The ins and outs of notch ligands and downstream events

Signaling through the Notch receptor constitutes an evolutionarily conserved pathway important for embryonic development of many organs, including the central nervous system and vasculature, in multicellular animals. The Notch receptor is a transmembrane protein that, upon binding of ligands presented on neighboring cells, undergoes proteolytic processing events leading to the release of the Notch intracellular domain (ICD). The Notch ICD translocates to the nucleus, where it interacts with a DNA-binding protein of the CSL family. Through the interaction with the Notch ICD, the CSL protein is converted from a transcriptional repressor to an activator of downstream target genes, most notably the Hes and Hey genes that encode basic helix-loop-helix proteins inhibiting expression of various proneural genes. Thus, a prevalent outcome of signaling through the Notch receptor is keeping cells in an undifferentiated state. In this thesis, I have studied three different aspects of Notch signaling. i) processing of ligands and receptor activation, ii) processing of receptors and iii) characterization of novel Notch downstream genes. Paper I is focused on the signaling cell. More specificaIly, the intracellular trafficking of the Notch ligand Jagged1 and its importance for binding to and activation of the Notch receptor was examined. The results show that cell surface expression of Jagged1 is sufficient for interaction with receptor, but internalization of ligand into the signaling cell is required for Notch receptor activation in the recieving cell. Secondly, we have studied the assembly and regulation of the gamma-secretase complex, an enzyme complex required for the last proteolytic cleavage event, occuring in the lipid bilayer, during Notch receptor activation. Four proteins, Presenilin, nicastrin, Aph-1, and Pen-2, have been shown to be required for gammasecretase activity, but the role of individual proteins was poorly understood when we started our studies. In an attempt to identify the proteins present in the gamma-secretase complex that cleaves Notch we show in paper II that presenilin, nicastrin, Aph-1, and Pen-2 are present as a complex on the cell surface, where Notch receptor-ligand interaction takes place. Furthermore, the presenilin-nicastrin-Aph-1 complex (presumably also containing Pen-2) interacts with Notch on the cell surface, indicating that Aph-1 is a part of the mature gamma-secretase complex. In paper III we have studied the membrane topology of Presenilin. We find that Presenilin has nine transmembrane domains, with the C terminus located in the lumen/ extracellular space. Importantly, we could show that Presenilin protein molecules with a lumenal orientation of the C terminus interact with the other components of the complex, and that such Presenilin molecules are found at the cell surface. This strongly implies that this membrane topology is compatible with gamma-secretase function. In paper IV, we present data showing that presenilin regulates the subcellular localization of Pen-2: in the absence of Presenilin, Pen-2 is sequestered in the endoplasmic reticulum (ER) and not transported to post-ER compartments, where the mature gamma-secretase complexes reside. Presenilin deficiency also leads to destabilization of Pen-2, which is alleviated by proteasome inhibitors. In keeping with this, we show that Pen-2 is ubiquitylated prior to degradation and presumably retrotranslocated from the ER to the cytoplasm. Collectively, our data suggest that failure to become incorporated into the gamma-secretase complex leads to degradation of Pen-2 through the ER-associated degradation (ERAD) -proteasome pathway. Finally, in paper V we have studied downstream effects of Notch receptor activation to try to identify novel Notch target genes. We have compared transcriptional profiles in cells before and after activation of Noth signaling, and have identified several genes that appear to be activated directly by the Notch ICD/CSL complex. We present data from a more detailed characterization of one of these genes; the gene encoding platelet-derived growth factor receptor beta (PDGFR-beta. This may shed new fight on the role of Notch signaling in vascular biology