The outcome of the Notch pathway on proliferation depends on cellular context, being growth promotion in some, including several cancers, and growth inhibition in others. Such disparate outcomes are evident in Drosophila wing discs, where Notch overactivation causes hyperplasia despite having localized inhibitory effects on proliferation. To understand the underlying mechanisms, we have used genomic strategies to identify the Notch-CSL target genes directly activated during wing disc hyperplasia. Among them were genes involved in both autonomous and non-autonomous regulation of proliferation, growth and cell death, providing molecular explanations for many characteristics of Notch induced wing disc hyperplasia previously reported. The Notch targets exhibit different response patterns, which are shaped by both positive and negative feed-forward regulation between the Notch targets themselves. We propose, therefore, that both the characteristics of the direct Notch targets and their cross-regulatory relationships are important in coordinating the pattern of hyperplasia.

5-Hydroxytryptamine (5-HT) 3 and ␥-aminobutyric acid, type C (GABA C ) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which also includes nicotinic acetylcholine, GABA A , and glycine receptors. The details of how agonist binding to these receptors results in channel opening is not fully understood but is known to involve charged residues at the extracellular/transmembrane interface. Here we have examined the roles of such residues in 5-HT 3 and GABA C receptors. Charge reversal experiments combined with data from activation by the partial agonist ␤-alanine show that in GABA C receptors there is a salt bridge between Glu-92 (in loop 2) and Arg-258 (in the pre-M1 region), which is involved in receptor gating. The equivalent residues in the 5-HT 3 receptor are important for receptor expression, but charge reversal experiments do not restore function, indicating that there is not a salt bridge here. There is, however, an interaction between Glu-215 (loop 9) and Arg-246 (pre-M1) in the 5-HT 3 receptor, although the coupling energy determined from mutant cycle analysis is lower than might be expected for a salt bridge. Overall the data show that charged residues at the extracellular/transmembrane domain interfaces in 5-HT 3 and GABA C receptors are important and that specific, but not equivalent, molecular interactions between them are involved in the gating process. Thus, we propose that the molecular details of interactions in the transduction pathway between the binding site and the pore can differ between different Cys-loop receptors.There have been many biochemical and functional studies on different members of the Cys-loop family of ligand-gated ion channels, but the prototypic member of this family, the neuromuscular nicotinic acetylcholine (nACh) 2 receptor, is still the best understood. This is the only receptor for which reasonably high resolution structural information (4 Å) is available from cryo-electron microscopy studies (1), and further knowledge has come from x-ray crystal structures of acetylcholine binding proteins (AChBP), which are homologous to the extracellular domain of this receptor (2, 3). Cys-loop receptors are composed of five pseudo-symmetrically arranged subunits surrounding a central ion-conducting pore. Each subunit is composed of an extracellular, a transmembrane, and an intracellular domain. The extracellular domain (ECD) contains the ligand binding site, which is formed at the interface of two adjacent subunits by the convergence of three amino acid loops (A-C) from one (the principal) subunit and three ␤-strands (D-F) from the adjacent (or complementary) subunit. The transmembrane domain (TMD) contains four membrane spanning ␣-helices (M1-M4) and a short C terminus. M2 from each subunit lines the pore and contains regions responsible for channel gating and ion selectivity. A large loop between M3-M4 forms the intracellular domain and is involved in channel conductance and modulation.Structural details extrapolated from AChBP have greatly enhanced o...

The ligand binding site of Cys-loop receptors is dominated by aromatic amino acids. In GABAC receptors, these are predominantly tyrosine residues, with a number of other aromatic residues located in or close to the binding pocket. Here we examine the roles of these residues using substitution with both natural and unnatural amino acids followed by functional characterization. Tyr198 (loop B) has previously been shown to form a cation−π interaction with GABA; the current data indicate that none of the other aromatic residues form such an interaction, although the data indicate that both Tyr102 and Phe138 may contribute to stabilization of the positively charged amine of GABA. Tyr247 (loop C) was very sensitive to substitution and, combined with data from a model of the receptor, suggest a π–π interaction with Tyr241 (loop C); here again functional data show aromaticity is important. In addition the hydroxyl group of Tyr241 is important, supporting the presence of a hydrogen bond with Arg104 suggested by the model. At position Tyr102 (loop D) size and aromaticity are important; this residue may play a role in receptor gating and/or ligand binding. The data also suggest that Tyr167, Tyr200, and Tyr208 have a structural role while Tyr106, Trp246, and Tyr251 are not critical. Comparison of the agonist binding site “aromatic box” across the superfamily of Cys-loop receptors reveals some interesting parallels and divergences.

Background Drosophila neuroblasts (NBs) are neural stem cells whose maintenance relies on Notch activity. NBs proliferate throughout larval stages to generate a large number of adult neurons. Their proliferation is protected under conditions of nutrition restriction but the mechanisms responsible are not fully understood. As amino acid transporters (Solute Carrier transporters, SLCs), such as SLC36, have important roles in coupling nutrition inputs to growth pathways, they may have a role in this process. For example, an SLC36 family transporter Pathetic (Path) that supports body size and neural dendrite growth in Drosophila , was identified as a putative Notch target in genome-wide studies. However, its role in sustaining stem cell proliferation and maintenance has not been investigated. This study aimed to investigate the function of Path in the larval NBs and to determine whether it is involved in protecting them from nutrient deprivation. Methods The expression and regulation of Path in the Drosophila larval brain was analysed using a GFP knock-in allele and reporter genes containing putative Notch regulated enhancers. Path function in NB proliferation and overall brain growth was investigated under different nutrition conditions by depleting it from specific cell types in the CNS, using mitotic recombination to generate mutant clones or by directed RNA-interference. Results Path is expressed in both NBs and glial cells in the Drosophila CNS. In NBs, path is directly targeted by Notch signalling via Su(H) binding at an intronic enhancer, PathNRE . This enhancer is responsive to Notch regulation both in cell lines and in vivo. Loss of path in neural stem cells delayed proliferation, consistent with it having a role in NB maintenance. Expression from pathNRE was compromised in conditions of amino acid deprivation although other Notch regulated enhancers are unaffected. However, NB-expressed Path was not required for brain sparing under amino acid deprivation. Instead, it appears that Path is important in glial cells to help protect brain growth under conditions of nutrient restriction. Conclusions We identify a novel Notch target gene path that is required in NBs for neural stem cell proliferation, while in glia it protects brain growth under nutrition restriction .