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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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Cdc42 and Rac1 have distinct but overlapping binding sites on PICK1.Upper panel: GST pulldowns were carried out using purified his6flagCdc42 or his6mycRac1 and truncation mutants of PICK1 as GST fusions as depicted. Bound proteins were detected by western blotting using anti-myc or anti-flag.Lower panel: diagram showing truncation mutants of PICK1 used, and a summary of the results. A tick indicates a positive interaction, whereas a cross indicates no binding.
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fig0010: Cdc42 and Rac1 have distinct but overlapping binding sites on PICK1.Upper panel: GST pulldowns were carried out using purified his6flagCdc42 or his6mycRac1 and truncation mutants of PICK1 as GST fusions as depicted. Bound proteins were detected by western blotting using anti-myc or anti-flag.Lower panel: diagram showing truncation mutants of PICK1 used, and a summary of the results. A tick indicates a positive interaction, whereas a cross indicates no binding.

Mentions: To further compare, the PICK1–Cdc42 interaction with that of PICK1–Rac1, we analysed the binding of purified his6-tagged flagCdc42 and mycRac1 to a range of PICK1 truncations. Wild-type GST–PICK1 binds both GTPases, demonstrating that the interactions are direct, with no requirement for intermediary protein components. Interestingly, the two GTPases show distinct patterns of binding to the PICK1 mutants, indicating that Cdc42 and Rac1 have overlapping, but not identical binding sites on PICK1 (Fig. 2). Both GTPases require the presence of the BAR domain, indeed Cdc42 binds the isolated BAR domain and binding is unaffected by deletion of either acidic region (ΔCT, ΔNT) or deletion of an extreme C-terminal region (1-379) of the full-length protein. However, Cdc42 binding is abolished in the absence of the PDZ domain when the C-terminal region is present (105-416), suggesting an intramolecular inhibition of the interaction. It has previously been suggested that PICK1 forms an intramolecular interaction between the PDZ and BAR domains [18,24], and also that the C-terminal tail interacts with the BAR domain [16]. In contrast, Rac1 does not bind the isolated BAR domain, but requires the presence of both BAR and C-terminal regions for the interaction (Fig. 2).


PICK1 links AMPA receptor stimulation to Cdc42.

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

Cdc42 and Rac1 have distinct but overlapping binding sites on PICK1.Upper panel: GST pulldowns were carried out using purified his6flagCdc42 or his6mycRac1 and truncation mutants of PICK1 as GST fusions as depicted. Bound proteins were detected by western blotting using anti-myc or anti-flag.Lower panel: diagram showing truncation mutants of PICK1 used, and a summary of the results. A tick indicates a positive interaction, whereas a cross indicates no binding.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4300402&req=5

fig0010: Cdc42 and Rac1 have distinct but overlapping binding sites on PICK1.Upper panel: GST pulldowns were carried out using purified his6flagCdc42 or his6mycRac1 and truncation mutants of PICK1 as GST fusions as depicted. Bound proteins were detected by western blotting using anti-myc or anti-flag.Lower panel: diagram showing truncation mutants of PICK1 used, and a summary of the results. A tick indicates a positive interaction, whereas a cross indicates no binding.
Mentions: To further compare, the PICK1–Cdc42 interaction with that of PICK1–Rac1, we analysed the binding of purified his6-tagged flagCdc42 and mycRac1 to a range of PICK1 truncations. Wild-type GST–PICK1 binds both GTPases, demonstrating that the interactions are direct, with no requirement for intermediary protein components. Interestingly, the two GTPases show distinct patterns of binding to the PICK1 mutants, indicating that Cdc42 and Rac1 have overlapping, but not identical binding sites on PICK1 (Fig. 2). Both GTPases require the presence of the BAR domain, indeed Cdc42 binds the isolated BAR domain and binding is unaffected by deletion of either acidic region (ΔCT, ΔNT) or deletion of an extreme C-terminal region (1-379) of the full-length protein. However, Cdc42 binding is abolished in the absence of the PDZ domain when the C-terminal region is present (105-416), suggesting an intramolecular inhibition of the interaction. It has previously been suggested that PICK1 forms an intramolecular interaction between the PDZ and BAR domains [18,24], and also that the C-terminal tail interacts with the BAR domain [16]. In contrast, Rac1 does not bind the isolated BAR domain, but requires the presence of both BAR and C-terminal regions for the interaction (Fig. 2).

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