Postsynaptic targeting of alternative postsynaptic density-95 isoforms by distinct mechanisms

Abstract

Members of the postsynaptic density-95 (PSD95)/synapse-associated protein-90 (SAP90) family of scaffolding proteins contain a common set of modular protein interaction motifs including PDZ (postsynaptic density-95/Discs large/zona occludens-1), Src homology 3, and guanylate kinase domains, which regulate signaling and plasticity at excitatory synapses. We report that N-terminal alternative splicing of PSD95 generates an isoform, PSD95beta that contains an additional "L27" motif, which is also present in SAP97. Using yeast two hybrid and coimmunoprecipitation assays, we demonstrate that this N-terminal L27 domain of PSD-95beta, binds to an L27 domain in the membrane-associated guanylate kinase calcium/calmodulin-dependent serine kinase, and to Hrs, an endosomal ATPase that regulates vesicular trafficking. By transfecting heterologous cells and hippocampal neurons, we find that interactions with the L27 domain regulate synaptic clustering of PSD95beta. Disrupting Hrs-regulated early endosomal sorting in hippocampal neurons selectively blocks synaptic clustering of PSD95beta but does not interfere with trafficking of the palmitoylated isoform, PSD95alpha. These studies identify molecular and functional heterogeneity in synaptic PSD95 complexes and reveal critical roles for L27 domain interactions and Hrs regulated vesicular trafficking in postsynaptic protein clustering.

Fig. 1.

PSD95β and SAP97 contain N-terminal L27 domains.A, Scheme showing 5′ genomic structure of PSD95 (top). Three exons (β1, β1′, andβ1") encode the N-terminal 53 amino acids of PSD95β; immediately downstream is a single exon (α1) that encodes the N-terminal 10 amino acids of PSD95α. These alternatively spliced isoforms of PSD95 splice into a common exon 2. The nucleotide and predicted amino acid sequences of these exons are shown (bottom); introns are in italics.B, Alignment of N termini of PSD95β and SAP97 with representative L27 domains. Identical residues are shaded darkly, and homologous residues are shaded lightly. Predicted helical regions underlie gray cylinders. C, Domain structures of PSD95α and L27-containing proteins. Jagged lines denote N-terminal palmitates on PSD95α. Note that DLG3 has a similar domain structure as PALS (protein associated with Lin-7). CaMK, Calcium–calmodulin kinase domain.

Fig. 2.

PSD95β and SAP97 can bind to CASK via L27 heteromultimerization. A, COS cells were transfected with PSD95α, PSD95β, or SAP97 in the presence or absence of CASK–GFP, cell lysates were prepared, and CASK–GFP was immunoprecipitated with an antibody to GFP. Whereas PSD95α showed no interaction with CASK–GFP, PSD95β and SAP97 coimmunoprecipitated with CASK–GFP. B, Lysates were prepared from COS cells cotransfected with MALS2 and PSD95α–GFP, PSD95β–GFP, SAP97–GFP, CASK–GFP, or empty vector. Western blotting after GFP immunoprecipitation shows that MALS2 binds only to CASK–GFP.C, Lysates were prepared from rat brain, and PSD95 was immunoprecipitated. Western blotting after immunoprecipitation shows that CASK binds to PSD95. D, Table showing domains of CASK that bind to SAP97, PSD95β, or MALS2 by yeast two-hybrid assay. Yeast were transformed with plasmids encoding the N termini of MAGUK proteins or full-length MALS2 and L27 domains of CASK. MAGUK N termini and MALS were fused to the GAL4 DNA binding domain, and CASK L27 domains were fused to the GAL4 activation domain. Colonies that grew on plates lacking Leu, Trp, adenine, and His were scored as positives (+).

Fig. 3.

Expression of PSD95β in rat brain.A–C, In situ hybridization in sagittal sections of rat brain shows that PSD95β is expressed in diverse populations of neurons. Both PSD95β and PSD95α occur at high levels in cerebellum (Cb), hippocampus (H), corpus striatum (S), and cerebral cortex (Ctx).D, Northern blotting shows that the unique region of PSD95β hybridizes to a single 4.2 kb band in poly(A⁺) RNA. E, Brain lysates were prepared and subjected to immunoprecipitation. A PSD95β-specific antibody recognizes a 95 kDa band in brain lysates immunoprecipitated with a general PSD95 antibody raised to the PDZ domains [anti-PSD-95(PDZ)]. Conversely, the PSD-95β antibody immunoprecipitates an appropriately sized band that is recognized by the general PSD-95(PDZ) antibody.

Fig. 4.

