Microcephaly with altered cortical layering in GIT1 deficiency revealed by quantitative neuroimaging.

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.

GIT1 regulates synaptic structural plasticity underlying learning.

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.

Presynaptic Deletion of GIT Proteins Results in Increased Synaptic Strength at a Mammalian Central Synapse.

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.

Structure-activity relationship studies of QS11, a small molecule Wnt synergistic agonist.

Both the Wnt/ß-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/ß-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/ß-catenin pathway. The results confirm the direct inhibition of ARFGAP1 by QS11, and also suggest the presence of other potential cellular targets of QS11.

Nuclear GIT2 is an ATM substrate and promotes DNA repair.

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.

GIT2 Acts as a Systems-Level Coordinator of Neurometabolic Activity and Pathophysiological Aging.

Aging represents one of the most complicated and highly integrated somatic processes. Healthy aging is suggested to rely upon the coherent regulation of hormonal and neuronal communication between the central nervous system and peripheral tissues. The hypothalamus is one of the main structures in the body responsible for sustaining an efficient interaction between energy balance and neurological activity and therefore likely coordinates multiple systems in the aging process. We previously identified, in hypothalamic and peripheral tissues, the G protein-coupled receptor kinase interacting protein 2 (GIT2) as a stress response and aging regulator. As metabolic status profoundly affects aging trajectories, we investigated the role of GIT2 in regulating metabolic activity. We found that genomic deletion of GIT2 alters hypothalamic transcriptomic signatures related to diabetes and metabolic pathways. Deletion of GIT2 reduced whole animal respiratory exchange ratios away from those related to primary glucose usage for energy homeostasis. GIT2 knockout (GIT2KO) mice demonstrated lower insulin secretion levels, disruption of pancreatic islet beta cell mass, elevated plasma glucose, and insulin resistance. High-dimensionality transcriptomic signatures from islets isolated from GIT2KO mice indicated a disruption of beta cell development. Additionally, GIT2 expression was prematurely elevated in pancreatic and hypothalamic tissues from diabetic-state mice (db/db), compared to age-matched wild type (WT) controls, further supporting the role of GIT2 in metabolic regulation and aging. We also found that the physical interaction of pancreatic GIT2 with the insulin receptor and insulin receptor substrate 2 was diminished in db/db mice compared to WT mice. Therefore, GIT2 appears to exert a multidimensional "keystone" role in regulating the aging process by coordinating somatic responses to energy deficits.

The cytoskeletal regulatory scaffold protein GIT2 modulates mesenchymal stem cell differentiation and osteoblastogenesis.

G protein-coupled receptor kinase interacting protein 2 (GIT2) is a signaling scaffold protein involved in the regulation of cytoskeletal structure, membrane trafficking, and G protein-coupled receptor internalization. Since dynamic cytoskeletal reorganization plays key roles both in osteoblast differentiation and in the maintenance of osteoclast polarity during bone resorption, we hypothesized that skeletal physiology would be altered in GIT2(-/-) mice. We found that adult GIT2(-/-) mice have decreased bone mineral density and bone volume in both the trabecular and cortical compartments. This osteopenia was associated with decreased numbers of mature osteoblasts, diminished osteoblastic activity, and increased marrow adiposity, suggesting a defect in osteoblast maturation. In vitro, mesenchymal stem cells derived from GIT2(-/-) mice exhibited impaired differentiation into osteoblasts and increased adipocyte differentiation, consistent with a role for GIT2 in mesenchymal stem cell fate determination. Despite elevated osteoclast inducing cytokines and osteoclast numbers, GIT2(-/-) mice also exhibit impaired bone resorption, consistent with a further role for GIT2 in regulating osteoclast function. Collectively, these findings underscore the importance of the cytoskeleton in both osteoblast and osteoclast function and demonstrate that GIT2 plays essential roles in skeletal metabolism, affecting both bone formation and bone resorption in vivo.

Metastasis: wherefore arf thou?

The small GTP-binding protein Arf6 is known to be an important regulator of the actin cytoskeleton and of cell motility associated with metastasis. A recent study identifies yet another role for Arf6 in metastasis - as a regulator of plasma-membrane-derived microvesicle release.

Impaired fear response in mice lacking GIT1.

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.

Anxiety-like behaviors in mice lacking GIT2.

G protein-coupled receptor kinase-interactor 2 (GIT2) is a signaling scaffold protein that also functions as GTPase-activating protein (GAPs) for ADP-ribosylation factor (Arf) small GTP-binding proteins. GIT2 has been implicated in the regulation of G protein-coupled receptor trafficking and cell adhesion and migration. To evaluate possible neurobehavioral functions of GIT2 in vivo, we evaluated GIT2-knockout (KO) mice for abnormalities in emotionality and mood. Male and female GIT2-KO mice presented with anxiety-like behaviors in the zero-maze and light-dark emergence tests. Immobility times in tail suspension were reduced in GIT2-KO males, but were normal in GIT2-KO females. Hence, GIT2-KO mice display anxiety-like behavior in an absence of depressive-like responses.

Differential expression of the ARF GAP genes GIT1 and GIT2 in mouse tissues.

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.

GIT1 utilizes a focal adhesion targeting-homology domain to bind paxillin.

The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins for the ADP-ribosylation factor family of small GTP-binding proteins, but also serve as adaptors to link signaling proteins to distinct cellular locations. One role for GIT proteins is to link the PIX family of Rho guanine nucleotide exchange factors and their binding partners, the p21-activated protein kinases, to remodeling focal adhesions by interacting with the focal adhesion adaptor protein paxillin. We here identified the C-terminal domain of GIT1 responsible for paxillin binding. Combining structural and mutational analyses, we show that this region folds into an anti-parallel four-helix domain highly reminiscent to the focal adhesion targeting (FAT) domain of focal adhesion kinase (FAK). Our results suggest that the GIT1 FAT-homology (FAH) domain and FAT bind the paxillin LD4 motif quite similarly. Since only a small fraction of GIT1 is bound to paxillin under normal conditions, regulation of paxillin binding was explored. Although paxillin binding to the FAT domain of FAK is regulated by tyrosine phosphorylation within this domain, we find that tyrosine phosphorylation of the FAH domain GIT1 is not involved in regulating binding to paxillin. Instead, we find that mutations within the FAH domain may alter binding to paxillin that has been phosphorylated within the LD4 motif. Thus, despite apparent structural similarity in their FAT domains, GIT1 and FAK binding to paxillin is differentially regulated.

The GIT/PIX complex: an oligomeric assembly of GIT family ARF GTPase-activating proteins and PIX family Rac1/Cdc42 guanine nucleotide exchange factors.

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.