notch activation – e.g. induction of wing margin tissue in a fruit fly. Cellular Migration – e.g. Axonal pathfinding in flies Cell Division – e.g. Mitosis in the C. elegans male gonad. Cell Adhesion – e.g. Adhesion between the Oocyte and Follicle cells in Drosophila. Mesenchymal/Epithelial Transitions – e.g. maintenance of epithelial tissues in Drosophila Cell Survival/Death – e.g. Regulates cell death in the developing fly eye

Question: Maybe Notch receptors control everything? Answer: Probably involved in most biological processes in complex animals. Development: We know from temperature sensitive mutants of Notch in both flies and worms that Notch receptors are required for development of every tissue (blood vessels, nervous system, white blood cells, epithelial tissues, etc.). Diseases: Cancer (Notch oncogenes and Anti- Oncogenes/Tumor Suppressor Genes: Dementia caused by mutations in Presenilin (PS) genes and in Notch3).

Notch: A large Transmembrane Receptor/Transcription factor that regulates development of every tissue in complex animals. First analyzed in Drosophila (~1930s). Extracellular domains: Mammalian Notch 1, 2, 3 and 4 contain 36, 36, 34 and 29 highly conserved EGF-like repeats respectively. EGF-like repeats are ~40 amino acids in length and contain 6 cysteine residues (forming 3 C-C bonds). Three LNR repeats (Lin-12, Notch Repeats) are also present in the Notch extracellular domain. LNR repeats are cysteine-rich domains found only in Notch family receptors (Note that Lin-12 is a C. elegans Notch receptor). EGF Repeats LNR Repeats

Notch: A large Transmembrane Receptor/Transcription factor. The Ram23 domain is ~50 amino acids in length. It binds to a CSL DNA-binding protein (CSL = CBF-1, Suppresser of Hairless, and Lag-1 which are the mammalian, fly and worm versions of this protein) – see below. Cdc10 repeats are ~33 amino acids in length and bind to a number of proteins involved in chromatin remodeling such as Mastermind, Lag-3/Sel-8, or EMB-5, and also to Deltex. Ram23 domainCdc10/ankyrin repeats NLSPEST Sequences

Notch: A large Transmembrane Receptor/Transcription factor. Each domain of Notch is important for at least one function: -Receptor Trafficking/Processing or Activation -Receptor Signaling -Receptor Regulation (+ or -)

Notch is made as a very large single-chain precursor that must be glycosylated and cleaved before it appears on the cell surface. Glycosylation of Notch starts in the ER and is finished in the Golgi. Cleavage to generate the mature heterodimeric form on the plasma membrane occurs in the Golgi. This cleavage reaction at site 1 (S1) is carried out by a Furin protease. Furin Protease S1 cleavage

Notch

Question: What is Notch? Answer: A Receptor, an Adhesion Molecule, and a Transcription factor. Question: How is Notch regulated? Answer: Just about every way that you can imagine: Synthesis Folding Glycosylation Processing Subcellular localization Ligand Activation Proteolytic cleavage at activation Endocytosis Translocation to the Nucleus Binding to partners DNA binding/Transcription Degradation

Notch Serrate Delta

Notch +OFUT1 (O-fucosyltransferase) Serrate +OFUT1 Delta +OFUT1 = O-linked Fucose

Notch +Fringe Serrate +Fringe Delta +Fringe = GlcNac - Galactose - Sialic Acid

Notch Serrate Delta Notch 1,2,3,4 Jagged 1,2 Dll 1, 4 Dll3, Dlk/Pref1 antagonists Fringe Lunatic Fringe Manic Fringe Radical Fringe The mammalian Notch receptors, ligands, and Fringes

TACE or Kuzbanian Disintegrin protease S2 cleavage Neuralized?

S3 cleavage by a membrane protease complex that includes PS proteins and Nicastrin (in endocytic compartment?) Endocytosis

Hes genes CSL/RBPJk

Hes genes CSL/RBPJk

Hes genes CSL/RBPJk

Hes genes CSL/RBPJk The Notch IC (intracellular domain) switches on genes that were repressed by CSL/RBPJk/Su(H) through conversion of a repressor complex into a transcriptional activation complex.

Prior to Notch activation, the CSL protein was sitting on DNA in a complex with Ski-interacting protein (SKIP) and a transcriptional co- repressor termed SMRT. The CSL-Skip-SMRT complex represses gene expression and therefore CSL was repressing expression of a set of genes prior to Notch activation. Once Notch IC arrives in the nucleus, it binds to the CSL-Skip complex, displacing SMRT and recruiting transcription activating proteins such as Mastermind, Lag- 3/Sel-8, and/or EMB-5. Consequently, Notch activation turns on the genes that were repressed by CSL. Many of these Notch target genes are themselves transcription factors that regulate differentiation (e.g. HES).

Notch signaling Notch 1 Receptor EGF-like repeats TAD Anky RAM LNR Pest LNR RAM TAD Pest Lin/Notch repeats RAM domain PEST domain Transactivation domain Anky Ankyrin repeats Nuclear localization signal DSL Delta/Serrate domain vWF Von Willebrand Factor cysteine-rich domain Jagged 1 Ligand Presenilin1 TAD Anky RAM Pest CSL/RBP-J Corepressor CSL/RBP-J CBP MAML CBP TAD Anky RAM Pest TBP TFIIE TFIID Numb vWF DSL AAAAA HES proteins mRNA  -secretase Nicastrin PEN2 APH1

Signal Integration

Lz Notch signalling completely changes the way that cells respond to other signaling pathways Flores et al., Cell 103:75-85 (2000) R7 Photoreceptor D-Pax2 Cone cell

Wingless Arrow/Frizzled Dishevelled Armadillo/  -catenin Notch Deltex ac-sc Delta Notch Su(H) E(spl) ac-sc Groucho

Notch controls developmental decisions together with: -Wnts -TGF  /BMPs -HH -FGFs -EGFs -VEGFs -Integrins -Hypoxia signaling -etc

Notch receptors and ligands (Human) Notch aa EGF-like repeats LNR RAM TAD Pest Notch aa Notch aa Notch aa Lin/Notch repeats RAM domain PEST domain Transactivation domain Anky Ankyrin repeats Nuclear localization signal TAD Anky LNR RAM LNR Pest TAD LNR RAM LNR Pest Anky LNR RAM Pest Anky LNR RAM LNR Pest Anky vWF DSL Delta/Serrate domain vWF Von Willebrand Factor cysteine-rich domain Jagged aa vWF DSL Jagged aa Delta-like aa DSL Delta-like aa Delta-like aa DSL