PICK1 links AMPA receptor stimulation to Cdc42.
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.
Affiliation: School of Biochemistry, Medical Sciences Building,University of Bristol, University Walk, Bristol BS8 1TD, UK.Show MeSH
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Mentions: Since PICK1 is a well-established AMPAR accessory protein , 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.
Affiliation: School of Biochemistry, Medical Sciences Building,University of Bristol, University Walk, Bristol BS8 1TD, UK.