Slit-Robo GTPase-activating Protein Research Articles

Srgap2 suppression ameliorates retinal ganglion cell degeneration in mice.

Slit-Robo GTPase-activating protein 2 (SRGAP2) plays important roles in axon guidance, neuronal migration, synapse formation, and nerve regeneration. However, the role of SRGAP2 in neuroretinal degenerative disease remains unclear. In this study, we found that SRGAP2 protein was first expressed in the retina of normal mice at the embryonic stage and was mainly located in the mature retinal ganglion cell layer and the inner nuclear layer. SRGAP2 protein in the retina and optic nerve increased after optic nerve crush. Then, we established a heterozygous knockout (Srgap2+/-) mouse model of optic nerve crush and found that Srgap2 suppression increased retinal ganglion cell survival, lowered intraocular pressure, inhibited glial cell activation, and partially restored retinal function. In vitro experiments showed that Srgap2 suppression activated the mammalian target of rapamycin signaling pathway. RNA sequencing results showed that the expression of small heat shock protein genes (Cryaa, Cryba4, and Crygs) related to optic nerve injury were upregulated in the retina of Srgap2+/- mice. These results suggest that Srgap2 suppression reduced the robust activation of glial cells, activated the mammalian target of rapamycin signaling pathway related to nerve protein, increased the expression of small heat shock protein genes, inhibited the degeneration of retinal ganglion cells, and partially restored optic nerve function.

C. elegans srGAP is an α-catenin M domain-binding protein that strengthens cadherin-dependent adhesion during morphogenesis.

The cadherin-catenin complex (CCC) is central to embryonic development and tissue repair, yet how CCC binding partners function alongside core CCC components remains poorly understood. Here, we establish a previously unappreciated role for an evolutionarily conserved protein, the slit-robo GTPase-activating protein SRGP-1/srGAP, in cadherin-dependent morphogenetic processes in the Caenorhabditis elegans embryo. SRGP-1 binds to the M domain of the core CCC component, HMP-1/α-catenin, via its C terminus. The SRGP-1 C terminus is sufficient to target it to adherens junctions, but only during later embryonic morphogenesis, when junctional tension is known to increase. Surprisingly, mutations that disrupt stabilizing salt bridges in the M domain block this recruitment. Loss of SRGP-1 leads to an increase in mobility and decrease of junctional HMP-1. In sensitized genetic backgrounds with weakened adherens junctions, loss of SRGP-1 leads to late embryonic failure. Rescue of these phenotypes requires the C terminus of SRGP-1 but also other domains of the protein. Taken together, these data establish a role for an srGAP in stabilizing and organizing the CCC during epithelial morphogenesis by binding to a partially closed conformation of α-catenin at junctions.

Effect of electroacupuncture on muscular atrophy and Slit/Robo signaling in sciatic nerve and lumbar spinal cord tissues in rats with sciatic nerve injury

To observe the effect of electroacupuncture (EA) of "Huantiao"(GB30) and" Zusanli"(ST36)on muscular atrophy and expression of Slit-Robo GTPase-activating protein(srGAP)1, 2 and 3 in the injured sciatic nerve and lumbar spinal cord tissues in sciatic nerve injury (SNI) rats, so as to reveal its mechanisms underlying improvement of peripheral nerve injury (PNI)．. A total of 120 healthy male SD rats were randomly divided into control, sham-operation, model and EA groups (n＝30 rats in each) which were further divided into 7, 15 and 23 d subgroups (n＝10 rats in each subgroup). The SNI model was established by transecting the right sciatic nerve beneath the piriformis and immediately subsequent end-to-end suture. Rats of the sham operation group received an incision of the corresponding skin and suture. EA (5 Hz/20 Hz, 2－3 mA) was applied to the right GB30 and ST36 for 15 min, once daily, 6 days a week separately for 1，2 and 3 weeks. Rats in the sham-operation and model groups were grasped in the similar procedure as the EA group. The wet weight of gastrocnemius muscles (WWG) on both sides was measured to calculate the recovery rate (weight of the right WWG/weight of the left WWG×100%), and the expression levels of srGAP1, srGAP2 and srGAP3 proteins in the sciatic nerve and the spinal cord (L4-L6) tissues were detected by Western blot. After modeling and compared with the control and sham-operation groups, the recovery rate of WWG was significantly reduced, and the expression levels of srGAP1, srGAP2 and srGAP3 proteins of the sciatic nerve and lumbar spinal cord on day 7, 15 and 23 were considerably increased in the model group (P<0.01). Following the EA treatment, the reco-very rate of WWG was obviously increased and the expression levels of srGAP1, srGAP2 and srGAP3 proteins of both sciatic nerve and spinal cord on day 7, 15 and 23 were further significantly up-regulated in the EA group relevant to the model group (P<0.05，P<0.01). In addition, the expression levels of the 3 proteins in both sciatic nerve and lumbar spinal cord peaked on day 15 and attenuated on day 23. EA of GB30 and ST36 may relieve gastrocnemius atrophy in SNI rats, which is related to its function in up-regulating the Slit/Robo signaling in the sciatic nerve and lumbar spinal cord to promote the axonal targeting regeneration and repair of axonal plasma nutrition transportation.

