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PICK1 links AMPA receptor stimulation to Cdc42.

Rocca DL, Hanley JG - Neurosci. Lett. (2014)

Bottom Line: The Rho-family member Cdc42 regulates dendritic spine morphology via its effector N-WASP, which activates the actin-nucleating Arp2/3 complex.Furthermore, AMPAR stimulation deactivates Cdc42 and alters its detergent solubility in neurons via a PICK1-dependent process.This work suggests a novel role for PICK1 in transducing AMPAR stimulation to Cdc42 function in neurons.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building,University of Bristol, University Walk, Bristol BS8 1TD, UK.

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PICK1 links AMPAR stimulation to Cdc42 deactivation.(A) Both Cdc42 and Rac1 form a triple complex with PICK1 and GluA2 in vitro. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing flag-tagged Cdc42(V12) or myc-tagged Rac1(V12) using GST-GluA2 C-terminus (GluA2C) in the absence or presence of purified his6PICK1, or GST alone. Bound proteins were detected by western blotting using anti-PICK1, anti-flag or anti-myc.(B) Cdc42 forms a triple complex with PICK1 and GluA2 in neurons. Lysates were prepared from dissociated cortical neurons, and immunoprecipitations carried out using anti-GluA2 or non-immune mouse IgG as control. Bound proteins were detected by western blotting using anti-GluA2, anti-PICK1 and anti-Cdc42.(C) AMPAR stimulation increases the detergent solubility of Cdc42 and reduces the proportion of GTP-bound Cdc42. Dissociated cortical neurons were treated with 100 μM AMPA or vehicle for 5 min. Lysates were prepared and GTP-bound Cdc42 was isolated by GST pulldown using GST–PAK. GST–PAK bound Cdc42–GTP and unbound Cdc42 in the lysate were detected by western blotting using anti-Cdc42. Tubulin serves as a loading control. Representative western blots are shown, and graphs show pooled data for total detergent-soluble Cdc42 (left graph) and for the proportion of Cdc42 that is GTP-bound (right graph). n = 5.(D) PICK1 PDZ domain interactions are involved in AMPAR-induced changes in detergent solubility of Cdc42. Dissociated cortical neurons were transduced with Sindbis virus expressing pep2-SVKE–IRES-EGFP, pep2-SVKI–IRES-EGFP or pep2-EVKI–IRES-EGFP. Cultures were treated with AMPA(+) or vehicle(−), and processed for biochemistry as in (B). A representative western blot is shown, and graphs show pooled data for total detergent-soluble Cdc42 (left graph) and for the proportion of Cdc42 that is GTP-bound (right graph). n = 5.
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fig0015: PICK1 links AMPAR stimulation to Cdc42 deactivation.(A) Both Cdc42 and Rac1 form a triple complex with PICK1 and GluA2 in vitro. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing flag-tagged Cdc42(V12) or myc-tagged Rac1(V12) using GST-GluA2 C-terminus (GluA2C) in the absence or presence of purified his6PICK1, or GST alone. Bound proteins were detected by western blotting using anti-PICK1, anti-flag or anti-myc.(B) Cdc42 forms a triple complex with PICK1 and GluA2 in neurons. Lysates were prepared from dissociated cortical neurons, and immunoprecipitations carried out using anti-GluA2 or non-immune mouse IgG as control. Bound proteins were detected by western blotting using anti-GluA2, anti-PICK1 and anti-Cdc42.(C) AMPAR stimulation increases the detergent solubility of Cdc42 and reduces the proportion of GTP-bound Cdc42. Dissociated cortical neurons were treated with 100 μM AMPA or vehicle for 5 min. Lysates were prepared and GTP-bound Cdc42 was isolated by GST pulldown using GST–PAK. GST–PAK bound Cdc42–GTP and unbound Cdc42 in the lysate were detected by western blotting using anti-Cdc42. Tubulin serves as a loading control. Representative western blots are shown, and graphs show pooled data for total detergent-soluble Cdc42 (left graph) and for the proportion of Cdc42 that is GTP-bound (right graph). n = 5.(D) PICK1 PDZ domain interactions are involved in AMPAR-induced changes in detergent solubility of Cdc42. Dissociated cortical neurons were transduced with Sindbis virus expressing pep2-SVKE–IRES-EGFP, pep2-SVKI–IRES-EGFP or pep2-EVKI–IRES-EGFP. Cultures were treated with AMPA(+) or vehicle(−), and processed for biochemistry as in (B). A representative western blot is shown, and graphs show pooled data for total detergent-soluble Cdc42 (left graph) and for the proportion of Cdc42 that is GTP-bound (right graph). n = 5.

