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Synapse formation is regulated by the signaling adaptor GIT1.

Zhang H, Webb DJ, Asmussen H, Horwitz AF - J. Cell Biol. (2003)

Bottom Line: Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines.The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac.These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908-0732, USA.

ABSTRACT
Dendritic spines in the central nervous system undergo rapid actin-based shape changes, making actin regulators potential modulators of spine morphology and synapse formation. Although several potential regulators and effectors for actin organization have been identified, the mechanisms by which these molecules assemble and localize are not understood. Here we show that the G protein-coupled receptor kinase-interacting protein (GIT)1 serves such a function by targeting actin regulators and locally modulating Rac activity at synapses. In cultured hippocampal neurons, GIT1 is enriched in both pre- and postsynaptic terminals and targeted to these sites by a novel domain. Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines. The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac. In addition, constitutively active Rac shows a phenotype similar to the GIT1 mutant, whereas dominant-negative Rac inhibits the dendritic protrusion formation induced by mislocalized GIT1. These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.

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Synaptic targeting of GIT1. (A) Schematic diagram of the full-length and deletion constructs of GIT1 (all GFP-tagged at the NH2 terminus except N-SLD and C-SLD, which are FLAG-tagged at the NH2 terminus). The indicated domains are as follows: ARF-GAP domain (ARF-GAP), ankyrin repeats (Ank), Spa2 homology domain 1(SHD1), and paxillin binding site (paxillin). The fusion proteins with synaptic localization are indicated with “+”. N-SLD shows weak localization to synapses which is indicated with “+/−”. (B) GIT1 fusion proteins that show specific localization to synapses. Hippocampal neurons were transfected with the various GIT1 fusion proteins (left column) and stained for synaptic markers (middle column). GIT1Δc and CD-GIT1 were coimmunostained for SV2. CDΔAnk was coimmunostained for PSD-95. CDΔAS/SLD was coimmunostained for synapsin1. Overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm. (C) FLAG–N-SLD– expressing neurons were coimmunostained for FLAG and synapsin1. N-SLD shows partial localization to synapses (arrows). The overlaid picture is shown in the right column. N-SLD was pseudocolored green, and synapsin1 was pseudocolored red. Bar, 2 μm. (D) GIT1 fusion proteins that fail to localize to synapses. Hippocampal neurons expressing the GFP-tagged GIT1 deletion constructs (left column) were stained for SV2 (middle column). The GIT1 constructs lacking SLD (cGIT1, GIT1ΔCD, nGIT1, and GIT1ΔSLD) do not show synaptic localization. Deletion of the NH2-terminal 32 aa of SLD (SLDΔ32) dramatically reduces localization to the synapse. For localization of C-SLD, neurons expressing FLAG–C-SLD were coimmunostained for FLAG and synapsin1. C-SLD shows a diffuse labeling pattern. The overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm.
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fig2: Synaptic targeting of GIT1. (A) Schematic diagram of the full-length and deletion constructs of GIT1 (all GFP-tagged at the NH2 terminus except N-SLD and C-SLD, which are FLAG-tagged at the NH2 terminus). The indicated domains are as follows: ARF-GAP domain (ARF-GAP), ankyrin repeats (Ank), Spa2 homology domain 1(SHD1), and paxillin binding site (paxillin). The fusion proteins with synaptic localization are indicated with “+”. N-SLD shows weak localization to synapses which is indicated with “+/−”. (B) GIT1 fusion proteins that show specific localization to synapses. Hippocampal neurons were transfected with the various GIT1 fusion proteins (left column) and stained for synaptic markers (middle column). GIT1Δc and CD-GIT1 were coimmunostained for SV2. CDΔAnk was coimmunostained for PSD-95. CDΔAS/SLD was coimmunostained for synapsin1. Overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm. (C) FLAG–N-SLD– expressing neurons were coimmunostained for FLAG and synapsin1. N-SLD shows partial localization to synapses (arrows). The overlaid picture is shown in the right column. N-SLD was pseudocolored green, and synapsin1 was pseudocolored red. Bar, 2 μm. (D) GIT1 fusion proteins that fail to localize to synapses. Hippocampal neurons expressing the GFP-tagged GIT1 deletion constructs (left column) were stained for SV2 (middle column). The GIT1 constructs lacking SLD (cGIT1, GIT1ΔCD, nGIT1, and GIT1ΔSLD) do not show synaptic localization. Deletion of the NH2-terminal 32 aa of SLD (SLDΔ32) dramatically reduces localization to the synapse. For localization of C-SLD, neurons expressing FLAG–C-SLD were coimmunostained for FLAG and synapsin1. C-SLD shows a diffuse labeling pattern. The overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm.

