Phosphorylation of the postsynaptic density-95 (PSD-95)/discs large/zona occludens-1 binding site of stargazin regulates binding to PSD-95 and synaptic targeting of AMPA receptors

Abstract

Dynamic regulation of AMPA-type receptors at the synapse is proposed to play a critical role in alterations of the synaptic strength seen in cellular models of learning and memory such as long-term potentiation in the hippocampus. Stargazin, previously identified as an AMPA receptor (AMPAR)-interacting protein, is critical for surface expression and synaptic targeting of AMPARs. Stargazin interacts with postsynaptic density-95/discs large/zona occludens-1 (PDZ) proteins via a C-terminal PDZ binding motif. Interestingly, the C terminal of stargazin also predicts phosphorylation at a threonine residue critical for PDZ protein binding. Because protein phosphorylation regulates synaptic plasticity, we characterized this site and the effects of stargazin phosphorylation on AMPAR function. In vitro peptide phosphorylation assays and Western blot analysis with phospho-stargazin-specific antibodies indicate that the critical threonine within the stargazin PDZ binding site is phosphorylated by protein kinase A. This phosphorylation disrupts stargazin interaction and clustering with postsynaptic density-95 (PSD-95) in transfected COS-7 cells. Furthermore, a stargazin construct with a Thr-to-Glu mutation that mimics phosphorylation fails to cluster at synaptic spines and downregulates synaptic AMPAR function in cultured hippocampal neurons. These data suggest that phosphorylation of the stargazin PDZ ligand can disrupt stargazin interaction with PSD-95 and thereby regulate synaptic AMPAR function.

Fig. 1.

Stargazin is phosphorylated at the PDZ binding site by PKA. A, The C-terminal amino acid sequence of stargazin (γ2) as well as other stargazin family members that contain predicted PDZ binding domains. The PDZ binding domain is highlighted ingray, the protein kinase consensus phosphorylation sequence is bordered by a rectangle, and the predicted phosphorylation site and critical PDZ-binding residue are inbold type. B, Peptide substrates were incubated with PKA and [γ-³²P]ATP in vitro. Although peptides corresponding to the N-terminal 20 aa of PSD-95 [PSD-95(1–20)] and the C terminal of stargazin phosphorylated at the −2 threonine residue [phospho-stargazin(313–323)] were not phosphorylated, the C terminus of stargazin [stargazin(313–323)] was phosphorylated. Data are presented as counts per minute and represent the average of four experiments ± SEM. *p < 0.001. C, Stargazin(313–323) and phospho-stargazin(313–323) were coupled to BSA, spotted on polyvinylidene difluoride membranes, and immunoprobed. Antibodies prepared to phospho-stargazin(313–323) were 100-fold more sensitive for the phosphorylated peptide compared with the nonphosphorylated peptide. D, COS-7 cells were transfected with stargazin-GFP (star-GFP) in the presence or absence of PKA-GFP; cell lysates were prepared and subjected to SDS-PAGE and Western blotting with stargazin and phospho-stargazin antibodies. Cotransfection with PKA dramatically increased the phosphorylation of stargazin, an effect that was eliminated by treatment of the lysates with λ-phosphatase.

Fig. 2.

The phospho-mimic stargazin does not interact with PSD-95. A, COS-7 cells were transfected alone or with the indicated combinations of stargazin-GFP (star-GFP), PSD-95, and stargazin(T321E)-GFP [star(T321E)-GFP]; cell lysates were prepared, and PSD-95 was immunoprecipitated (IP). Whereas stargazin-GFP interacted strongly with PSD-95, stargazin(T321E)-GFP failed to coimmunoprecipitate.B, Interaction of stargazin constructs with the PDZ domains of PSD-95. Yeasts were transformed with plasmids encoding the C terminal of stargazin [stargazin(201–323)], stargazin missing the last four amino acids [stargazin(201–319)], or the phospho-mimic stargazin [stargazin(201–323,T321E)], together with PDZ domains I–III of PSD-95. The stargazin C termini were fused to the galactosidase-4 (GAL4) DNA binding domain and the PDZ domains were fused to the GAL4 activation domain. Colonies that grew on plates lacking Leu, Trp, adenine, and His were scored as positive (+).

Fig. 3.

Phosphorylation of the stargazin PDZ ligand disrupts clustering with PSD-95. COS-7 cells were transfected with combinations of stargazin-GFP, stargazin-HA, PSD-95, stargazin(T321E)-GFP, stargazin(R318,319A)-GFP, and PKA-GFP.A–C, When cotransfected, PSD-95 and stargazin-GFP cocluster in large plasma membrane patches. D–F, Stargazin(T321E)-GFP remains diffusely localized when transfected with PSD-95. G–I, Transfection of PKA-GFP with stargazin-HA and PSD-95 results in the diffuse localization of stargazin.J–L, Stargazin(R318,319A)-GFP, a construct that contains a mutated PKA recognition site, is clustered by PSD-95 in COS-7 cells. Merged images are shown in the panels on the right (C, F, I, L). Scale bar, 10 μm. M, COS-7 cells were transfected with PSD-95 and the indicated constructs. star, Stargazin-HA;star(T-E), stargazin(T321E)-HA;star(R-A), stargazin(R318,319A)-HA. Surface clustering was assessed in 100 randomly selected cells in four separate experiments. Although PSD-95-mediated clustering of stargazin-HA is reduced by PKA-GFP, there is no effect on PSD-95-mediated clustering of stargazin(R318,319A)-HA or K_v1.4-HA. *p < 0.001.

Fig. 4.

Phospho-mimic stargazin does not traffic to the synapse and attenuates synaptic AMPAR currents. Constructs encoding stargazin-GFP, stargazin(T321E)-GFP, or stargazin(R318,319A)-GFP (green) were transfected into neurons, which were then fixed and stained for PSD-95 (red) after 11–17 din vitro. Stargazin-GFP clusters at synaptic sites (A–C). The phospho-mimic stargazin, stargazin(T321E)-GFP, does not target to synapses (D–F), whereas stargazin(R318,319A)-GFP, a construct that contains a mutated PKA recognition site, retains synaptic localization (G–I). Merged images are shown in the panels on the right(C, F, I). Scale bar, 10 μm. In separate experiments, cultured neurons were transfected with stargazin(T321E)-GFP or stargazin(R318,319A)-GFP and synaptic currents were measured by whole-cell patch-clamp recording at a holding potential of −60 mV. Stargazin(T321E)-GFP transfection downregulates AMPAR mEPSC amplitude (p < 0.002) in hippocampal neurons (J), whereas stargazin(R318,319A)-GFP has no effect (K).