# Roco Proteins: GTPases with a Baroque Structure and Mechanism
## 6 Roco Proteins: Conventional G-Proteins, GADs or Yet Another Type of G Proteins?
Rho guanine nucleotide exchange factor ARHGEF7 was shown to interact and partially co-localize with LRRK2 in vitro and in vivo. It enhances the exchange of GDP for GTP and in this way, might function as a GEF for the GTPase activity of LRRK2. It should, however, be noted that ARHGEF7 does not bind directly to the Roc domain of LRRK2, unlike conventional GEFs. Strikingly, ARHGEF7 is also reported to be an in vitro substrate of LRRK2, with two threonine residues at the N-terminus of ARHGEF7 being phosphorylated by LRRK2 [116; 117]. Two potential LRRK2 GAPs were identified as well: ArfGAP1 and RGS2 [118; 119]. The ArfGAP1-LRK2 interaction has been demonstrated both in vitro and in vivo in brain tissue [118]. Binding of ArfGAP1 has no effect on LRRK2's GTP binding capacity but mediates LRRK2 toxicity by increasing the GTP hydrolysis rate. However, ArfGAP1 binds LRRK2 primarily via the WD40 and kinase domain, rather than via the Roc GTPase domain [120]. In accordance, LRRK2 has been shown to phosphorylate ArfGAP1. Studies concerning the effect of ArfGAP1 on LRRK2 kinase activity and the other way around, have however given controversial results. In different conflicting studies, ArfGAP1 has been shown to either reduce or induce LRRK2 kinase activity. In addition, the role of phosphorylation of ArfGAP1 is unclear [118; 120]. In 2014, [PERSON] et al. identified a second possible LRRK2 GAP, named RGS2. RGS2 increases the GTPase activity of LRRK2 in vitro. Surprisingly, independent of its influence on the GTPase activity, RGS2 was shown to have an inhibitory effect on LRRK2 kinase activity and in this way controls neurite length. RGS2 is also a substrate for phosphorylation by LRRK2 in vitro. Future studies are needed to investigate whether RGS2 is also a substrate of LRRK2 in vivo and whether RGS2 is a physiological GAP for LRRK2 in vivo, or mediates its function via another mechanism [117; 121]. In fact, for all GEFs and GAPs reported, their direct interaction with the LRRK2 Roc domain is still not proven. Hence, ARHGEF7, RGS2, and ArfGAP1 could modulate the GTPase activity of LRRK2, but most likely function in a manner distinct from classical GEFs and GAPs [115]. On the other hand, based on the above-discussed structures and the dimeric nature of Roco family proteins, including LRRK2, it was postulated that Roco proteins belong to the GAD family of proteins [33; 34; 108; 122]. Moreover, we recently showed that full length prokaryotic Roco proteins have a low, micromolar affinity for GDP (9-55 uM) associated with fast GDP dissociation rates (12.8-230 min\({}^{-1}\)) [18]. This is in line with the reported affinity of the human LRRK2 Roc domain for GTP and GDP of, respectively, 7.85 and 0.47 uM [105]. Based on these studies it can be concluded that GEFs are not strictly necessary for the functioning of Roco proteins. The in vitro GTP hydrolysis rate of Roco proteins (0.06-0.5 min\({}^{-1}\)) [18] lies well within the range of the basal activity of GALs, like MnmE (0.33 min\({}^{-1}\)), as well as within the intrinsic GTP hydrolysis activity of conventional G-proteins, such as Ras (0.03 min\({}^{-1}\)) [123; 124]. Whereas the basal activity of Ras is stimulated up to 300 to 600 min\({}^{-1}\) by RasGAP, the GTP hydrolysis rate of MnmE increases up to 9.3 min\({}^{-1}\) upon interaction with K\({}^{+}\) ions and its interaction partner MnmG [125]. It is, thus, possible that the moderate GTPase activity of Roco proteins is indeed triggered by interaction with cofactors, including RGS2 and ARFGAP1 [119; 121; 126]. The kinetic data, combined with the dimeric prokaryotic RocCOR protein crystal structures, thus, argue against a model where Roco proteins function as conventional G-proteins.