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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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GIT1ΔSHD-expressing neurons show a phenotype similar to SLD-expressing neurons. (A) Hippocampal neurons were transfected with either GFP-GIT1 or GFP-GIT1ΔSHD at day 7 in culture and imaged at day 14 in culture. Note the increase in dendritic protrusions in GIT1ΔSHD-expressing neurons. Bar, 2 μm. (B) Quantification of the number of spines and dendritic protrusions in GIT1- and GIT1ΔSHD-expressing neurons. (C) Quantification of the synaptic density in GIT1- and GIT1ΔSHD-expressing neurons. The difference between GIT1 and GIT1ΔSHD was statistically significant as determined by Student's t test (*P < 0.0001).
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fig4: GIT1ΔSHD-expressing neurons show a phenotype similar to SLD-expressing neurons. (A) Hippocampal neurons were transfected with either GFP-GIT1 or GFP-GIT1ΔSHD at day 7 in culture and imaged at day 14 in culture. Note the increase in dendritic protrusions in GIT1ΔSHD-expressing neurons. Bar, 2 μm. (B) Quantification of the number of spines and dendritic protrusions in GIT1- and GIT1ΔSHD-expressing neurons. (C) Quantification of the synaptic density in GIT1- and GIT1ΔSHD-expressing neurons. The difference between GIT1 and GIT1ΔSHD was statistically significant as determined by Student's t test (*P < 0.0001).

Mentions: How does GIT1 affect synapse formation? Since CD-GIT1, which contains the PIX binding domain in addition to SLD, behaved like wild-type GIT1, it is likely that the PIX binding domain is a functional domain. To test this hypothesis, we constructed a GIT1 mutant that has the PIX binding domain deleted (GIT1ΔSHD). As expected, GIT1ΔSHD localized to synapses when expressed at relatively low levels. Neurons expressing high levels of GIT1ΔSHD showed a phenotype similar to the SLD-expressing neurons. The cells had numerous dendritic protrusions (Fig. 4 A) with a decrease in spine and synaptic density (Fig. 4, B and C). This suggests that PIX is acting downstream of GIT1 to affect spine morphology and synapse formation.


Synapse formation is regulated by the signaling adaptor GIT1.

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

GIT1ΔSHD-expressing neurons show a phenotype similar to SLD-expressing neurons. (A) Hippocampal neurons were transfected with either GFP-GIT1 or GFP-GIT1ΔSHD at day 7 in culture and imaged at day 14 in culture. Note the increase in dendritic protrusions in GIT1ΔSHD-expressing neurons. Bar, 2 μm. (B) Quantification of the number of spines and dendritic protrusions in GIT1- and GIT1ΔSHD-expressing neurons. (C) Quantification of the synaptic density in GIT1- and GIT1ΔSHD-expressing neurons. The difference between GIT1 and GIT1ΔSHD was statistically significant as determined by Student's t test (*P < 0.0001).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172873&req=5

fig4: GIT1ΔSHD-expressing neurons show a phenotype similar to SLD-expressing neurons. (A) Hippocampal neurons were transfected with either GFP-GIT1 or GFP-GIT1ΔSHD at day 7 in culture and imaged at day 14 in culture. Note the increase in dendritic protrusions in GIT1ΔSHD-expressing neurons. Bar, 2 μm. (B) Quantification of the number of spines and dendritic protrusions in GIT1- and GIT1ΔSHD-expressing neurons. (C) Quantification of the synaptic density in GIT1- and GIT1ΔSHD-expressing neurons. The difference between GIT1 and GIT1ΔSHD was statistically significant as determined by Student's t test (*P < 0.0001).
Mentions: How does GIT1 affect synapse formation? Since CD-GIT1, which contains the PIX binding domain in addition to SLD, behaved like wild-type GIT1, it is likely that the PIX binding domain is a functional domain. To test this hypothesis, we constructed a GIT1 mutant that has the PIX binding domain deleted (GIT1ΔSHD). As expected, GIT1ΔSHD localized to synapses when expressed at relatively low levels. Neurons expressing high levels of GIT1ΔSHD showed a phenotype similar to the SLD-expressing neurons. The cells had numerous dendritic protrusions (Fig. 4 A) with a decrease in spine and synaptic density (Fig. 4, B and C). This suggests that PIX is acting downstream of GIT1 to affect spine morphology and synapse formation.

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