SRGAP1, a crucial target of miR-340 and miR-124, functions as a potential oncogene in gastric tumorigenesis

Slit-Robo GTPase-activating protein 1 (SRGAP1) functions as a GAP for Rho-family GTPases and downstream of Slit-Robo signaling. We aim to investigate the biological function of SRGAP1 and reveal its regulation by deregulated microRNAs (miRNAs) in gastric cancer (GC). mRNA and protein expression of SRGAP1 were examined by quantitative reverse transcription PCR (qRT-PCR) and western blot. The biological role of SRGAP1 was demonstrated through siRNA-mediated knockdown experiments. The regulation of SRGAP1 by miR-340 and miR-124 was confirmed by western blot, dual luciferase activity assays and rescue experiments. SRGAP1 is overexpressed in 9 out of 12 (75.0%) GC cell lines. In primary GC samples from TCGA cohort, SRGAP1 shows gene amplification in 5/258 (1.9%) of cases and its mRNA expression demonstrates a positive correlation with copy number gain. Knockdown of SRGAP1 in GC cells suppressed cell proliferation, reduced colony formation, and significantly inhibited cell invasion and migration. Luciferase reporter assays revealed that SRGAP1 knockdown significantly inhibited Wnt/β-catenin pathway. In addition, SRGAP1 was found to be a direct target of two tumor-suppressive miRNAs, miR-340 and miR-124. Concordantly, these two miRNAs were downregulated in primary gastric tumors and these decreasing levels w5ere associated with poor outcomes. Expression of miR-340 and SRGAP1 displayed a reverse relationship in primary samples and re-expressed SRGAP1, rescued the anti-cancer effects of miR-340. Taken together, these data strongly suggest that, apart from gene amplification and mutation, the activation of SRGAP1 in GC is partly due to the downregulation of tumor-suppressive miRNAs, miR-340 and miR-124. Thus SRGAP1 is overexpressed in gastric carcinogenesis and plays an oncogenic role through activating Wnt/β-catenin pathway.

Slit-Robo GTPase-Activating Protein 2 as a metastasis suppressor in osteosarcoma.

Osteosarcoma is the most common primary bone tumor, with metastatic disease responsible for most treatment failure and patient death. A forward genetic screen utilizing Sleeping Beauty mutagenesis in mice previously identified potential genetic drivers of osteosarcoma metastasis, including Slit-Robo GTPase-Activating Protein 2 (Srgap2). This study evaluates the potential role of SRGAP2 in metastases-associated properties of osteosarcoma cell lines through Srgap2 knockout via the CRISPR/Cas9 nuclease system and conditional overexpression in the murine osteosarcoma cell lines K12 and K7M2. Proliferation, migration, and anchorage independent growth were evaluated. RNA sequencing and immunohistochemistry of human osteosarcoma tissue samples were used to further evaluate the potential role of the Slit-Robo pathway in osteosarcoma. The effects of Srgap2 expression modulation in the murine OS cell lines support the hypothesis that SRGAP2 may have a role as a suppressor of metastases in osteosarcoma. Additionally, SRGAP2 and other genes in the Slit-Robo pathway have altered transcript levels in a subset of mouse and human osteosarcoma, and SRGAP2 protein expression is reduced or absent in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in osteosarcoma metastasis, with loss of SRGAP2 potentially contributing to a more aggressive phenotype.

