p250GAP, a novel brain-enriched GTPase-activating protein for Rho family GTPases, is involved in the N-methyl-d-aspartate receptor signaling

Abstract

N-methyl-d-aspartate (NMDA) receptors regulate structural plasticity by modulating actin organization within dendritic spines. Herein, we report identification and characterization of p250GAP, a novel GTPase-activating protein for Rho family proteins that interacts with the GluRepsilon2 (NR2B) subunit of NMDA receptors in vivo. The p250GAP mRNA was enriched in brain, with high expression in cortex, corpus striatum, hippocampus, and thalamus. Within neurons, p250GAP was highly concentrated in the postsynaptic density and colocalized with the GluRepsilon2 (NR2B) subunit of NMDA receptors and with postsynaptic density-95. p250GAP promoted GTP hydrolysis of Cdc42 and RhoA in vitro and in vivo. When overexpressed in neuroblastoma cells, p250GAP suppressed the activities of Rho family proteins, which resulted in alteration of neurite outgrowth. Finally, NMDA receptor stimulation led to dephosphorylation and redistribution of p250GAP in hippocampal slices. Together, p250GAP is likely to be involved in NMDA receptor activity-dependent actin reorganization in dendritic spines.

Figure 1.

Sequence of human p250GAP. (A) Protein sequence and domain structure of p250GAP. The RhoGAP domain is boxed, proline-rich sequences are double underlined, and the cDNA sequence obtained by two-hybrid screening (amino acids 1056–1738) is underlined. (B) Homology between RhoGAP domains of p250GAP, p190RhoGAP, and p50RhoGAP. Identical residues are highlighted in black, and the predicted critical residues for GAP activity are marked with asterisks.

Figure 2.

Interaction between p250GAP and GluRε2 (NR2B). (A) Interaction between p250GAP and GluRε2 (NR2B) in HEK293T cells. HEK293T cells were transfected with combinations of expression plasmids for FLAG-tagged p250GAP and GluRε2 (NR2B). Two days later, the cells were lysed and anti-FLAG immunoprecipitates (IP, a) and cell lysates (b) were subjected to immunoblotting (IB) with anti-GluRε2 (NR2B) mAb (top) and anti-FLAG mAb (bottom). (B) Mapping of the regions in p250GAP required for interaction with GluRε2 (NR2B). HEK293T cells were transfected with the expression plasmid for GluRε2 (NR2B). The lysates were incubated with 1 μg of the GST or GST-fusion proteins (GST1–5) immobilized on glutathione-Sepharose. After centrifugation, the beads were washed and subjected to immunoblotting with anti-GluRε2 (NR2B) mAb. (C) Interaction between p250GAP and GluRε2 (NR2B) in mouse brain. Brain lysates were immunoprecipitated with preimmune sera, anti-p250GAP antibodies, or anti-p250GAP antibodies preabsorbed with immunogen. The immunoprecipitates and input were immunoblotted (IB) with anti-GluRε2 (NR2B) (top) mAb and anti-p250GAP antibodies (bottom).

Figure 3.

Expression pattern of p250GAP. (A) Northern blot analysis. A blot with RNAs from adult mouse tissues was probed with a fragment of mouse p250GAP cDNA. The amounts and quality of RNAs were verified by EtBr staining. (B–D) In situ hybridization of p250GAP mRNA in mouse tissue sections. A parasagittal section of adult brain (B), a coronal section of adult brain (C), or a sagittal section of E18.5 embryo (D) was hybridized with an α-³⁵S-UTP–labeled mouse p250GAP cRNA probe.

Figure 4.

Enrichment of p250GAP in postsynaptic density. (A) Localization of p250GAP at punctate structures arrayed along dendrites. Hippocampal neurons were dissociated at embryonic day 17.5, cultured for 20 d, and stained with anti-p250GAP antibodies (green) and anti-MAP2 mAb (red). Bars, 25 μm (top). Lower panels show higher magnification images. Bar, 10 μm (bottom). (B) Colocalization of p250GAP with GluRε2 (NR2B). The hippocampal neurons were stained with antibodies against p250GAP (green) and GluRε2 (NR2B) (red). Bars, 25 μm (top). Lower panels show higher magnification images. Arrows indicate colocalization of p250GAP with GluRε2 (NR2B). Bars, 5 μm. (C) p250GAP in isolated PSD fraction. A sample of 50 μg (left) or 5 μg (right) of mouse forebrain lysates and synaptosomes, and the PSD extracted once with Triton X-100 (5 μg), were subjected to immunoblotting (IB) with antibodies against p250GAP, GluRε2 (NR2B), PSD-95, Synaptophysin (a presynaptic marker), RhoA, Cdc42, and Rac1.

Figure 5.

