Annual Meeting of the Society for Neuroscience edition:38 location:Washington DC, USA date:15-19 November 2008
The leucine rich repeat kinase 2 gene (LRRK2) codes a large protein (2527 amino acids) encompassing several predicted functional domains: an armadillo/ankyrin repeat domain, a leucine rich repeat domain (LRR), a domain homologous to small GTPases of the Ras family termed Roc (for Ras of complex proteins), a COR domain (for C-terminal of Roc) of unknown function, a kinase domain as well as a WD40 domain. Autosomal dominant pathogenic mutations which segregate with Parkinson’s disease are found in all domains. The WD40 also harbors a prevalent clinical mutation, the G2385R, which is a PD risk factor in asian populations. Although cellular studies using full length LRRK2 confirm a role for mutant LRRK2 in neurodegeneration, the precise role of the WD40 domain in these mechanisms is not yet fully understood. Via homology modeling we confirm a 7 bladed beta-propeller fold for the WD40 domain, and it has been proposed that the WD40 domain may mediate protein-protein interactions. The LRRK2 WD40 domain has also previously been shown to interact with LRRK2 Roc domain in co-immunoprecipitation experiments. In the present study, we examine in further detail the properties of the WD40 domain and its relevance to the functioning of LRRK2. For this we used lentiviral vectors to overexpress in HEK293T and SHSY-5Y cells various LRRK2 fragments containing the WD40 domain, ranging from WD40 domain alone to full length. We also purified recombinant LRRK2 WD40 protein from induced E. coli cell extracts in a multi-step purification protocol. Using these tools, we are currently examining the biochemical properties of the WD40 domain in more detail, including the interaction of WD40 to LRRK2 Roc domain. We are also evaluating the properties of mutant LRRK2 WD40 as well as the cell biological consequences of overexpression of LRRK2 fragments containing WD40 domain in neuroblastoma cells.