Mentions: Since PICK1 is a well-established AMPAR accessory protein [12], we explored an association between AMPAR stimulation and Cdc42. Initially, we investigated whether PICK1 can form a triple complex with Cdc42 or Rac1 and GluA2 C-terminus. GST–GluA2 C-terminus (GluA2C) does not bind Cdc42 or Rac1 in the absence of PICK1, but when his6-PICK1 is added, a robust interaction with both GTPases is observed (Fig. 3A). Furthermore, both Cdc42 and PICK1 are present in GluA2 immunoprecipitations from neuronal lysate, strongly suggesting the presence of a GluA2–PICK1–Cdc42 tripartite complex in vivo (Fig. 3B). These experiments demonstrate that Cdc42 can associate with AMPARs via PICK1, and suggest that either Cdc42 regulates AMPAR trafficking, or AMPARs regulate Cdc42 function via PICK1. To test the latter hypothesis, we used GST-PAK pulldown assays to determine the effect of AMPAR stimulation on Cdc42 activation in cultured neurons. Bath application of AMPA for 5 min causes a significant reduction in GTP-bound Cdc42 (Fig. 3C). In addition, we noted an increase in the detergent solubility of Cdc42 after AMPAR stimulation (Fig. 3C), suggesting that AMPAR stimulation displaces Cdc42 from specific membrane compartments or protein complexes. Since cell lysis and western analysis were carried out after just 5 min of drug treatment, it is highly unlikely that this difference in Cdc42 immunoreactivity could be explained by an increase in protein translation or a reduction in protein degradation.


PICK1 links AMPA receptor stimulation to Cdc42.

Rocca DL, Hanley JG - Neurosci. Lett. (2014)

