Rho GTPases, including RhoA, Rac1, and Cdc42, are key regulators of actin cytoskeleton dynamics. Since BDNF stimulation has been observed to activate Rac1 and Cdc42 in neurons [15], we were interested to delineate if Rho GTPases contribute to BDNF-stimulated dendritic growth. To investigate if Rho GTPases are involved, and to identify the Rho GTPase(s) implicated, hippocampal neurons were transfected with WT or DN Rac1, Cdc42, or RhoA. We found that while overexpression of WT and DN Rac1 increased the basal number of dendrites in the absence of BDNF treatment, overexpression of both forms of Rac1 abolished BDNF-stimulated dendritic growth. On the other hand, while overexpression of DN RhoA slightly enhanced primary dendrites irrespective of BDNF stimulation, overexpression of both WT and DN forms of RhoA inhibited BDNF-stimulated dendritic growth. Remarkably, in contrast to the inhibition of BDNF-stimulated dendritic growth in cells overexpressing WT Rac1 and RhoA, BDNF stimulation of hippocampal neurons overexpressing WT Cdc42 resulted in an increase in primary dendrites, which was nearly abolished by overexpression of DN Cdc42 (Figure 6A). Our observations therefore suggest that while Rac1 and RhoA may also modulate BDNF-stimulated dendritic growth, it is the activation of Cdc42 following BDNF stimulation that most likely mediates the increase in primary dendrites by BDNF.
In the current study, we demonstrated that Ser478 phosphorylation of TrkB by Cdk5 is essential for the Cdc42-dependent increase in primary dendrites triggered by BDNF, thus adding a new regulatory component to the mechanisms involved in Rho GTPase activation by neurotrophin. Although the precise downstream pathways by which this phosphorylation affects Cdc42 activation remains to be determined, our observations provide some interesting insights.Interestingly, APPL1 would bind to Rab21 in a GTP-dependent manner (Figure 4), indicating that APPL1 is an effector for both Rab5 and Rab21.
GTPase binding has emerged as a major function of PH domains in addition to lipid binding (Lemmon, 2004). For example, PH domains in some guanine nucleotide-exchange factors (GEF) have been shown to bind directly to their cognate small GTPases (Rossman et al, 2002, 2003; Lu et al, 2004), and our data now show direct interaction between the APPL1 PH domain and Rab5. So far, only two crystal structures of small GTPase−PH domain complexes are available. One is Ran−RanBD1 (PDB file 1RRP). The interactions between the Ran GTPase domain and RanBD1 PH core domain is fairly minor, occurring between the switch I region of the GTPase (equivalent to the 40's in Rab5) and strand β2 of the PH domain. This interaction alone is unlikely to be sufficient to form a stable complex. Indeed, Ran has a long C-terminal peptide beside the GTPase domain, while the PH domain of RanBD1 has an extra N-terminal peptide. These two terminal peptides wrap around the partner proteins forming the major interaction between Ran and RanBD1. Such an interaction seems not to be required for Rab5 and APPL1, because the GTPase domain of Rab5 and BAR-PH domain of APPL1 are sufficient to mediate their interaction. The second published small GTPase−PH complex is that of Ral−Exo84 (PDB file 1ZC3). In this complex, the PH domain of Exo84 uses L1, β5, and L6 to interact with the interswitch and switch II regions of Ral forming an intermolecular β-sheet extension mediated by the PH β5 strand and GTPase β2 strand (Jin et al, 2005). Our mutagenesis analysis points to a different surface region (β3, L3, and β4) of the PH domain for Rab5 binding. Therefore, the Rab5−APPL1 interaction represents a new GTPase−PH binding mode.