PSD95β is not palmitoylated and fails to form plasma membrane clusters with Kv1.4. A, COS cells were transfected with constructs encoding PSD95α fused to GFP (PSD95α–GFP), PSD95β–GFP, or empty vector, and were metabolically labeled with [³H]palmitate. Cells were then lysed, proteins were separated by SDS-PAGE, and gels were analyzed by autoradiography (left panel) or Western blotting (right panel). Arrows indicate the predicted molecular weight for PSD95α–GFP and PSD95β–GFP.B, COS cells were transfected with Kv1.4 and PSD95β–GFP, SAP97–GFP, or PSD95α–GFP. Cells were fixed 48 hr after transfection and labeled with antibodies to Kv1.4. When coexpressed with PSD95β–GFP (a–c) or SAP97–GFP (d–f), Kv1.4 (red) accumulates on the nuclear membrane and on round perinuclear vesicles. In contrast, when transfected with PSD95α–GFP (g–i), Kv1.4 clusters in plasma membrane patches. Merged images are shown inpanels on right (c, f, i). Scale bar, 10 μm.

Fig. 5.

PSD95β and PSD95α synaptically cluster in primary hippocampal neurons. Constructs encoding PSD95β–GFP or PSD95α–GFP were transfected into neurons that were then fixed and stained for synaptophysin (red). After 11–17 din vitro, PSD95α–GFP (A–C) and PSD95β–GFP (D–F) cluster at synaptic sites. PSD95β–GFP lacking the N-terminal 19 residues [PSD95β(Δ1–19)–GFP] retains synaptic localization (G–I), whereas deleting the N-terminal 44 residues from [PSD95β-Δ(1–44)–GFP] disrupts synaptic targeting (J–L). Merged images are shown in panels onright (C, F, I, L). Scale bar, 10 μm.

Fig. 6.

The L27 domains of PSD95β and SAP97 bind the coiled-coil domain of Hrs. A, Yeast two-hybrid screening with PSD95β(1–53) yielded a clone of Hrs containing the second coiled-coil domain and flanking sequences. Deletions of up to 228 residues from the C terminus of this clone preserve binding whereas removing as few as 20 residues from the N terminus disrupts binding.B, Hrs binds to PSD95β and SAP97 but not PSD95α or SAP102. COS cells were cotransfected with constructs encoding Hrs and a GFP-tagged MAGUK protein or fragment. Cells were lysed, and immunoprecipitation with anti-GFP was followed by immunoblotting. Hrs coimmunoprecipitates with full-length PSD95β–GFP and SAP97–GFP, as well as N-terminal GFP-fusion constructs of PSD95β [PSD95β(1–53)–GFP, PSD95β(11–53)–GFP, and PSD95β(21–53)–GFP], but does not interact with PSD95α–GFP or SAP102–GFP (top panel). INPUTrepresents 10% of the COS cell lysate used for immunoprecipitation (bottom panels). C, Brain lysates were prepared and subjected to immunoprecipitation. Antibodies to PSD95 coimmunoprecipitated HRS, and an HRS-specific antibody coimmunoprecipitated PSD95.

Fig. 7.

PSD95β colocalizes with Hrs in neurons and transfected COS Cells. A, When expressed alone in COS cells, PSD95β shows perinuclear and diffuse cellular staining.B, In contrast, Hrs–GFP localizes to large intracellular vesicles. C–E, When cotransfected, Hrs–GFP colocalizes with PSD95β at these large vesicles.F–H, Deleting the first 19 amino acids of PSD95β maintains its colocalization with Hrs, whereas deleting the first 44 amino acids of PSD95β disrupts this colocalization (I–K). L–N, PSD95α does not colocalize with cotransfected Hrs–GFP. O–Q,Immunohistochemistry of endogenous Hrs and PSD95 shows colocalization to a subpopulation of puncta in dendrites of hippocampal neurons (arrows). Merged images are shown in E, H, K, N, and Q. Scale bar, 10 μm.

Fig. 8.

Hrs overexpression disrupts synaptic clustering of PSD95β and SAP97 in hippocampal neurons. A–C, Hrs expressed in hippocampal neurons (green) localizes to the surface of large vesicles in the proximal dendrites that do not overlap with synaptophysin-staining synaptic clusters (red). D–F, These proximal Hrs-positive vesicles are positive for PSD95, which is also clustered at distal synaptic sites that lack Hrs. G–I, When cotransfected with Hrs (red), PSD95α–GFP (green) staining retains its synaptically clustered distribution. J–L, In contrast, Hrs disrupts synaptic clustering of PSD95β–GFP, which instead colocalizes with the proximal Hrs-positive vesicles. M–O, SAP97–GFP also accumulates at the site of Hrs-positive vesicles. Merged images are shown in C, F, I, andL. Scale bar, 10 μm.