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Despite tremendous effort, the molecular and cellular basis of cognitive deficits in schizophrenia remain poorly understood. Recent progress in elucidating the genetic architecture of schizophrenia has highlighted the association of multiple loci and rare variants that may impact susceptibility. One key example, given their potential etiopathogenic and therapeutic relevance, is a set of genes that encode proteins that regulate excitatory glutamatergic synapses in brain. A critical next step is to delineate specifically how such genetic variation impacts synaptic plasticity and to determine if and how the encoded proteins interact biochemically with one another to control cognitive function in a convergent manner. Towards this goal, here we study the roles of GPCR-kinase interacting protein 1 (GIT1), a synaptic scaffolding and signaling protein with damaging coding variants found in schizophrenia patients, as well as copy number variants found in patients with neurodevelopmental disorders. We generated conditional neural-selective GIT1 knockout mice and found that these mice have deficits in fear conditioning memory recall and spatial memory, as well as reduced cortical neuron dendritic spine density. Using global quantitative phospho-proteomics, we revealed that GIT1 deletion in brain perturbs specific networks of GIT1-interacting synaptic proteins. Importantly, several schizophrenia and neurodevelopmental disorder risk genes are present within these networks. We propose that GIT1 regulates the phosphorylation of a network of synaptic proteins and other critical regulators of neuroplasticity, and that perturbation of these networks may contribute specifically to cognitive deficits observed in schizophrenia and neurodevelopmental disorders., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

G Protein-Coupled Receptor Kinase-Interacting Protein-1 (GIT1) regulates neuronal functions, including cell and axon migration and synapse formation and maintenance, and GIT1 knockout (KO) mice exhibit learning and memory deficits. We noted that male and female GIT1-KO mice exhibit neuroimaging phenotypes including microcephaly, and altered cortical layering, with a decrease in neuron density in cortical layer V. Micro-CT and magnetic resonance microscopy (MRM) were used to identify morphometric phenotypes for the skulls and throughout the GIT1-KO brains. High field MRM of actively-stained mouse brains from GIT1-KO and wild type (WT) controls (n = 6 per group) allowed segmenting 37 regions, based on co-registration to the Waxholm Space atlas. Overall brain size in GIT1-KO mice was ~32% smaller compared to WT controls. After correcting for brain size, several regions were significantly different in GIT1-KO mice relative to WT, including the gray matter of the ventral thalamic nuclei and the rest of the thalamus, the inferior colliculus, and pontine nuclei. GIT1-KO mice had reduced volume of white matter tracts, most notably in the anterior commissure (~26% smaller), but also in the cerebral peduncle, fornix, and spinal trigeminal tract. On the other hand, the basal ganglia appeared enlarged in GIT1-KO mice, including the globus pallidus, caudate putamen, and particularly the accumbens - supporting a possible vulnerability to addiction. Volume based morphometry based on high-resolution MRM (21.5 μm isotropic voxels) was effective in detecting overall, and local differences in brain volumes in GIT1-KO mice, including in white matter tracts. The reduced relative volume of specific brain regions suggests a critical, but not uniform, role for GIT1 in brain development, conducive to brain microcephaly, and aberrant connectivity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Since its discovery, G protein-coupled receptor kinase-interacting protein 2, GIT2, and its family member, GIT1, have received considerable interest concerning their potential key roles in regulating multiple inter-connected physiological and pathophysiological processes. GIT2 was first identified as a multifunctional protein that is recruited to G protein-coupled receptors (GPCRs) during the process of receptor internalization. Recent findings have demonstrated that perhaps one of the most important effects of GIT2 in physiology concerns its role in controlling multiple aspects of the complex ageing process. Ageing can be considered the most prevalent pathophysiological condition in humans, affecting all tissue systems and acting as a driving force for many common and intractable disorders. The ageing process involves a complex interplay among various deleterious activities that profoundly disrupt the body's ability to cope with damage, thus increasing susceptibility to pathophysiologies such as neurodegeneration, central obesity, osteoporosis, type 2 diabetes mellitus and atherosclerosis. The biological systems that control ageing appear to function as a series of interconnected complex networks. The inter-communication among multiple lower-complexity signaling systems within the global ageing networks is likely coordinated internally by keystones or hubs, which regulate responses to dynamic molecular events through protein-protein interactions with multiple distinct partners. Multiple lines of research have suggested that GIT2 may act as one of these network coordinators in the ageing process. Identifying and targeting keystones, such as GIT2, is thus an important approach in our understanding of, and eventual ability to, medically ameliorate or interdict age-related progressive cellular and tissue damage., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The signaling scaffold protein GIT1 is expressed widely throughout the brain, but its function in vivo remains elusive. Mice lacking GIT1 have been proposed as a model for attention deficit-hyperactivity disorder, due to alterations in basal locomotor activity as well as paradoxical locomotor suppression by the psychostimulant amphetamine. Since we had previously shown that GIT1-knockout mice have normal locomotor activity, here we examined GIT1-deficient mice for ADHD-like behavior in more detail, and find neither hyperactivity nor amphetamine-induced locomotor suppression. Instead, GIT1-deficient mice exhibit profound learning and memory defects and reduced synaptic structural plasticity, consistent with an intellectual disability phenotype. We conclude that loss of GIT1 alone is insufficient to drive a robust ADHD phenotype in distinct strains of mice. In contrast, multiple learning and memory defects have been observed here and in other studies using distinct GIT1-knockout lines, consistent with a predominant intellectual disability phenotype related to altered synaptic structural plasticity.

