The LDL receptor family and signal transduction

Research project P 13931 The LDL receptor family and signal transduction Johannes NIMPF 11.10.1999 The LDL receptor family is a group of modular cell surface receptors mediating the uptake of many different ligands in many kinds of cells and organs. Their functions span from maintaining cholesterol homeostasis, mainly mediated by the LDL receptor, over removal of proteinases and their inhibitors from the circulation by LDL receptor related protein to the uptake of yolk precursors `in egg-laying species which is carried out by a protein homologous to the mammalian VLDL receptor. Recent studies on these receptors in our laboratory, however, laid ground to a whole new direction reaching far beyond simply taking part in receptor-mediated endocytosis. We identified two novel intracellular proteins interacting with members of the LDL receptor family. One (JIP-2) is a homologue to JNK-interacting protein- 1 (JIP-1) which acts as scaffolding protein for the JNK-specified signal transduction pathway, leading to the activation of the AP1 transcription complex. JIP-2 binds to a splice variant of LR7/8B, a member of the receptor family specifically expressed in the brain. The other protein which shows a homology to sorting nexin-1 interacts with the intracellular tail of LR7/8B and most likely of the other members of the family also. This protein may regulate the intracellular sorting of the receptors by directing them for lysosomal degradation. The major aims of the project are i) the characterization of novel signal transduction pathways mediated by LR7/8B and JIP-2 and ii) defining new mechanisms in regulating the surface expression and intracellular pathways of LDLR family members in general. To reach the first goal, we will primarily focus on the molecular and functional analysis of JIP-2. This will include to study its interaction with the receptor in close detail, to search for target genes of JIP-2, and to screen for additional interacting proteins. Targeted disruption of the JIP-2 gene in mice should relate the obtained in vitro data to the physiological context of the entire organism. The second aim will include the molecular characterization of KI, and subsequently to study its interaction not only with LR7/8B, but with all receptor family members sharing highly homologous intracellular regions. We are confident that results obtained within this project will not only deepen our existing understanding of receptor biology, but will highlight yet unknown aspects regarding their role in normal and pathophysiological conditions.

The LDL receptor gene family specifies a group of highly related composite membrane proteins engaged in receptor-mediated endocytosis of a variety of independent ligands. For many years these receptors have been studied in the context of receptor mediated endocytosis and the LDL-receptor now serves as the textbook model for this process. At the time this project was proposed however, it became evident that, at least two members of this family, namely VLDL Receptor and ApoER2 serve other functions as well. These receptors bind Reelin and are involved in signaling processes orchestrating neuronal migration. This exciting development in the functional characterization of ApoER2 and VLDL Receptor suggested a complicated molecular network at the crossroad of endocytosis and signaling. In order to sort out mechanisms which orchestrate these two independent functions of the receptors, namely endocytosis and signaling, we set out to screen for adapter proteins, which bind to the cytoplasmic domains of the receptors. Besides a list of known candidate proteins we focused on JNK-interacting protein (JIP) and SNX17, a member of the so-called sorting nexin family. JIP might linke the Reelin-pathway to the JNK pathway suggesting a complicated signaling network originating from ApoER2 and VLDL Receptor. SNX17, on the other hand, was shown to enhance the endocytosis rate of the receptors and seems to play an important role in endocytosis. In addition we have made great progress in understanding the molecular mechanism of the Reelin signaling pathway. We could demonstrate that a splice variant of ApoER2 can be cleaved and the resulting soluble receptor fragment is able to inhibit Reelin signaling. In addition we figured out that phosphorylation of Dab1, which is the first event of the signaling cascade, does not require any co-receptors but is mediated by receptor clustering at the surface of target neurons. In summary, the results obtained during this project significantly extended our knowledge of molecular events, which guides neuronal migration, and development of the brain.