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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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PIX is targeted to the synapses by GIT1. (A) HA-tagged βPIX localizes to the synapses. Hippocampal neurons were transfected with βPIX-HA and stained for HA and synapsin1. Arrows indicate PIX puncta that colocalize with synapsin1 puncta. Bar, 20 μm. (B) The localization of PIX to synapses is inhibited by coexpression of GFP-SLD. Hippocampal neurons were cotransfected with either GFP-GIT1 and PIX-HA or GFP-SLD and PIX-HA. They were fixed and stained for HA and synapsin1. Note the localization of PIX in the synapses when coexpressed with GIT1 (arrows, top) and the decreased localization of PIX to synapses when coexpressed with SLD (arrows, bottom). Bar, 20 μm. (C) A GIT1 binding-deficient PIX mutant (PIXΔGBD) does not localized to synapses. Hippocampal neurons were transfected with HA-PIXΔGBD and coimmunostained for HA and synapsin1. Note the lack of localization to synapses with PIXΔGBD (arrows). Bar, 2 μm.
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fig5: PIX is targeted to the synapses by GIT1. (A) HA-tagged βPIX localizes to the synapses. Hippocampal neurons were transfected with βPIX-HA and stained for HA and synapsin1. Arrows indicate PIX puncta that colocalize with synapsin1 puncta. Bar, 20 μm. (B) The localization of PIX to synapses is inhibited by coexpression of GFP-SLD. Hippocampal neurons were cotransfected with either GFP-GIT1 and PIX-HA or GFP-SLD and PIX-HA. They were fixed and stained for HA and synapsin1. Note the localization of PIX in the synapses when coexpressed with GIT1 (arrows, top) and the decreased localization of PIX to synapses when coexpressed with SLD (arrows, bottom). Bar, 20 μm. (C) A GIT1 binding-deficient PIX mutant (PIXΔGBD) does not localized to synapses. Hippocampal neurons were transfected with HA-PIXΔGBD and coimmunostained for HA and synapsin1. Note the lack of localization to synapses with PIXΔGBD (arrows). Bar, 2 μm.

Mentions: To confirm the protein expression of PIX in hippocampal neurons, we performed immunoblot analysis on a hippocampal lysate using a βPIX antibody. We detected a band at ∼78 kD, which is the expected mobility of βPIX (Koh et al., 2001) (unpublished data), indicating that endogenous βPIX is present in hippocampal neurons. In addition to the 78-kD band, another band at ∼55 kD could be detected, which is the expected mobility of an alternative spliced isoform, p50Cool. To see if PIX also localizes to synapses, we transfected HA-tagged βPIX into the neurons and looked at its localization by coimmunostaining with synapsin1. PIX and synapsin1 colocalized in clusters along the processes, suggesting that PIX is synaptic as well (Fig. 5 A). Furthermore, immunostaining of endogenous PIX in hippocampal neurons with a polyclonal βPIX antibody showed that endogenous PIX also localized to synapses (unpublished data). This is consistent with the localization of dPIX, the Drosophila homologue of mammalian PIX, to the postsynaptic density of Drosophila neuromuscular junctions (Parnas et al., 2001).


Synapse formation is regulated by the signaling adaptor GIT1.

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

PIX is targeted to the synapses by GIT1. (A) HA-tagged βPIX localizes to the synapses. Hippocampal neurons were transfected with βPIX-HA and stained for HA and synapsin1. Arrows indicate PIX puncta that colocalize with synapsin1 puncta. Bar, 20 μm. (B) The localization of PIX to synapses is inhibited by coexpression of GFP-SLD. Hippocampal neurons were cotransfected with either GFP-GIT1 and PIX-HA or GFP-SLD and PIX-HA. They were fixed and stained for HA and synapsin1. Note the localization of PIX in the synapses when coexpressed with GIT1 (arrows, top) and the decreased localization of PIX to synapses when coexpressed with SLD (arrows, bottom). Bar, 20 μm. (C) A GIT1 binding-deficient PIX mutant (PIXΔGBD) does not localized to synapses. Hippocampal neurons were transfected with HA-PIXΔGBD and coimmunostained for HA and synapsin1. Note the lack of localization to synapses with PIXΔGBD (arrows). Bar, 2 μm.
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Related In: Results  -  Collection

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fig5: PIX is targeted to the synapses by GIT1. (A) HA-tagged βPIX localizes to the synapses. Hippocampal neurons were transfected with βPIX-HA and stained for HA and synapsin1. Arrows indicate PIX puncta that colocalize with synapsin1 puncta. Bar, 20 μm. (B) The localization of PIX to synapses is inhibited by coexpression of GFP-SLD. Hippocampal neurons were cotransfected with either GFP-GIT1 and PIX-HA or GFP-SLD and PIX-HA. They were fixed and stained for HA and synapsin1. Note the localization of PIX in the synapses when coexpressed with GIT1 (arrows, top) and the decreased localization of PIX to synapses when coexpressed with SLD (arrows, bottom). Bar, 20 μm. (C) A GIT1 binding-deficient PIX mutant (PIXΔGBD) does not localized to synapses. Hippocampal neurons were transfected with HA-PIXΔGBD and coimmunostained for HA and synapsin1. Note the lack of localization to synapses with PIXΔGBD (arrows). Bar, 2 μm.
Mentions: To confirm the protein expression of PIX in hippocampal neurons, we performed immunoblot analysis on a hippocampal lysate using a βPIX antibody. We detected a band at ∼78 kD, which is the expected mobility of βPIX (Koh et al., 2001) (unpublished data), indicating that endogenous βPIX is present in hippocampal neurons. In addition to the 78-kD band, another band at ∼55 kD could be detected, which is the expected mobility of an alternative spliced isoform, p50Cool. To see if PIX also localizes to synapses, we transfected HA-tagged βPIX into the neurons and looked at its localization by coimmunostaining with synapsin1. PIX and synapsin1 colocalized in clusters along the processes, suggesting that PIX is synaptic as well (Fig. 5 A). Furthermore, immunostaining of endogenous PIX in hippocampal neurons with a polyclonal βPIX antibody showed that endogenous PIX also localized to synapses (unpublished data). This is consistent with the localization of dPIX, the Drosophila homologue of mammalian PIX, to the postsynaptic density of Drosophila neuromuscular junctions (Parnas et al., 2001).

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