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

Abstract

jMutations 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. Parkinson's disease (PD) is the second most common age-related neurodegenerative disease and is clinically characterized by movement impairments, bradykinesia, rigidity, and resting tremor and pathologically by the progressive loss of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies (1, 2). Although the majority of cases are sporadic, mutations in the leucine-rich repeat kinase 2 (LRRK2) gene (PARK8; Online Mendelian Inheritance in Man [OMIM] accession number 609007) had been unequivocally linked to late-onset autosomal dominant PD. LRRK2 mutations account for up to 13% of familial PD cases compatible with dominant inheritance and are also found in 1 to 2% of sporadic PD patients (62-64). LRRK2 is a complex 286-kDa protein that consists of multiple domains, including (in order, from the amino to carboxyl terminus) armadillo, ankyrin, and the namesake leucine-rich repeats (LRRs), followed by an ROC (Ras of complex proteins) GTPase domain, a COR (C-terminal of ROC) dimerization domain, a kinase domain, and a predicted C-terminal WD40 repeat domain (4-6). Several single-nucleotide alterations have been identified in LRRK2, but only five missense mutations within the ROC, COR, and kinase domains clearly segregate with PD in large family studies (7,8). It has recently been shown that the WD40 domain is required to stabilize the LRRK2 dimer and to execute LRRK2-associated kinase activity as well as neurotoxicity (9, 10), but the role of this domain within LRRK2 physiological and pathological function has not yet been completely defined. The beta-propellerforming WD40 domains are among the 10 most abundant domain types across eukaryotic proteomes (11) and constitute platforms where multiprotein complexes assemble reversibly (12). Here, we systematically analyzed the protein-protein interactions conferred by the LRRK2 WD40 domain. The nature of the LRRK2 WD40 interactors and the finding that the LRRK2 WD40 domain is able to bind to synaptic...

LRRK2 functions in synaptic vesicle endocytosis through a kinase-dependent mechanism

“…Substrates are involved in different processes, such as neuronal survival, cytoskeletal rearrangement, autophagy and apoptosis (Berwick and Harvey, 2011;Gillardon, 2009;Plowey and Chu, 2011;Martin et al, 2014). LRRK2 has also been shown to regulate neurotransmission, by interacting with synaptic proteins that modulate clathrin-mediated endocytosis of synaptic vesicles, such as Rab5b, snapin and N-ethylmaleimide-sensitive factor (NSF) (Piccoli et al, 2011;Shin et al, 2008;Yun et al, 2013;Piccoli et al, 2014). Moreover, we revealed an essential role for LRRK in synaptic vesicle endocytosis at Drosophila melanogaster neuromuscular junctions through phosphorylation of endophilin A (EndoA) (Matta et al, 2012).…”

Synaptic function is modulated by LRRK2 and glutamate release is increased in cortical neurons of G2019S LRRK2 knock-in mice

“…LRRK2 binds several synaptic vesicle cycle proteins, including adaptor proteins 1 and 2, alpha-actinin 2, the clathrin coat assembly protein AP180, synapsin 1, VAMP2, SNAP25, dynamin 1 and synaptophysin (Piccoli et al, 2011, 2014). In cultured cortical neurons, acute RNAi knock-down of LRRK2 increases glutamatergic release probability (Pr), vesicle motility and recycling (Piccoli et al, 2011); conversely decreased glutamate release is reported in LRRK2 KO mouse pups at postnatal day 15 (Parisiadou et al, 2014).…”

Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation

“…Although the function of the Lrrk2 WD40 domain has been well studied, little is known on the function of a putative Lrrk1 WD40. It has been reported that the WD40 domain of Lrrk2 can bind and sequester synaptic vesicles via interaction with vesicle-associated proteins, and a G2385R point mutation in the WD40 domain correlated with a reduced binding affinity of Lrrk2 WD40 to synaptic vesicles (25). Molecular modeling also suggests that the G2385 residue is located on the outer surface of the WD40 domain toward the COOH terminus, and the substitution of the neutral and flexible glycine for a passively changed arginine at this position could interfere with interdomain interaction, causing 50% reduction in Lrrk2 kinase activity.…”