Characterization of GAP activity of p250GAP. (A) GAP activity of recombinant GAP domain of p250GAP. Equal concentrations (10 nM) of recombinant GST (circle), GST-GAP domain of wild-type p250GAP (square), and R58I mutant of p250GAP (diamond) were added to in vitro GAP assay with 20 nM [γ-³²P]GTP-loaded RhoA, Cdc42, or Rac1. (B) GAP activity of p250GAP in HEK293T cells was analyzed by Rho GTPase effector pulldown assays. HEK293T cells were transiently transfected with expression plasmids for Myc-tagged RhoA, Cdc42, or Rac1 together with or without (-) FLAG-tagged wild-type (WT) or R58I mutant (RI) of p250GAP. Lysates of the cells were pulled down by GST-CRIB (for Cdc42 and Rac1) or GST-RBD (for RhoA) immobilized on glutathione-Sepharose. Levels of GTP-loaded RhoA, Cdc42, and Rac1 were analyzed by immunoblotting with anti-Myc mAb (a). Whole-cell lysates were verified by immunoblotting with anti-Myc mAb (b, top) and anti-p250GAP antibodies (b, bottom). (C) GAP activity of endogenous p250GAP in mouse brain. Lysates of HEK293T cells expressing Myc-tagged RhoA, Cdc42, or Rac1 were incubated with (+) or without (-) p250GAP immunoprecipitates from brain lysates (IP). GTP-loaded RhoA, Cdc42, or Rac1 in the lysates was then collected by GST-CRIB or GST-RBD and detected by anti-Myc mAb (top). p250GAP immunoprecipitates were verified by anti-p250GAP antibodies (bottom). All experiments were repeated more than three times.

Figure 6.

Regulation of neuritogenesis in Neuro-2A cells by overexpression of p250GAP. (A) Suppression of neuritogenesis after serum withdrawal by overexpression of wild-type p250GAP. Neuro-2A cells were transiently transfected with expression plasmids for mock (Aa and antibody), Myc-tagged N17Cdc42 inactive mutant (Ac and Ad), FLAG-tagged wild-type (WT) p250GAP (Ae and Af), or R58I mutant (RI) of p250GAP (Ag and Ah). (B) Induction of neuritogenesis by overexpression of wild-type p250GAP in the presence of serum. Neuro-2A cells were transiently transfected with an expression plasmid for mock (Bi and Bj), Myc-tagged N19RhoA inactive mutant (Bk and Bl), FLAG-tagged wild-type (WT) p250GAP (Bm and Bn), and R58I mutant (RI) of p250GAP (Bo and Bp). (A and B) The cells were fixed and viewed using a Nomarski microscope (antibody, Ad, Af, Ah, Bj, Bl, Bn, and Bp). Exogenous expression of the proteins was confirmed by anti-Myc mAb or anti-FLAG antibodies staining (Aa, Ac, Ae, Ag, Bi, Bk, Bm, and Bo). Bar, 20 μm. (C and D) The neurite length of 20 cells was calculated using TI workbench software (kindly provided by T. Inoue, University of Tokyo). Results were shown as the mean from three independent experiments. Values are mean ± SEM, ^*p < 0.01, mock transfected versus N17Cdc42, N19RhoA, wild-type (WT) p250GAP, or RI mutant of p250GAP (Student's t test).

Figure 7.

Redistribution and dephosphorylation of p250GAP after NMDA receptor stimulation in hippocampal slices. (A and C) Redistribution of p250GAP after NMDA receptor stimulation. Shown are representative blots of TNE buffer-soluble (A) or whole (C) lysates with antibodies against p250GAP, PSD-95, phospho-ERK, and ERK. The samples were prepared from mouse hippocampal slices that were mock stimulated (lane 3 in A and lane 1 in C), or stimulated with 50 μM NMDA for 5 min, in the absence (lane 4 in A and lane2 in C) or presence (lane 5 in A and lane 3 in C) of 100 μM DL-APV. In parallel experiments, the lysates of mock-stimulated slices were treated with (lane 2 in A) or without (lane 1 in A) BAP and were subjected to immunoblotting with antibodies against p250GAP, PSD-95, phospho-ERK, and ERK. (B and D) Quantification of the p250GAP level. Results were shown as the mean from three independent experiments. Values are mean ± SEM, ^*p < 0.0001, mock stimulated versus NMDA stimulation (Student's t test). (E) Dephosphorylation of p250GAP after NMDA receptor stimulation. The anti-p250GAP immunoprecipitates from mock-stimulated or NMDA stimulated slices were blotted with anti-pY mAb (RC20) (top) and then with anti-p250GAP antibodies (bottom). (F) Quantification of p250GAP phosphorylation level. The Student's t test value (0.39 ± 0.05: mean ± SEM, ^*p < 0.01) was from three independent experiments.