The most common known cause of inherited parkinsonism is the gene Leucine rich repeat kinase 2 (LRRK2). Depending on the population, LRRK2 accounts for between 1 and 30% of all cases of Parkinson disease, including some apparently sporadic cases. The LRRK2 protein is large and complex, containing kinase and GTPase domains linked by an intervening sequence called the COR region. Most of the confirmed pathogenic mutations are in this tridomain region, suggesting that enzyme regulation and activity are likely important for understanding how LRRK2 mutations cause disease. Furthermore, given the frequency of mutations and the presence of two domains that could be targeted by small molecules, LRRK2 is an attractive target for novel therapeutics for Parkinson disease.? In the past year, we have concentrated on understanding some of the basic biochemistry of LRRK2. Specifically, we have identified that the large protein forms dimers in a variety of situations and have begun to map the critical regions responsible for dimer formation. We have shown that both transfected and endogenous LRRK2 are present in native protein complexes (identified using non-denaturing techniques) slightly larger than the predicted size of a LRRK2 homodimer. Using complementary techniques, including yeast two hybrid immunoprecipitation and in vitro pulldowns, we confirmed that the central portion of LRRK2 mediates much of the dimer formation. Furthermore, we showed that using full length LRRK2 protein, phosphorylation of LRRK2 by itself (autophosphorylation) occurs within each dimer. This has implications for the design of experiments to test the idea that LRRK2 might regulate itself and we are currently attempting to map the specific regions of the protein that act as phosphorylation acceptors.? We have also begun to approach this problem using structural tools. Our collaborators have solved the structure of the GTPase or ROC (for Ras of Complex proteins) domain. We have shown that this is an active enzyme using protein prepared from mammalian cells or as a single domain produced as a recombinant protein. Importantly, we found that a mutation within the ROC domain (R1441C) will weaken the formation of a dimer within LRRK2, thus potentially changing regulation of the protein. We suggest that this altered intramolecular regulation results in an overstimulated kinase activity and, eventually, to toxic effects in neurons. We plan to further explore these phenomena by developing a more detailed map of the various interactions of LRRK2 and identifying how this impacts protein function.