Molecular symmetry-constrained systematic search approach to structure solution of the coiled-coil SRGAP2 F-BARx domain.

SRGAP2 (Slit-Robo GTPase-activating protein 2) is a cytoplasmic protein found to be involved in neuronal branching, restriction of neuronal migration and restriction of the length and density of dendritic postsynaptic spines. The extended F-BAR (F-BARx) domain of SRGAP2 generates membrane protrusions when expressed in COS-7 cells, while most F-BARs induce the opposite effect: membrane invaginations. As a first step to understand this discrepancy, the F-BARx domain of SRGAP2 was isolated and crystallized after co-expression with the carboxy domains of the protein. Diffraction data were collected from two significantly non-isomorphous crystals in the same monoclinic C2 space group. A correct molecular-replacment solution was obtained by applying a molecular symmetry-constrained systematic search approach that took advantage of the conserved biological symmetry of the F-BAR domains. It is shown that similar approaches can solve other F-BAR structures that were previously determined by experimental phasing. Diffraction data were reprocessed with a high-resolution cutoff of 2.2 Å, chosen using less strict statistical criteria. This has improved the outcome of multi-crystal averaging and other density-modification procedures.

Abstract 1621: SRGAP2 expression levels may induce a biphasic metastatic phenotype in osteosarcoma cell lines

Abstract The purpose of this study is to characterize the role of Slit-Robo GTPase-Activating Protein 2 (SRGAP2) in osteosarcoma (OS) metastasis. OS is the most common primary bone tumor and the eighth leading pediatric cancer. Despite combined therapies, treatment failure is experienced within 5 years of diagnosis by over 40% of patients, generally due to metastatic disease. Using the conditional Sleeping Beauty transposon mutagenesis system, our laboratory recently identified candidate tumor promoting genes in transgenic mice that develop OS, including the potential tumor suppressor SRGAP2 involved in regulating actin dynamics. We hypothesized that loss of SRGAP2 would increase the metastatic potential of OS cell lines in vitro and in vivo, whereas gain would decrease metastatic potential. Gene expression was amplified with a stably integrated, tetracycline-inducible over expression vector containing SRGAP2 cDNA and gene silencing was accomplished with a clustered regularly interspaced short palindromic repeat (CRISPR) targeting the gene. Studies were conducted in the well-characterized murine osteosarcoma cell lines K12 and its more aggressive derivative K7M2 and human osteosarcoma cell lines HOS and its more aggressive derivative 143B. Compared to luciferase-controls, overexpression and knockout of SRGAP2 had no effect on cell proliferation in K12, K7M2, HOS, and 143B (n = 4). A reduction of soft agar colony formation was observed in knockout cell lines but not in cell lines with SRGAP2 overexpression for K12 and 143B (n = 3); HOS cell lines did not form colonies in soft agar. Interestingly, overexpression of SRGAP2 decreased wound-healing closure in 143B and K12 lines, but increased closure time in HOS (n = 4). Tail vein injections in NRG mice are currently being investigated. Overexpression of SRGAP2 in K12 decreased development of macrometastases in lungs, whereas knockout of SRGAP2 eliminated gross detection of lung masses (n = 3). Future studies include imaging fixed cells to determine if SRGAP2 knockout and overexpression affects cell morphology and completing studies with K7M2 lines. These results suggest that SRGAP2 has a biphasic phenotype in osteosarcoma cell lines, with an optima in expression for efficient metastasis. It's dual role of deforming cell membranes independent of its ability to activate Rac GTPases and alter actin dynamics may be responsible for the observed results. Citation Format: Tracy Marko, Ghaidan Shamsan, David Largaespada. SRGAP2 expression levels may induce a biphasic metastatic phenotype in osteosarcoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1621.