PICK1 links AMPAR stimulation to Cdc42 deactivation.(A) Both Cdc42 and Rac1 form a triple complex with PICK1 and GluA2 in vitro. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing flag-tagged Cdc42(V12) or myc-tagged Rac1(V12) using GST-GluA2 C-terminus (GluA2C) in the absence or presence of purified his6PICK1, or GST alone. Bound proteins were detected by western blotting using anti-PICK1, anti-flag or anti-myc.(B) Cdc42 forms a triple complex with PICK1 and GluA2 in neurons. Lysates were prepared from dissociated cortical neurons, and immunoprecipitations carried out using anti-GluA2 or non-immune mouse IgG as control. Bound proteins were detected by western blotting using anti-GluA2, anti-PICK1 and anti-Cdc42.(C) AMPAR stimulation increases the detergent solubility of Cdc42 and reduces the proportion of GTP-bound Cdc42. Dissociated cortical neurons were treated with 100 μM AMPA or vehicle for 5 min. Lysates were prepared and GTP-bound Cdc42 was isolated by GST pulldown using GST–PAK. GST–PAK bound Cdc42–GTP and unbound Cdc42 in the lysate were detected by western blotting using anti-Cdc42. Tubulin serves as a loading control. Representative western blots are shown, and graphs show pooled data for total detergent-soluble Cdc42 (left graph) and for the proportion of Cdc42 that is GTP-bound (right graph). n = 5.(D) PICK1 PDZ domain interactions are involved in AMPAR-induced changes in detergent solubility of Cdc42. Dissociated cortical neurons were transduced with Sindbis virus expressing pep2-SVKE–IRES-EGFP, pep2-SVKI–IRES-EGFP or pep2-EVKI–IRES-EGFP. Cultures were treated with AMPA(+) or vehicle(−), and processed for biochemistry as in (B). A representative western blot is shown, and graphs show pooled data for total detergent-soluble Cdc42 (left graph) and for the proportion of Cdc42 that is GTP-bound (right graph). n = 5.
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fig0015: PICK1 links AMPAR stimulation to Cdc42 deactivation.(A) Both Cdc42 and Rac1 form a triple complex with PICK1 and GluA2 in vitro. GST-pulldowns were carried out from lysates prepared from COS7 cells expressing flag-tagged Cdc42(V12) or myc-tagged Rac1(V12) using GST-GluA2 C-terminus (GluA2C) in the absence or presence of purified his6PICK1, or GST alone. Bound proteins were detected by western blotting using anti-PICK1, anti-flag or anti-myc.(B) Cdc42 forms a triple complex with PICK1 and GluA2 in neurons. Lysates were prepared from dissociated cortical neurons, and immunoprecipitations carried out using anti-GluA2 or non-immune mouse IgG as control. Bound proteins were detected by western blotting using anti-GluA2, anti-PICK1 and anti-Cdc42.(C) AMPAR stimulation increases the detergent solubility of Cdc42 and reduces the proportion of GTP-bound Cdc42. Dissociated cortical neurons were treated with 100 μM AMPA or vehicle for 5 min. Lysates were prepared and GTP-bound Cdc42 was isolated by GST pulldown using GST–PAK. GST–PAK bound Cdc42–GTP and unbound Cdc42 in the lysate were detected by western blotting using anti-Cdc42. Tubulin serves as a loading control. Representative western blots are shown, and graphs show pooled data for total detergent-soluble Cdc42 (left graph) and for the proportion of Cdc42 that is GTP-bound (right graph). n = 5.(D) PICK1 PDZ domain interactions are involved in AMPAR-induced changes in detergent solubility of Cdc42. Dissociated cortical neurons were transduced with Sindbis virus expressing pep2-SVKE–IRES-EGFP, pep2-SVKI–IRES-EGFP or pep2-EVKI–IRES-EGFP. Cultures were treated with AMPA(+) or vehicle(−), and processed for biochemistry as in (B). A representative western blot is shown, and graphs show pooled data for total detergent-soluble Cdc42 (left graph) and for the proportion of Cdc42 that is GTP-bound (right graph). n = 5.
Mentions: Since PICK1 is a well-established AMPAR accessory protein [12], we explored an association between AMPAR stimulation and Cdc42. Initially, we investigated whether PICK1 can form a triple complex with Cdc42 or Rac1 and GluA2 C-terminus. GST–GluA2 C-terminus (GluA2C) does not bind Cdc42 or Rac1 in the absence of PICK1, but when his6-PICK1 is added, a robust interaction with both GTPases is observed (Fig. 3A). Furthermore, both Cdc42 and PICK1 are present in GluA2 immunoprecipitations from neuronal lysate, strongly suggesting the presence of a GluA2–PICK1–Cdc42 tripartite complex in vivo (Fig. 3B). These experiments demonstrate that Cdc42 can associate with AMPARs via PICK1, and suggest that either Cdc42 regulates AMPAR trafficking, or AMPARs regulate Cdc42 function via PICK1. To test the latter hypothesis, we used GST-PAK pulldown assays to determine the effect of AMPAR stimulation on Cdc42 activation in cultured neurons. Bath application of AMPA for 5 min causes a significant reduction in GTP-bound Cdc42 (Fig. 3C). In addition, we noted an increase in the detergent solubility of Cdc42 after AMPAR stimulation (Fig. 3C), suggesting that AMPAR stimulation displaces Cdc42 from specific membrane compartments or protein complexes. Since cell lysis and western analysis were carried out after just 5 min of drug treatment, it is highly unlikely that this difference in Cdc42 immunoreactivity could be explained by an increase in protein translation or a reduction in protein degradation.

Bottom Line: The Rho-family member Cdc42 regulates dendritic spine morphology via its effector N-WASP, which activates the actin-nucleating Arp2/3 complex.Furthermore, AMPAR stimulation deactivates Cdc42 and alters its detergent solubility in neurons via a PICK1-dependent process.This work suggests a novel role for PICK1 in transducing AMPAR stimulation to Cdc42 function in neurons.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building,University of Bristol, University Walk, Bristol BS8 1TD, UK.

Show MeSH
Related in: MedlinePlus