Leucine-Rich Repeat Kinase 2 Binds to Neuronal Vesicles through Protein Interactions Mediated by Its C-Terminal WD40 Domain

Abstract

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) are associated with familial and sporadic Parkinson's disease (PD). LRRK2 is a complex protein that consists of multiple domains, including predicted C-terminal WD40 repeats. In this study, we analyzed functional and molecular features conferred by the WD40 domain. Electron microscopic analysis of the purified LRRK2 C-terminal domain revealed doughnut-shaped particles, providing experimental evidence for its WD40 fold. We demonstrate that LRRK2 WD40 binds and sequesters synaptic vesicles via interaction with vesicle-associated proteins. In fact, a domain-based pulldown approach combined with mass spectrometric analysis identified LRRK2 as being part of a highly specific protein network involved in synaptic vesicle trafficking. In addition, we found that a C-terminal sequence variant associated with an increased risk of developing PD, G2385R, correlates with a reduced binding affinity of LRRK2 WD40 to synaptic vesicles. Our data demonstrate a critical role of the WD40 domain within LRRK2 function.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00914-13.

ACKNOWLEDGMENTS

We are grateful to Nathalie Théret (INSERM, Rennes, France) for reagents, to Sandra Helm for technical assistance, and to Peijian Zou and Arie Geerlof for initial bacterial expression clones of the WD40 domain. In addition we thank Pablo Porras and Henning Hermjakob (EMBL-EBI) for data integration into the IntAct database.

This work was supported by the National Genome Research Framework program NGFN-Plus (grant 01GS08140, subproject 12), the Helmholtz Alliance for Mental Health in an Aging Society (grant HA-215, topic 3 WP11), the European Community's Seventh Framework Program FP7/2009 under grant agreement number 241955, number 278568, PRIMES, and number 241481, AFFINOMICS (to M.U.), and the LRRK2 Biology LEAPS 2012 award of the Michael J. Fox Foundation to G.P., C.J.G., M.S., and M.U. G.P. and F.O. are supported by Fondazione Cariplo (grant 2011-0540), MJFF, and Fondazione Telethon (grant GGP12237). G.P. is supported also by the FIRB program (grant RBFR08F82X_002) and Fondazione Grigioni per il morbo di Parkinson. F.O. is grateful to PRIN 2010-11. M.M. is supported by Fondazione Cariplo (grant 2008-3184).

Author contributions are as follows. G.P., C.J.G., F.O. and M.U. designed the study. G.P., C.J.G., and M.U. wrote the paper. G.P., M.D.C., A.M., F.P., F.A., F.G., P.J., C.J.K., F.V.Z., and A.K. performed experiments. G.P., C.J.G., C.S., C.J.O.K., M.M., A.V., C.J.O.K., L.P., M.Z, S.W., M.S., and M.U. analyzed the data and/or provided data analysis expertise.