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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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GIT1 is expressed in cultured hippocampal neurons and enriched in synapses. (A) Western blot of a lysate from day 10 cultured hippocampal neurons. The blot was probed with a GIT1 antibody. A specific band at ∼95 kD confirms the presence of the GIT1 protein in these neurons. (B) Hippocampal neurons at 2–3 wk in culture were double immunostained for endogenous GIT1 (left column) and various synaptic proteins (right column). GIT1 colocalizes with the presynaptic marker SV2 (top). GIT1 shows colocalization with PSD-95 in some puncta (arrowheads), but some GIT1 puncta, especially those on the cell body, do not overlap with PSD-95 (middle, arrows). These puncta show colocalization with the inhibitory synapse marker GAD-6 (bottom). Enlargements of the boxed regions are shown in insets at the bottom right of each panel. Bar, 20 μm. (C) Hippocampal neurons were transfected with either GFP-GIT1 (top) or GIT1-FLAG (bottom) and immunostained for the presynaptic marker synapsin1 at 3 wk in culture. Both GFP-GIT1 and GIT1-FLAG colocalize with synapsin1 in dendritic spines and shafts (arrows). Bar, 20 μm. (D) Hippocampal neurons were transfected with GFP-GIT1 and immunostained for the appropriate synaptic markers at 2–3 wk in culture. The GIT1 clusters on the dendrites (Dendritic) almost completely merge with the postsynaptic marker PSD-95 and are in close apposition to the presynaptic marker synapsin1 (Overlay). The GIT1 clusters on the axons (Axonal) completely merge with the presynaptic marker SV2 and are in close apposition to the postsynaptic marker PSD-95 (Overlay). Note the colocalization of GIT1 clusters with the synaptic markers (arrowheads). Enlargements of individual synapses are shown in the right column. GFP-GIT1 is pseudocolored green, and the synaptic markers are pseudocolored red. Bar, 2 μm.
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fig1: GIT1 is expressed in cultured hippocampal neurons and enriched in synapses. (A) Western blot of a lysate from day 10 cultured hippocampal neurons. The blot was probed with a GIT1 antibody. A specific band at ∼95 kD confirms the presence of the GIT1 protein in these neurons. (B) Hippocampal neurons at 2–3 wk in culture were double immunostained for endogenous GIT1 (left column) and various synaptic proteins (right column). GIT1 colocalizes with the presynaptic marker SV2 (top). GIT1 shows colocalization with PSD-95 in some puncta (arrowheads), but some GIT1 puncta, especially those on the cell body, do not overlap with PSD-95 (middle, arrows). These puncta show colocalization with the inhibitory synapse marker GAD-6 (bottom). Enlargements of the boxed regions are shown in insets at the bottom right of each panel. Bar, 20 μm. (C) Hippocampal neurons were transfected with either GFP-GIT1 (top) or GIT1-FLAG (bottom) and immunostained for the presynaptic marker synapsin1 at 3 wk in culture. Both GFP-GIT1 and GIT1-FLAG colocalize with synapsin1 in dendritic spines and shafts (arrows). Bar, 20 μm. (D) Hippocampal neurons were transfected with GFP-GIT1 and immunostained for the appropriate synaptic markers at 2–3 wk in culture. The GIT1 clusters on the dendrites (Dendritic) almost completely merge with the postsynaptic marker PSD-95 and are in close apposition to the presynaptic marker synapsin1 (Overlay). The GIT1 clusters on the axons (Axonal) completely merge with the presynaptic marker SV2 and are in close apposition to the postsynaptic marker PSD-95 (Overlay). Note the colocalization of GIT1 clusters with the synaptic markers (arrowheads). Enlargements of individual synapses are shown in the right column. GFP-GIT1 is pseudocolored green, and the synaptic markers are pseudocolored red. Bar, 2 μm.

Mentions: Previous studies showed that GIT1 is highly expressed in the brain both at the mRNA and protein levels (Premont et al., 1998; Zhao et al., 2000). To determine whether GIT1 is expressed in neurons, we performed RT-PCR with hippocampal total RNA using GIT1-specific primers. The sequence of the PCR product was identical to the previously described rat GIT1 sequence, showing the presence of the GIT1 mRNA in these neurons. To confirm the protein expression of GIT1, we subjected a lysate from hippocampal neurons to immunoblot analysis using a GIT1 antibody. We detected a specific band at ∼95 kD, which is the expected mobility of GIT1 (Fig. 1 A). This confirms the presence of GIT1 in the neurons at the protein level since our antibody is specific for GIT1; however, we cannot exclude the possibility that other GIT family members may also be present.


Synapse formation is regulated by the signaling adaptor GIT1.