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins (inactivators) for the ADP-ribosylation factor (Arf) small GTP-binding proteins, and function to limit the activity of Arf proteins. The PIX proteins, α-PIX and β-PIX (also known as ARHGEF6 and ARHGEF7, respectively), are guanine nucleotide exchange factors (activators) for the Rho family small GTP-binding protein family members Rac1 and Cdc42. Through their multi-domain structures, GIT and PIX proteins can also function as signaling scaffolds by binding to numerous protein partners. Importantly, the constitutive association of GIT and PIX proteins into oligomeric GIT-PIX complexes allows these two proteins to function together as subunits of a larger structure that coordinates two distinct small GTP-binding protein pathways and serves as multivalent scaffold for the partners of both constituent subunits. Studies have revealed the involvement of GIT and PIX proteins, and of the GIT-PIX complex, in numerous fundamental cellular processes through a wide variety of mechanisms, pathways and signaling partners. In this Commentary, we discuss recent findings in key physiological systems that exemplify current understanding of the function of this important regulatory complex. Further, we draw attention to gaps in crucial information that remain to be filled to allow a better understanding of the many roles of the GIT-PIX complex in health and disease., (© 2016. Published by The Company of Biologists Ltd.)

A cytomatrix of proteins at the presynaptic active zone (CAZ) controls the strength and speed of neurotransmitter release at synapses in response to action potentials. However, the functional role of many CAZ proteins and their respective isoforms remains unresolved. Here, we demonstrate that presynaptic deletion of the two G protein-coupled receptor kinase-interacting proteins (GITs), GIT1 and GIT2, at the mouse calyx of Held leads to a large increase in AP-evoked release with no change in the readily releasable pool size. Selective presynaptic GIT1 ablation identified a GIT1-specific role in regulating release probability that was largely responsible for increased synaptic strength. Increased synaptic strength was not due to changes in voltage-gated calcium channel currents or activation kinetics. Quantitative electron microscopy revealed unaltered ultrastructural parameters. Thus, our data uncover distinct roles for GIT1 and GIT2 in regulating neurotransmitter release strength, with GIT1 as a specific regulator of presynaptic release probability., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Both the Wnt/β-catenin signaling pathway and small GTPases of the ADP-ribosylation factors (ARF) family play important roles in regulating cell development, homeostasis and fate. The previous report of QS11, a small molecule Wnt synergist that binds to ARF GTPase-activating protein 1 (ARFGAP1), suggests a role for ARFGAP1 in the Wnt/β-catenin pathway. However, direct inhibition of enzymatic activity of ARFGAP1 by QS11 has not been established. Whether ARFGAP1 is the only target that contributes to QS11's Wnt synergy is also not clear. Here we present structure-activity relationship (SAR) studies of QS11 analogs in two assays: direct inhibition of enzymatic activity of purified ARFGAP1 protein and cellular activation of the Wnt/β-catenin pathway. The results confirm the direct inhibition of ARFGAP1 by QS11, and also suggest the presence of other potential cellular targets of QS11., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Insults to nuclear DNA induce multiple response pathways to mitigate the deleterious effects of damage and mediate effective DNA repair. G-protein-coupled receptor kinase-interacting protein 2 (GIT2) regulates receptor internalization, focal adhesion dynamics, cell migration, and responses to oxidative stress. Here we demonstrate that GIT2 coordinates the levels of proteins in the DNA damage response (DDR). Cellular sensitivity to irradiation-induced DNA damage was highly associated with GIT2 expression levels. GIT2 is phosphorylated by ATM kinase and forms complexes with multiple DDR-associated factors in response to DNA damage. The targeting of GIT2 to DNA double-strand breaks was rapid and, in part, dependent upon the presence of H2AX, ATM, and MRE11 but was independent of MDC1 and RNF8. GIT2 likely promotes DNA repair through multiple mechanisms, including stabilization of BRCA1 in repair complexes; upregulation of repair proteins, including HMGN1 and RFC1; and regulation of poly(ADP-ribose) polymerase activity. Furthermore, GIT2-knockout mice demonstrated a greater susceptibility to DNA damage than their wild-type littermates. These results suggest that GIT2 plays an important role in MRE11/ATM/H2AX-mediated DNA damage responses., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

