Abstract
GTPase-activating proteins (GAPs) and guanine exchange factors (GEFs) play essential roles in regulating the activity of small GTPases. Several GAPs and GEFs have been shown to be present at the postsynaptic density (PSD) within excitatory glutamatergic neurons and regulate the activity of glutamate receptors. However, it is not known how synaptic GAP and GEF proteins are organized within the PSD signaling machinery, if they have overlapping interaction networks, or if they associate with proteins implicated in contributing to psychiatric disease. Here, we determine the interactomes of three interacting GAP/GEF proteins at the PSD, including the RasGAP Syngap1, the ArfGAP Agap2, and the RhoGEF Kalirin, which includes a total of 280 interactions. We describe the functional properties of each interactome and show that these GAP/GEF proteins are highly associated with and cluster other proteins directly involved in GTPase signaling mechanisms. We also utilize Agap2 as an example of GAP/GEFs localized within multiple neuronal compartments and determine an additional 110 interactions involving Agap2 outside of the PSD. Functional analysis of PSD and non-PSD interactomes illustrates both common and unique functions of Agap2 determined by its subcellular location. Furthermore, we also show that these GAPs/GEFs associate with several proteins involved in psychiatric disease.
Highlights
Small GTPases can regulate a variety of cellular functions
With Agap[2], Kalirin, and Syngap[1] being implicated in contributing to the risk of psychiatric disease, we show that they interact with and cluster proteins involved in autism spectrum disorder (ASD), schizophrenia (SCZ), and intellectual disability (ID)
Through immunoisolation followed by HPLC-MS/MS we were able to determine protein complexes involving the interacting GTPase-activating proteins (GAPs)/guanine exchange factors (GEFs) proteins Agap[2], Syngap[1], and Kalirin which comprised a total of 281 interactions
Summary
Small GTPases can regulate a variety of cellular functions. In neurons, they have been shown to be involved in different aspects of synaptic function such as regulation of the actin cytoskeleton, spine remodeling, and synaptic plasticity[6,7,8,9]. PSD protein complexes link glutamate receptors to downstream signaling pathways through a number of scaffold proteins with multi-modular protein domain architectures[11, 13] It is not known how synaptic GAPs and GEFs are organized within this signaling machinery, if they have overlapping binding partners, and if they associate to brain disease risk factors. Using immmunoisolation and high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS), we determined the interactomes of the RasGAP Syngap[1], the RhoGEF Kalirin, and the ArfGAP Agap[2] in PSD fractions of adult mouse cortex These proteins are able to interact with each other at the PSD and have been described to be involved in intellectual disability and psychiatric disease[17,18,19,20,21]. With Agap[2], Kalirin, and Syngap[1] being implicated in contributing to the risk of psychiatric disease, we show that they interact with and cluster proteins involved in autism spectrum disorder (ASD), schizophrenia (SCZ), and intellectual disability (ID)
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