Identification of novel ligands of WDR47, using yeast two-hybrid analysis
Thesis (MScMedSc (Biomedical Sciences. Molecular Biology and Human Genetics. Medical Biochemistry))--University of Stellenbosch, 2009.
The mammalian neocortex contributes to the increasing functional complexity of the mammalian brain, partly because of its striking organisation into distinct neuronal layers. The development of the neocortex has been well studied because disrupted neurodevelopment results in several human diseases. The basic principles of neocortical development have been well established for some time; however the molecular mechanisms have only recently been identified. One major advance in our understanding of these molecular mechanisms was the discovery of Reelin, an extracellular matrix protein that directs the migration of neurons to their final positions in the developing neocortex. Reelin is a large multi-domain protein that exerts its functions by binding to its ligands on the cell surface and initiating a signal transduction cascade that ultimately results in cytoskeletal rearrangements. Several investigations have been undertaken to elucidate the functions of each of these domains to gain a better understanding reelin’s functions. We have previously identified the WR40 repeat protein 47 (WDR47), a protein of unknown function, as a novel putative ligand for the N-terminal reeler domain of reelin. To gain better understanding into the functional significance of this interaction, the present study sought to identify novel WDR47- interacting proteins. In order to achieve this, a cDNA encoding a polypeptide that contains the two N-terminal domains of WDR47, i.e. the Lis homology and the C-terminal Lis homology domain (CTLH) was used as bait in a Y2H screen of a foetal brain cDNA library. Putative WDR47 ligands were subsequently verified using 3D in vivo co-localisation. Results of these analyses showed that SCG10, a microtubule destabilizing protein belonging to the stathmin family of proteins, interacted with the N-terminal of WDR47. The identification of SCG10 as a novel WDR47 interacting protein not only sheds some light on the role and function of WDR47 but also aids in a better understanding of the reelin pathway and cortical lamination. Moreover, the data presented here, may also provide researchers with new avenues of research into molecular mechanisms involved in neuronal migration disorders.