G protein-coupled receptor kinase interacting protein 1 (GIT1) belongs to the family of Arf GAP proteins and has been implicated in the regulation of G protein-coupled receptor (GPCR) sequestration, cell migration, synapse formation and dendritic spine morphogenesis in neurons. To extend these cellular studies on GIT1 to an in vivo system, we generated mice with globally inactivated Git1 gene by breeding mice carrying a conditional Git1(flox) allele with mice expressing the CMV-Cre transgene. Although many GIT1 knockout (GIT1-KO) animals died shortly after birth, homozygous mutants that survived the early post-partum period developed normally into adulthood and were fertile. Behavioral analyses of adult GIT1-KO mice revealed normal exploratory, anxiety- and depressive-like behaviors. However, GIT1-KO mice show impaired responses to fear conditioning and fear-potentiated startle. Overall, these findings suggest that GIT1 is involved in the regulation of amygdala-mediated experience-based emotional behaviors.

At the FASEB summer research conference on "Arf Family GTPases", held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.

GIT1 and GIT2 belong to the family of ADP-ribosylation factor GTPase-activating proteins (ARF-GAP) and have been implicated in the regulation of G protein-coupled receptor sequestration, cell migration, T-cell activation, neuronal spine formation, and aggregate formation in Huntington's disease. Examination of endogenous GIT protein expression in tissues, however, has been hampered by the lack of GIT2-specific antibodies. To visualize GIT1 and GIT2 gene expression in mouse tissues, we created mice with beta-galactosidase (beta-Gal) reporters inserted into the two GIT genes. beta-Gal staining confirmed the broad tissue distribution of GIT1 and GIT2 in the mouse but also revealed striking differences. GIT2 is expressed in most cells of the body, whereas GIT1 is restricted to only a subset of cells. For example, GIT2 is uniformly expressed throughout lung and liver, whereas GIT1 is restricted to cells lining blood vessels, bronchi, and bile ducts. Expression of GIT1 and GIT2 is mutually exclusive in the testes, where a developmental expression shift occurs, with GIT2 present in spermatogonia but GIT1 in mature spermatids. In conclusion, analysis of endogenous GIT expression revealed a nearly ubiquitous distribution of GIT2, whereas GIT1 is restricted to specific cell types even in tissues with apparently high GIT1 expression and is entirely absent from some tissues.

Arf1 is a GTP binding protein that functions at a number of cellular sites to control membrane traffic and actin remodeling. Arf1 is regulated by site-specific GTPase-activating proteins (GAPs). The combined results of crystallographic and biochemical studies have led to the proposal that Arf1 GAPs differ in the specific interface formed with Arf1. To test this hypothesis, we have used mutagenesis to examine the interaction of three Arf GAPs (ASAP1, AGAP1, and ArfGAP1) with switch 1, switch 2, and alpha helix3 of Arf1. The GAPs were similar in being affected by mutations in switch 1 and 2. However, effects of a mutation within alpha helix3 and specific mutations within switch 1 and 2 differed among the GAPs. The largest differences were observed with a change of isoleucine 46 to aspartate ([I46D]Arf1), which reduced ASAP1-induced catalysis by approximately 10,000-fold but had a 3-fold effect on AGAP1. The reduction was due to an isolated effect on the catalytic rate, k(cat). In vivo [I46D]Arf1 had no detectable effect on the Golgi apparatus but, instead, functioned as a constitutively active mutant in the cell periphery, affecting the localization of ASAP1 and paxillin. Based on our results, we conclude that the contribution of specific residues within switch 1 of Arf to binding and achieving a transition state toward GTP hydrolysis differs among Arf GAPs.