Fasting and Systemic Insulin Signaling Regulate Phosphorylation of Brain Proteins That Modulate Cell Morphology and Link to Neurological Disorders

Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser858 of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease.

A single PXXP motif in the C-terminal region of srGAP3 mediates binding to multiple SH3 domains

The Slit-Robo GTPase-activating protein 3 (srGAP3) has been implicated in different critical aspects of neuronal development. These findings have mainly been based on the characterisation of the three conserved globular N-terminal domains, while the function of the C-terminal region (CTR) is still unknown. We show that this predicted unstructured region acts as an adaptor by binding to the endocytic proteins Amphiphysin, Endophilin-A2, Endophilin-A1, as well as the Ras signalling protein Grb2. All these interactions depend on a single proline-rich motif in the CTR and the Src-homology 3 domains of the binding partners. Via these interactions srGAP3 could link receptor signalling events to the endocytic machinery.

Differential expression of microRNAs in dorsal root ganglia after sciatic nerve injury.

This study investigated the possible involvement of microRNAs in the regulation of genes that participate in peripheral neural regeneration. A microRNA microarray analysis was conducted and 23 microRNAs were identified whose expression was significantly changed in rat dorsal root ganglia after sciatic nerve transection. The expression of one of the downregulated microRNAs, microRNA-214, was validated using quantitative reverse transcriptase-PCR. MicroRNA-214 was predicted to target the 3′-untranslated region of Slit-Robo GTPase-activating protein 3. In situ hybridization verified that microRNA-214 was located in the cytoplasm of dorsal root ganglia primary neurons and was downregulated following sciatic nerve transection. Moreover, a combination of in situ hybridization and immunohistochemistry revealed that microRNA-214 and Slit-Robo GTPase-activating protein 3 were co-localized in dorsal root ganglion primary neurons. Western blot analysis suggested that Slit-Robo GTPase-activating protein 3 was upregulated in dorsal root ganglion neurons after sciatic nerve transection. These data demonstrate that microRNA-214 is located and differentially expressed in dorsal root ganglion primary neurons and may participate in regulating the gene expression of Slit-Robo GTPase-activating protein 3 after sciatic nerve transection.

The F-BAR domains from srGAP1, srGAP2, and srGAP3 differentially regulate membrane deformation

Coordination of membrane deformation and cytoskeletal dynamics lies at the heart of many biological processes critical for cell polarity, motility and morphogenesis. We have recently shown that Slit-Robo GTPase-activating protein 2 (srGAP2) regulates neuronal morphogenesis through the ability of its F-BAR domain to regulate membrane deformation and induce filopodia formation. Here, we demonstrate that the F-BAR domains of two closely related family members, srGAP1 and srGAP3 [designated F-BAR(1) and F-BAR(3), respectively] display significantly different membrane deformation properties in non-neuronal COS7 cells and in cortical neurons. F-BAR(3) induces filopodia in both cell types, though less potently than F-BAR(2), whereas F-BAR(1) prevents filopodia formation in cortical neurons and reduces plasma membrane dynamics. These three F-BAR domains can heterodimerize, and they act synergistically towards filopodia induction in COS7 cells. As measured by fluorescence recovery after photobleaching, F-BAR(2) displays faster molecular dynamics than F-BAR(3) and F-BAR(1) at the plasma membrane, which correlates well with its increased potency to induce filopodia. We also show that the molecular dynamic properties of F-BAR(2) at the membrane are partially dependent on F-Actin. Interestingly, acute phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] depletion in cells does not interfere with plasma membrane localization of F-BAR(2), which is compatible with our result showing that F-BAR(2) binds to a broad range of negatively-charged phospholipids present at the plasma membrane, including phosphatidylserine (PtdSer). Overall, our results provide novel insights into the functional diversity of the membrane deformation properties of this subclass of F-BAR-domains required for cell morphogenesis.

