Regulation of neuronal RhoA guanine-nucleotide exchange factor, Tech, and its interaction with synaptic scaffold protein, MUPP1

Tech is a RhoA guanine nucleotide exchange factor (GEF) that is highly enriched in hippocampal and cortical neurons. To help define its function, we have conducted two sets of studies. Firstly, we aimed to identify partner proteins that bind to the C-terminal PDZ ligand motif of Tech. Yeast two hybrid studies using the Tech C-terminal segment as bait identified MUPP1, a protein that contains 13 PDZ domains and has been localized to the post-synaptic compartment, as a candidate partner protein for Tech. Co-transfection of Tech and MUPP1 in hEK 293 cells confirmed that these full-length proteins interact in a PDZ-dependent fashion. Furthermore, we confirmed that endogenous Tech co-precipitates with MUPP1, but not PSD-95, from hippocampal and cortical extracts prepared from rat brain. In addition, immunostaining of primary cortical cultures revealed co-localization of MUPP1 and Tech puncta in the vicinity of synapses. In assessing which PDZ domains of MUPP1 mediate binding to Tech, we found that Tech can bind to either PDZ domain 10 or 13 of MUPP1 as mutation of both these domains is needed to disrupt their interaction. In a second set of studies, we examined the means of Tech regulation. Here we found that Tech's C terminus has an inhibitory impact on Tech activation of RhoA, as assayed by SRF-mediated transcription. Moreover, we pinpointed a putative SH3-binding region in Tech's C terminus that mediates this inhibitory property. Taken together, these findings demonstrate that Tech is able to bind to MUPP1 via its PDZ ligand motif, and to regulate its activity via an autoinhibitory domain. Also, as our findings point to synaptic localization of Tech, they suggest that Tech regulates RhoA signaling pathways in the vicinity of synapses.