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

GIT1 is expressed in cultured hippocampal neurons and enriched in synapses. (A) Western blot of a lysate from day 10 cultured hippocampal neurons. The blot was probed with a GIT1 antibody. A specific band at ∼95 kD confirms the presence of the GIT1 protein in these neurons. (B) Hippocampal neurons at 2–3 wk in culture were double immunostained for endogenous GIT1 (left column) and various synaptic proteins (right column). GIT1 colocalizes with the presynaptic marker SV2 (top). GIT1 shows colocalization with PSD-95 in some puncta (arrowheads), but some GIT1 puncta, especially those on the cell body, do not overlap with PSD-95 (middle, arrows). These puncta show colocalization with the inhibitory synapse marker GAD-6 (bottom). Enlargements of the boxed regions are shown in insets at the bottom right of each panel. Bar, 20 μm. (C) Hippocampal neurons were transfected with either GFP-GIT1 (top) or GIT1-FLAG (bottom) and immunostained for the presynaptic marker synapsin1 at 3 wk in culture. Both GFP-GIT1 and GIT1-FLAG colocalize with synapsin1 in dendritic spines and shafts (arrows). Bar, 20 μm. (D) Hippocampal neurons were transfected with GFP-GIT1 and immunostained for the appropriate synaptic markers at 2–3 wk in culture. The GIT1 clusters on the dendrites (Dendritic) almost completely merge with the postsynaptic marker PSD-95 and are in close apposition to the presynaptic marker synapsin1 (Overlay). The GIT1 clusters on the axons (Axonal) completely merge with the presynaptic marker SV2 and are in close apposition to the postsynaptic marker PSD-95 (Overlay). Note the colocalization of GIT1 clusters with the synaptic markers (arrowheads). Enlargements of individual synapses are shown in the right column. GFP-GIT1 is pseudocolored green, and the synaptic markers are pseudocolored red. Bar, 2 μm.
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Related In: Results  -  Collection

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fig1: GIT1 is expressed in cultured hippocampal neurons and enriched in synapses. (A) Western blot of a lysate from day 10 cultured hippocampal neurons. The blot was probed with a GIT1 antibody. A specific band at ∼95 kD confirms the presence of the GIT1 protein in these neurons. (B) Hippocampal neurons at 2–3 wk in culture were double immunostained for endogenous GIT1 (left column) and various synaptic proteins (right column). GIT1 colocalizes with the presynaptic marker SV2 (top). GIT1 shows colocalization with PSD-95 in some puncta (arrowheads), but some GIT1 puncta, especially those on the cell body, do not overlap with PSD-95 (middle, arrows). These puncta show colocalization with the inhibitory synapse marker GAD-6 (bottom). Enlargements of the boxed regions are shown in insets at the bottom right of each panel. Bar, 20 μm. (C) Hippocampal neurons were transfected with either GFP-GIT1 (top) or GIT1-FLAG (bottom) and immunostained for the presynaptic marker synapsin1 at 3 wk in culture. Both GFP-GIT1 and GIT1-FLAG colocalize with synapsin1 in dendritic spines and shafts (arrows). Bar, 20 μm. (D) Hippocampal neurons were transfected with GFP-GIT1 and immunostained for the appropriate synaptic markers at 2–3 wk in culture. The GIT1 clusters on the dendrites (Dendritic) almost completely merge with the postsynaptic marker PSD-95 and are in close apposition to the presynaptic marker synapsin1 (Overlay). The GIT1 clusters on the axons (Axonal) completely merge with the presynaptic marker SV2 and are in close apposition to the postsynaptic marker PSD-95 (Overlay). Note the colocalization of GIT1 clusters with the synaptic markers (arrowheads). Enlargements of individual synapses are shown in the right column. GFP-GIT1 is pseudocolored green, and the synaptic markers are pseudocolored red. Bar, 2 μm.
Mentions: Previous studies showed that GIT1 is highly expressed in the brain both at the mRNA and protein levels (Premont et al., 1998; Zhao et al., 2000). To determine whether GIT1 is expressed in neurons, we performed RT-PCR with hippocampal total RNA using GIT1-specific primers. The sequence of the PCR product was identical to the previously described rat GIT1 sequence, showing the presence of the GIT1 mRNA in these neurons. To confirm the protein expression of GIT1, we subjected a lysate from hippocampal neurons to immunoblot analysis using a GIT1 antibody. We detected a specific band at ∼95 kD, which is the expected mobility of GIT1 (Fig. 1 A). This confirms the presence of GIT1 in the neurons at the protein level since our antibody is specific for GIT1; however, we cannot exclude the possibility that other GIT family members may also be present.

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