# Roco Proteins: GTPases with a Baroque Structure and Mechanism
## 4 Physiological Functions of Roco Proteins
### Roco Proteins from Vertebrates
Finally, recent research in LRRK1 knockout mice revealed a negative regulatory role of LRRK1 for bone mass, thus, providing a link between LRRK1 and osteoporosis [28, 59]. Since mutations in the gene coding for LRRK2 have been linked to PD, LRRK2 has become the most intensively investigated Roco protein [15]. LRRK2 localizes both in the cytosol and at specific membrane compartments, where it interacts with a wide variety of proteins. Over the last few years, the protein has been linked to numerous cellular and molecular pathways and functions, including autophagy, cytoskeletal regulation, mitochondrial function, protein translation and degradation, neurite outgrowth and vesicular trafficking. Moreover, accumulating evidence suggests that LRRK2 plays an important role in both the innate and adaptive immunity [64, 65]. Both endogenous and exogenously expressed LRRK2 is reported to localize to the mitochondrial outer membrane [66]. Patients with PD-mutated LRRK2 have a decreased mitochondrial membrane potential and reduced cellular ATP levels. Their mitochondria are elongated and have an increased interconnectivity, suggesting an involvement of LRRK2 in mitochondrial fusion and fission [67]. Research by [PERSON] et al. later proved that LRRK2 directly interacts with mitochondrial dynamin-like protein (DLP1) and regulates mitochondrial dynamics using a DLP-mediated pathway [68, 69]. LRRK2 was also found to localize to autophagic vacuoles (AVs) and multivesicular bodies (MVBs) [70]. Moreover, expression of PD-mutated LRRK2 in human-derived cell lines leads to an accumulation of AVs and MVBs [70]. This LRRK2-mediated autophagy is suggested to be regulated by the MAPK-ERK signaling pathway and by the calcium-dependent AMPK pathway via LRRK2 activation of NAADP receptors that are involved in the calcium efflux of endosomes [71, 72]. A role of LRRK2 in maintaining the neuronal development process has also been reported [73]. Transfection of cortical cultures with PD-mutated LRRK2 with increased kinase activity dramatically reduces neurite length and branching [73, 74, 75]. Inversely, cells transfected with kinase-deficient LRRK2, show extended structures [73]. LRRK2 regulates neuronal development by, most likely indirectly, stimulating the phosphorylation level of ERM (ezrin, radixin and moesin) proteins, that subsequently regulate axonal growth, cytoskeletal organization and microtubule assembly [76, 77, 78]. Furthermore, it has been shown that LRRK2 localizes to endosomes where it plays a role in vesicular membrane trafficking [28, 79]. Only recently, [PERSON] et al. convincingly identified a subset of Rab GTPases as genuine in vivo LRRK2 substrates [80, 81]. Phosphorylation by endogenous LRRK2 has been confirmed for 10 Rab GTPases: Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35, and Rab43 [80]. Surprisingly, looking at the Rab phylogenetic tree, these Rab proteins are actually widely dispersed. Thus, it might be that the Rabs that are phosphorylated by LRRK2, co-localize with LRRK2 and that this co-localization is the actual phosphorylation determinant [81]. Phosphorylation by LRRK2 influences the cycling of these Rab GTPases between the cytosol and the membrane. Inactive, guanosine-5'-diphosphate (GDP)-bound Rabs are bound by GDP-dissociation inhibitors (GDIs) locking them in the cytosol. Upon prenylation by Rab GGTases, Rab proteins dissociate from their GDI and insert in the membrane, where GDP is exchanged for GTP and the highly conserved threonine residue located in the switch II loop of Rab GTPases is phosphorylated by LRRK2. After GTP hydrolysis and dephosphorylation, the inactive Rabs again bind GDIs and dissociate from the membrane. Overactivation of LRRK2 by PD-mutations result in an increased phosphorylation of Rabs and a reduced affinity for GDIs. This, thus, leads to an accumulation of membrane-bound Rab GTPases which are functionally impaired due to disrupted interaction with effector proteins [80, 82, 83, 84].