Mentions: To identify the region that targets GIT1 to synapses, we expressed various domains of GIT1 as GFP fusion proteins in hippocampal neurons (Fig. 2 A). Their synaptic localization was assayed by GFP fluorescence and coimmunostaining with synaptic markers. We began by examining the localization of cGIT1, since it contains the binding site for paxillin and is responsible for the adhesion targeting of GIT1 in fibroblasts (Manabe et al., 2002). However, in the neurons, cGIT1 did not concentrate in synapses but distributed diffusely throughout the cytoplasm of the soma and processes (Fig. 2 D). Thus, the domain that targets GIT1 to synapses is different than the adhesion localization domain in fibroblasts. As expected, GIT1Δc, which lacks the COOH-terminal paxillin binding domain, localized in synapses (Fig. 2 B). To further localize the targeting domain within GIT1Δc, we first examined whether the NH2-terminal ARF-GAP domain is necessary for synaptic targeting. A central domain deletion mutant, GIT1ΔCD, which contains the ARF-GAP domain and the paxillin binding domain, did not accumulate in synapses (Fig. 2 D). Another mutant that comprises the central domain, CD-GIT1, localized in synapses (Fig. 2 B). Therefore, neither the ARF-GAP nor the paxillin binding domain is necessary for synaptic targeting, pointing to the central domain of GIT1 as the targeting domain.


Synapse formation is regulated by the signaling adaptor GIT1.

Zhang H, Webb DJ, Asmussen H, Horwitz AF - J. Cell Biol. (2003)

