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. 2013 Jul 31;33(31):12586-98.
doi: 10.1523/JNEUROSCI.0341-13.2013.

AGAP3 and Arf6 Regulate Trafficking of AMPA Receptors and Synaptic Plasticity

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Free PMC article

AGAP3 and Arf6 Regulate Trafficking of AMPA Receptors and Synaptic Plasticity

Yuko Oku et al. J Neurosci. .
Free PMC article

Abstract

During NMDA receptor-mediated long-term potentiation (LTP), synapses are strengthened by trafficking AMPA receptors to the synapse through a calcium-dependent kinase cascade following activation of NMDA receptors. This process results in a long-lasting increase in synaptic strength that is thought to be a cellular mechanism for learning and memory. Over the past 20 years, many signaling pathways have been shown to be involved in the induction and maintenance of LTP including the MAPK cascade. However, the crucial link between NMDA receptors and the signaling cascades involved in AMPA receptor trafficking during LTP remains elusive. In this study, we aimed to identify and characterize NMDA receptor signaling proteins that link NMDA receptor activation to downstream signaling pathways that lead to trafficking of AMPA receptors. We have identified a novel NMDA receptor interacting signaling protein, AGAP3. AGAP3 contains multiple signaling domains, a GTPase-like domain, a pleckstrin homology domain, and an ArfGAP domain, and exists as a component of the NMDA receptor complex. In addition, we found that AGAP3 regulates NMDA receptor-mediated Ras/ERK and Arf6 signaling pathways during chemically induced LTP in rat primary neuronal cultures. Finally, knocking down AGAP3 expression leads to occlusion of AMPA receptor trafficking during chemically induced LTP. Together, AGAP3 is an essential signaling component of the NMDA receptor complex that links NMDA receptor activation to AMPA receptor trafficking.