ADP ribosylation factors (Arf) regulate membrane trafficking at multiple intracellular sites by recruiting coat proteins to membranes. The site-specific regulation of Arf is thought to be mediated by regulatory proteins including the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Here, we test this hypothesis by comparing the site of action of the Arf GAP AGAP2 to the closely related AGAP1. AGAP1 has previously been found to associate with the adaptor protein complex AP-3 and regulate the function of AP-3 endosomes. We found that AGAP2 directly interacted with AP-1. AGAP2 colocalized with AP-1, transferrin receptor and Rab4 on endosomes. Overexpression of AGAP2 changed the intracellular distribution of AP-1 and promoted Rab4-dependent fast recycling of transferrin. Based on these results, we concluded that the closely related Arf GAPs, AGAP1 and AGAP2, distinguish between these related heterotetrameric adaptor protein complexes to specifically regulate AP-3 endosomes and AP-1 recycling endosomes.

Examining how key components of coat protein I (COPI) transport participate in cargo sorting, we find that, instead of ADP ribosylation factor 1 (ARF1), its GTPase-activating protein (GAP) plays a direct role in promoting the binding of cargo proteins by coatomer (the core COPI complex). Activated ARF1 binds selectively to SNARE cargo proteins, with this binding likely to represent at least a mechanism by which activated ARF1 is stabilized on Golgi membrane to propagate its effector functions. We also find that the GAP catalytic activity plays a critical role in the formation of COPI vesicles from Golgi membrane, in contrast to the prevailing view that this activity antagonizes vesicle formation. Together, these findings indicate that GAP plays a central role in coupling cargo sorting and vesicle formation, with implications for simplifying models to describe how these two processes are coupled during COPI transport.

Cargo sorting that promotes the transport of cargo proteins from a membrane compartment has been predicted to be unlikely in the endocytic recycling pathways. We now show that ACAP1 binds specifically and directly to recycling cargo proteins. Reducing this interaction for TfR inhibits its recycling. Moreover, ACAP1 binds to two distinct phenylalanine-based sequences in the cytoplasmic domain of TfR that function as recycling sorting signals to promote its transport from the recycling endosome. Taken together, these findings indicate that ACAP1 promotes cargo sorting by recognizing recycling sorting signals.

GIT proteins are GTPase-activating proteins (GAPs) for ADP-ribosylation factor (ARF) small GTP-binding proteins, and interact with the PIX family of Rac1/Cdc42 guanine nucleotide exchange factors. GIT and PIX transiently localize p21-activated protein kinases (PAKs) to remodeling focal adhesions through binding to paxillin. To understand the role of these interactions, the association of GIT and PIX proteins was examined in detail. Two separable binding interactions link GIT and PIX proteins, GIT and PIX proteins each dimerize and a beta-PIX fragment containing the GIT-binding region failed to inhibit the association of the GIT and PIX proteins. Endogenous GIT and PIX co-fractionate at a very high molecular size. Purified 6xHis-tagged beta-PIX from Sf9 cells co-expressing untagged GIT1 yields recombinant GIT1/beta-PIX complexes that have equal amounts of beta-PIX and GIT1 and co-fractionate at the same large size as native GIT/PIX complexes. Thus, GIT and PIX proteins are tightly associated as a multimeric nexus capable of linking together important signaling molecules, including PAKs.

The Arf GAPs are a structurally diverse group of proteins that catalyze the hydrolysis of GTP bound to Arf1. Here, we directly compare the role of amino acids 2-17 of Arf1, a GTP- and phospholipid-sensitive switch, for interaction with three Arf GAPs: Arf GAP1, AGAP1 and ASAP1. Sequestration of amino acids 2-17 with an antibody inhibited interaction with the three tested Arf GAPs. Examination of Arf1 mutants also indicated that [2-17]Arf1 is a critical structural determinant of interaction with all three Arf GAPs; however, the effect of specific mutations differed among the GAPs. Compared to wild-type Arf1, Arf1 with the amino terminal 13 ([Delta13]Arf1) and 17 amino acids ([Delta17]Arf1) deleted had 200- and 4000-fold reduced interaction with ASAP1 and 150-fold reduced interaction with AGAP1. In contrast, deletion of the amino terminus of Arf reduced interaction with Arf GAP1 by 5-fold. By analysis of point mutants, we found that lysines 15 and 16 had a greater contribution to productive interaction between Arf1, ASAP1 and AGAP1 than between Arf1 and Arf GAP1. Leucine 8 contributed to the interaction with Arf GAP1 but not with ASAP1 and AGAP1. Amino acids 2-17 of Arf1, isolated from the protein, inhibited GAP activity of Arf GAP1, ASAP1 and AGAP1 and bound directly to ASAP1. Taken together, our results indicate that (i) Arf GAPs interact with amino acids 2-17 of Arf1 and (ii) each subgroup of Arf GAPs has a unique interface with Arf1.