Synaptic targeting of GIT1. (A) Schematic diagram of the full-length and deletion constructs of GIT1 (all GFP-tagged at the NH2 terminus except N-SLD and C-SLD, which are FLAG-tagged at the NH2 terminus). The indicated domains are as follows: ARF-GAP domain (ARF-GAP), ankyrin repeats (Ank), Spa2 homology domain 1(SHD1), and paxillin binding site (paxillin). The fusion proteins with synaptic localization are indicated with “+”. N-SLD shows weak localization to synapses which is indicated with “+/−”. (B) GIT1 fusion proteins that show specific localization to synapses. Hippocampal neurons were transfected with the various GIT1 fusion proteins (left column) and stained for synaptic markers (middle column). GIT1Δc and CD-GIT1 were coimmunostained for SV2. CDΔAnk was coimmunostained for PSD-95. CDΔAS/SLD was coimmunostained for synapsin1. Overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm. (C) FLAG–N-SLD– expressing neurons were coimmunostained for FLAG and synapsin1. N-SLD shows partial localization to synapses (arrows). The overlaid picture is shown in the right column. N-SLD was pseudocolored green, and synapsin1 was pseudocolored red. Bar, 2 μm. (D) GIT1 fusion proteins that fail to localize to synapses. Hippocampal neurons expressing the GFP-tagged GIT1 deletion constructs (left column) were stained for SV2 (middle column). The GIT1 constructs lacking SLD (cGIT1, GIT1ΔCD, nGIT1, and GIT1ΔSLD) do not show synaptic localization. Deletion of the NH2-terminal 32 aa of SLD (SLDΔ32) dramatically reduces localization to the synapse. For localization of C-SLD, neurons expressing FLAG–C-SLD were coimmunostained for FLAG and synapsin1. C-SLD shows a diffuse labeling pattern. The overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm.
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fig2: Synaptic targeting of GIT1. (A) Schematic diagram of the full-length and deletion constructs of GIT1 (all GFP-tagged at the NH2 terminus except N-SLD and C-SLD, which are FLAG-tagged at the NH2 terminus). The indicated domains are as follows: ARF-GAP domain (ARF-GAP), ankyrin repeats (Ank), Spa2 homology domain 1(SHD1), and paxillin binding site (paxillin). The fusion proteins with synaptic localization are indicated with “+”. N-SLD shows weak localization to synapses which is indicated with “+/−”. (B) GIT1 fusion proteins that show specific localization to synapses. Hippocampal neurons were transfected with the various GIT1 fusion proteins (left column) and stained for synaptic markers (middle column). GIT1Δc and CD-GIT1 were coimmunostained for SV2. CDΔAnk was coimmunostained for PSD-95. CDΔAS/SLD was coimmunostained for synapsin1. Overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm. (C) FLAG–N-SLD– expressing neurons were coimmunostained for FLAG and synapsin1. N-SLD shows partial localization to synapses (arrows). The overlaid picture is shown in the right column. N-SLD was pseudocolored green, and synapsin1 was pseudocolored red. Bar, 2 μm. (D) GIT1 fusion proteins that fail to localize to synapses. Hippocampal neurons expressing the GFP-tagged GIT1 deletion constructs (left column) were stained for SV2 (middle column). The GIT1 constructs lacking SLD (cGIT1, GIT1ΔCD, nGIT1, and GIT1ΔSLD) do not show synaptic localization. Deletion of the NH2-terminal 32 aa of SLD (SLDΔ32) dramatically reduces localization to the synapse. For localization of C-SLD, neurons expressing FLAG–C-SLD were coimmunostained for FLAG and synapsin1. C-SLD shows a diffuse labeling pattern. The overlays are shown in the right column. The GIT1 constructs were pseudocolored green, and the synaptic markers were pseudocolored red. Bar, 2 μm.
Mentions: To identify the region that targets GIT1 to synapses, we expressed various domains of GIT1 as GFP fusion proteins in hippocampal neurons (Fig. 2 A). Their synaptic localization was assayed by GFP fluorescence and coimmunostaining with synaptic markers. We began by examining the localization of cGIT1, since it contains the binding site for paxillin and is responsible for the adhesion targeting of GIT1 in fibroblasts (Manabe et al., 2002). However, in the neurons, cGIT1 did not concentrate in synapses but distributed diffusely throughout the cytoplasm of the soma and processes (Fig. 2 D). Thus, the domain that targets GIT1 to synapses is different than the adhesion localization domain in fibroblasts. As expected, GIT1Δc, which lacks the COOH-terminal paxillin binding domain, localized in synapses (Fig. 2 B). To further localize the targeting domain within GIT1Δc, we first examined whether the NH2-terminal ARF-GAP domain is necessary for synaptic targeting. A central domain deletion mutant, GIT1ΔCD, which contains the ARF-GAP domain and the paxillin binding domain, did not accumulate in synapses (Fig. 2 D). Another mutant that comprises the central domain, CD-GIT1, localized in synapses (Fig. 2 B). Therefore, neither the ARF-GAP nor the paxillin binding domain is necessary for synaptic targeting, pointing to the central domain of GIT1 as the targeting domain.

Bottom Line: Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines.The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac.These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia, Charlottesville, VA 22908-0732, USA.

ABSTRACT
Dendritic spines in the central nervous system undergo rapid actin-based shape changes, making actin regulators potential modulators of spine morphology and synapse formation. Although several potential regulators and effectors for actin organization have been identified, the mechanisms by which these molecules assemble and localize are not understood. Here we show that the G protein-coupled receptor kinase-interacting protein (GIT)1 serves such a function by targeting actin regulators and locally modulating Rac activity at synapses. In cultured hippocampal neurons, GIT1 is enriched in both pre- and postsynaptic terminals and targeted to these sites by a novel domain. Disruption of the synaptic localization of GIT1 by a dominant-negative mutant results in numerous dendritic protrusions and a significant decrease in the number of synapses and normal mushroom-shaped spines. The phenotype results from mislocalized GIT1 and its binding partner PIX, an exchange factor for Rac. In addition, constitutively active Rac shows a phenotype similar to the GIT1 mutant, whereas dominant-negative Rac inhibits the dendritic protrusion formation induced by mislocalized GIT1. These results demonstrate a novel function for GIT1 as a key regulator of spine morphology and synapse formation and point to a potential mechanism by which mutations in Rho family signaling leads to decreased neuronal connectivity and cognitive defects in nonsyndromic mental retardation.

Show MeSH
Related in: MedlinePlus