Figures

Figure 1.
Figure 1.
AGAP3 was identified as a SynGAP interactor. A, Diagram of interacting AGAP3 cDNA clones identified from a yeast 2-hybrid screen using SynGAP GAP domain as bait. The clones identified in the screen contain GLD in AGAP3 and CRAG. B, Immunoprecipitation (IP) with anti-myc antibody was performed from myc-SynGAP and HA-AGAP3-transfected HEK293T cells IB, immunoblot. C, In vitro GTPase activity assay using purified recombinant SynGAP RasGAP domain and AGAP3 GLD was performed. The graph plots the ratio of GDP/(GTP + GDP) 32P signal over time. Error bars are standard error of mean from three experiments. ▾, AGAP3 GLD with SynGAP's RasGAP domain; ○, AGAP3 GLD domain only; and ●, heat-inactivated AGAP3 GLD only. n = 3. D, AGAP3 was immunoprecipitated (IP) from cortical cultures with and without peptide antigen using anti-AGAP3 antibody. IB, immunoblot.
Figure 2.
Figure 2.
AGAP3 is found at postsynaptic sites in the adult brain. A, A diagram depicting AGAP3 splice variants and their domain structure. EST, expressed sequence tag found at NCBI database. B, AGAP3 and CRAG expression were detected in rat tissue homogenates using an antibody derived from the N terminus of AGAP3. C, AGAP3 and its splice variants were detected in subcellular fractionation of the adult rat brain. AGAP3-N, antibody derived from N terminus of AGAP3; AGAP3-C, antibody derived from C terminus of AGAP3. Fractions: S, supernatant; P, pellet; PSD, postsynaptic density. D, AGAP3 expression was detected in a developmental profile of rat brain homogenate using antibody derived against AGAP3 N terminus. E, Representative dendritic and spine image from Banker culture costained with indicated antibodies at 21 DIV. Scale bar, 5 μm. F, AGAP3 staining in hippocampal cultures at 21 DIV with transfection of Venus_shRNA#2 to knockdown AGAP3 expression. Scale bars: 50 and 10 μm.
Figure 3.
Figure 3.
AGAP3 is a component of the NMDA receptor signaling complex. A, NR2A- and NR2B-subunits were immunoprecipitated (IP) in the presence and absence of peptide antigen from brain. IB, immunoblot. B, Schematic of AGAP3 domain structure indicates the location of the target sequence of the AGAP3 shRNAs. C, Representative expression of AGAP3 with electroporation of shRNA constructs #1 and #2. AGAP3-N, antibody derived against AGAP3 N-terminus. D, Top, Phospho-ERK signal was detected in shRNA#1 and shRNA#2 electroporated cortical cultures under unstimulated, chemical LTP stimulated, and chemical LTP stimulated with APV conditions. Bottom, Quantification of Phospho-ERK2 signal normalized to unstimulated GFPshRNA control. One-way ANOVA, p = 3.71e–14, n = 6 (unstim), 12 (stim), 6 (APV). Gray, unstimulated; black, stimulated; white, APV; *p ≤ 0.05 after Tukey's HSD post hoc test. Error bars are standard error of mean. E, Top, Phospho-ERK signal detected after chemical LTP stimulation in SynGAP knock-out mice with and without AGAP3 knockdown. Bottom, Quantification of Phospho-ERK2 signal normalized to unstimulated vector control. One-way ANOVA, p = 6.0e–4, n = 4. Gray, unstimulated; black, stimulated; white, APV; *p ≤ 0.05 after Tukey's HSD post hoc test. Error bars are standard error of mean. F, Western blot of total protein expression with electroporation of shRNA#2 in primary rat cortical neurons.
Figure 4.
Figure 4.
AGAP3 regulates both Ras and Arf6 signaling pathways. A, Arf6 and Ras basal activity was detected using Active GTPase Pull-down kit (Thermo Scientific) with lentiviral expression of vector or shRNA#2 in cortical neurons. IB, immunoblot. B, Immunoprecipitation (IP) of Arf6 WT, Arf6 GTP-binding (Q67L), or GDP-binding (T44N) mutant was performed from HEK293T cells cotransfected with AGAP3. Asterisk indicates signal from Ig light chain. Arrow indicates position of Arf6 protein. IB, immunoblot. C, Active Ras and Arf6 were detected with electroporation of AGAP3 signaling mutants in cortical cells. Asterisk indicates nonspecific band present from addition of GST recombinant protein. Arrow indicates position of Ras protein. IB, immuno-blot. D, Quantification of basal Ras and Arf6 activity after overexpression of AGAP3 WT and signaling domain point mutants (mut). One-way ANOVA, p = 0.049, n = 6 (Ras); p = 0.042, n = 5 (Arf6); * p ≤ 0.05 after Tukey's HSD post hoc test. Error bars are standard error of mean.
Figure 5.
Figure 5.
AGAP3 regulates NMDA receptor-mediated Ras/ERK and Arf6 signaling pathways during chemically induced LTP. A, Active Ras and Arf6 was detected using Active GTPase Pull-down and Detection kit (Thermo Scientific) from cortical cultures in unstimulated, chemical LTP stimulated or stimulated with APV conditions. IB, immunoblot. B, Active Ras and Arf6 were detected after chemical LTP stimulation in Scrambled (Ctl.) or shRNA#2 (sh#2) electroporated cortical neurons with and without chemical LTP stimulation. Asterisk indicates nonspecific band present from addition of GST recombinant protein. Arrow indicates position of Ras protein. IB, immunoblot. C, Unstimulated and stimulated phospho-ERK2 signal after electroporation of AGAP3 shRNA#2 and/or AGAP3 mutant (mut) rescue constructs. IB, immunoblot. D, Quantification of phopho-ERK2 signals are normalized to GFPshRNA unstimulated control. One-way ANOVA, p = 0.0028, n = 3. Gray, unstimulated; black, stimulated. *p ≤ 0.05 after Tukey's HSD post hoc test compared with unstimulated GFPshRNA control. Error bars are standard error of mean.
Figure 6.
Figure 6.
AGAP3 regulates AMPA receptor trafficking through the ArfGAP domain. A, Representative images of GluA1 staining in hippocampal cultures with coexpression of AGAP3 shRNA#2 and AGAP3 rescue constructs that are insensitive to the shRNA knockdown. AGAP3 expression is rescued using either AGAP3 WT or signaling domain mutants (mut). Scale bars: 50 and 10 μm. B, Quantification of normalized surface/total GluA1. One-way ANOVA, p = 2.82e–14, dendritic regions = 138, 127, 137, 124, 121 from three independent culture sets. *p ≤ 0.05 after Tukey's HSD post hoc test compared with vector control. Error bars are standard error of mean. C, Quantification of NR2A surface staining in hippocampal cultures with coexpression of AGAP3 shRNA#2 and AGAP3 rescue constructs insensitive to the shRNA knockdown. AGAP3 expression is rescued using either AGAP3 WT or signaling domain mutants. One-way ANOVA, p = 0.062, dendritic regions = 75, 85, 85, 111, 90 from two independent culture sets. *p ≤ 0.05 after Tukey's HSD post hoc test compared with vector control. Error bars are standard error of mean.
Figure 7.
Figure 7.
AGAP3 and Arf6 together regulate trafficking of GluA1. A, Representative images of GluA1 in hippocampal staining after cotransfection of AGAP3 shRNA and Arf6 T44N (dominant-negative). Scale bars: 50 and 10 μm. B, Quantification of surface/total GluA1. One-way ANOVA, p = 2.2–16, dendritic regions = 128, 101, 85, 87 from two independent cultures sets. *p ≤ 0.05 after Tukey's HSD post hoc test compared with vector control. Error bars are standard error of mean.
Figure 8.
Figure 8.
AGAP3 regulates trafficking of GluA1 during chemical LTP. A, Representative images of GluA1 staining with and without chemical LTP in hippocampal cultures cotransfected with AGAP3 shRNA#2 and AGAP3 rescue constructs insensitive to the knockdown by shRNA#2. AGAP3 expression is rescued using either AGAP3 WT or the signaling domain mutants (mut). Scale bar: 50 and 10 μm. B, Quantification of surface/total GluA1 with and without chemical LTP stimulation. One-way ANOVA, p = 2.2e–16, dendritic regions = 242, 313, 199, 193, 238, 198, 216, 198, 187, 218 from four independent culture sets. Significant *p ≤ 0.05 after Tukey's HSD post hoc test is indicated compared with vector control. Error bars are standard error of mean.

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