Drosophila Scribble is implicated in the development of normal synapse structure and epithelial tissues, but it remains unclear how it plays a role and which process it controls. The mammalian homolog of Scribble, hScrib, has a primary structure and subcellular localization similar to that of its fly homolog, but its function remains unknown. Here we have used tandem mass spectrometry to identify major components of the hScrib network. We show that it includes betaPIX (also called Cool-1), a guanine nucleotide exchange factor (GEF), and its partner GIT1 (also called p95-APP1), a GTPase activating protein (GAP). betaPIX directly binds to the hScrib PDZ domains, and the hScrib/betaPIX complex is efficiently recovered in epithelial and neuronal cells and tissues. In cerebellar granule cell cultures, hScrib and betaPIX are both partially localized at neuronal presynaptic compartments. Furthermore, we show that hScrib is required to anchor betaPIX at the cell cortex and that dominant-negative betaPIX or hScrib proteins can each inhibit Ca2+-dependent exocytosis in neuroendocrine PC12 cells, demonstrating a functional relationship between these proteins. These data reveal the existence of a tight hScrib/betaPIX interaction and suggest that this complex potentially plays a role in neuronal transmission., (Copyright 2004 Elsevier Ltd.)

Liprin-alpha is a multidomain protein that interacts with the LAR family of receptor protein tyrosine phosphatases and the GRIP/ABP family of AMPA receptor-interacting proteins. Previous studies have indicated that liprin-alpha regulates the development of presynaptic active zones and that the association of liprin-alpha with GRIP is required for postsynaptic targeting of AMPA receptors. However, the underlying molecular mechanisms are not well understood. Here we report that liprin-alpha directly interacts with GIT1, a multidomain protein with GTPase-activating protein activity for the ADP-ribosylation factor family of small GTPases known to regulate protein trafficking and the actin cytoskeleton. Electron microscopic analysis indicates that GIT1 distributes to the region of postsynaptic density (PSD) as well as presynaptic active zones. GIT1 is enriched in PSD fractions and forms a complex with liprin-alpha, GRIP, and AMPA receptors in brain. Expression of dominant-negative constructs interfering with the GIT1-liprin-alpha interaction leads to a selective and marked reduction in the dendritic and surface clustering of AMPA receptors in cultured neurons. These results suggest that the GIT1-liprin-alpha interaction is required for AMPA receptor targeting and that GIT1 may play an important role in the organization of presynaptic and postsynaptic multiprotein complexes.

The cytoskeletal matrix assembled at active zones (CAZ) is implicated in defining neurotransmitter release sites. However, little is known about the molecular mechanisms by which the CAZ is organized. Here we report a novel interaction between Piccolo, a core component of the CAZ, and GIT proteins, multidomain signaling integrators with GTPase-activating protein activity for ADP-ribosylation factor small GTPases. A small region (approximately 150 amino acid residues) in Piccolo, which is not conserved in the closely related CAZ protein Bassoon, mediates a direct interaction with the Spa2 homology domain (SHD) domain of GIT1. Piccolo and GIT1 colocalize at synaptic sites in cultured neurons. In brain, Piccolo forms a complex with GIT1 and various GIT-associated proteins, including betaPIX, focal adhesion kinase, liprin-alpha, and paxillin. Point mutations in the SHD of GIT1 differentially interfere with the association of GIT1 with Piccolo, betaPIX, and focal adhesion kinase, suggesting that these proteins bind to the SHD by different mechanisms. Intriguingly, GIT proteins form homo- and heteromultimers through their C-terminal G-protein-coupled receptor kinase-binding domain in a tail-to-tail fashion. This multimerization enables GIT1 to simultaneously interact with multiple SHD-binding proteins including Piccolo and betaPIX. These results suggest that, through their multimerization and interaction with Piccolo, the GIT family proteins are involved in the organization of the CAZ.

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPgammaS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.