Slit2 Regulates Attractive Eosinophil and Repulsive Neutrophil Chemotaxis through Differential srGAP1 Expression during Lung Inflammation

Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However, the molecular mechanisms governing directional chemotaxis of leukocytes remain poorly understood. The Slit family of guidance cues has been implicated for inhibition of leuocyte migration. We report that Clara cells in the bronchial epithelium secreted Slit2, whereas eosinophils and neutrophils expressed its cell-surface receptor, Robo1. Compared to neutrophils, eosinophils exhibited a significantly lower level of Slit-Robo GTPase-activating protein 1 (srGAP1), leading to activation of Cdc42, recruitment of PI3K to Robo1, enhancment of eotaxin-induced eosinophil chemotaxis, and exaggeration of allergic airway inflammation. Notably, OVA sensitization elicited a Slit2 gradient at so-called bronchus-alveoli axis, with a higher level of Slit2 in the bronchial epithelium and a lower level in the alveolar tissue. Aerosol administration of rSlit2 accelerated eosinophil infiltration, whereas i.v. administered Slit2 reduced eosinophil deposition. In contrast, Slit2 inactivated Cdc42 and suppressed stromal cell-derived factor-1α-induced chemotaxis of neutrophils for inhibiting endotoxin-induced lung inflammation, which were reversed by blockade of srGAP1 binding to Robo1. These results indicate that the newly identified Slit2 gradient at the bronchus-alveoli axis induces attractive PI3K signaling in eosinophils and repulsive srGAP1 signaling in neutrophils through differential srGAP1 expression during lung inflammation.

Structural Basis of Robo Proline-rich Motif Recognition by the srGAP1 Src Homology 3 Domain in the Slit-Robo Signaling Pathway

The Slit-Robo (sr) GTPase-activating protein (GAPs) are important components in the intracellular pathway mediating Slit-Robo signaling in axon guidance and cell migration. We report the first crystal structure of the srGAP1 SH3 domain at 1.8-A resolution. The unusual side chain conformation of the conserved Phe-13 in the P1 pocket renders the ligand binding pocket shallow and narrow, which contributes toward the low binding affinity. Moreover, the opposing electrostatic charge and the hydrophobic properties of the P3 specificity pocket are consistent with the observed binding characteristics of the srGAP1 SH3 domain to its ligand. Surface plasmon resonance experiments indicate that the srGAP1 SH3 domain interacts with its natural ligand inaCtoN orientation. The srGAP1 SH3 domain can bind to both the CC2 and CC3 motifs in vitro. The N-terminal two acidic residues in the CC3 motif recognition site are necessary for srGAP1 SH3 domain binding. A longer CC3 peptide (CC3-FL) binds with greater affinity than its shorter counterpart, suggesting that the residues surrounding the proline-rich core are important for protein-peptide interactions. Our study reveals previously unknown properties of the srGAP-Robo interaction. Our data provide a structural basis for the srGAP-Robo interaction, consistent with the role of the Robo intracellular domain in interacting with other downstream signaling molecules and mediating versatile and dynamic responses to axon guidance and cell migration cues.

Changes in mRNA of Slit–Robo GTPase-activating protein 2 following facial nerve transection

Complex processes following peripheral nerve injury integrate a number of various external cues and their intracellular responses resulting in the cytoskeletal remodeling. One of these cues, Slit protein, plays an important role in neuronal migration and axonal guidance through the interaction with Roundabout (Robo) receptor. It was reported that the signal from Robo is transmitted to a specific family of GTPase-activating proteins (GAPs) named Slit–Robo GAPs. The Slit–Robo GAPs (srGAPs) further transmit the signal to the actin cytoskeleton controlling Rho GTPases and thus provide a direct link between Slit–Robo signaling and actin cytoskeleton. We examined the effects of facial nerve transection on srGAP2 mRNA expression in the facial nerve nuclei by in situ hybridization. SrGAP2 mRNA was initially expressed, and its expression increased from 3 to 28 days after transection, with the peak at the seventh day after axotomy. The upregulation was found mostly in the neuronal cells and only to a small extent in the glial cells. Our results suggest that srGAP2, as a part of Slit–Robo pathway, plays an important role in the axonal regeneration after axotomy.