Roles of stargazin and IRSp53 in synaptic transmission and synapse formation

Abstract

The PSD-95 family of PDZ-containing proteins is involved in the organization of synaptic structure through their interaction with several membrane, scaffolding and signaling proteins. The functions of two synaptic proteins, which are associated with PSD-95, were investigated in this study. One is stargazin and the other is insulin receptor substrate p53 (IRSp53). Stargazin, the first transmembrane protein known to associate AMPA (a-amino-3-hydoroxy-5-methyl-isoxazole-4-propionate) glutamate receptors (AMPARs), interacts with PSD-95 through the C-terminal PDZ-binding motif. The stargazin C terminus also contains a consensus sequence for phosphorylation by protein kinase A (PKA). It was tested if C-terminus of stargazin is phosphoryated by PKA and this phophorylation may regulate its interaction with PSD-95. Phospho-specific antibodies reveal that the Thr-321 residue of stargazin is phosphorylated in COS cells and in vivo. Cotransfection of stargazin with the catalytic subunit of PKA dramatically increased phosphorylation level of stargazin in COS cells. Mutations mimicking phosphorylation (T321 D and T321 E) eliminated the interaction of stargazin with PSD-95, which was confirmed by yest two-hybrid assay, in vitro coimmunoprecipitation and coclustering assay. Phosphorylated stargazin showed a selective loss of coimmunoprecipitation with PSD-95 in COS cells and limited enrichment in postsynaptic density fractions of rat brain. These results suggest that phophorylation of the stargazin C terminus by PKA regulates its interaction with PSD-95 and synaptic targeting of AMPARs. The small GTPases Rac1 and Cdc42 are key regulators of the morphogenesis of actin-rich dendritic spines in neurons. However, little is known about how activated Rac l /Cdc42 regulates dendritic spines. Insulin receptor substrate 53 (IRSp53), which is highly expressed in the postsynaptic density (PSD), is known to link activated Racl/Cdc42 to downstream effectors for actin regulation in non-neur...