Localization of postsynaptic density-93 to dendritic microtubules and interaction with microtubule-associated protein 1A

Abstract

Postsynaptic density-93 (PSD-93)/Chapsyn-110 is a member of the membrane-associated guanylate kinase (MAGUK) family of PDZ domain-containing proteins. MAGUKs are widely expressed in the brain and are critical elements of the cytoskeleton and of certain synapses. In the ultrastructural studies that are described here, PSD-93 localizes to both postsynaptic densities and dendritic microtubules of cerebellar Purkinje neurons. The microtubule localization is paralleled by a high-affinity in vivo interaction of PSD-93 via its guanylate kinase (GK) domain with microtubule-associated protein 1A (MAP1A). GK domain truncations that mimic genetically identified mutations of a Drosophila MAGUK, discs-large, disrupt the GK/MAP-1A interaction. Additional biochemical experiments demonstrate that intact MAGUKs do not bind to MAP1A as effectively as do isolated GK domains. This appears to be attributable to an intramolecular inhibition of the GK domain by the PDZs, because GK binding activity of full-length MAGUKs is partially restored by a variety of PDZ ligands, including the C termini of NMDA receptor 2B, adenomatous polyposis coli (APC), and CRIPT. Beyond demonstrating a novel cytoskeletal link for PSD-93, these experiments support a model in which intramolecular interactions between the multiple domains of MAGUKs regulate intermolecular associations and thereby may play a role in the proper targeting and function of MAGUK proteins.

Fig. 1.

Ultrastructural localization of PSD-93 in cerebellar Purkinje neurons. Immunoperoxidase staining of rat cerebellum demonstrates that PSD-93 is enriched in Purkinje cell bodies and dendritic arbors in the molecular layer (A); no labeling is seen in sections incubated with preimmune serum (B). Electron micrography of a thin section from themolecular layer (C) shows specific labeling of microtubules (arrows), smooth endoplasmic reticulum, and membranous subsurface cisterns in a Purkinje cell (PC) dendrite cut in cross section. The unstained profiles at the left of the Purkinje cell provide examples of unstained microtubules seen both lengthwise and in cross section (arrowheads). Inset shows at higher magnification a portion of the Purkinje cell dendrite containing stained endoplasmic reticulum and microtubules (arrow) as well as some unstained microtubules. D, Stained Purkinje cell spines in the outer molecular layer. Immunoreaction product labels surface membranes of spine and postsynaptic densities. Scale bars: 0.5 μm in C; 0.3 μm in C,inset; 0.1 μm in D.

Fig. 2.

PSD-95 interacts with MAP1A and a set of proteins of 100–140 kDa. A, Overlay assay (OL) with [³²P]PSD-95 identifies PSD-95 binding proteins in rat brain (B), but not liver (L), extracts. The corresponding Coomassie blue-stained (CB) gel is shown on theleft. B, Expression cloning from a λgt11 rat brain cDNA library with [³²P]PSD-95 yielded a set of PSD-95 interacting clones encoding either MAP1A or APC. Autoradiographs of nitrocellulose filters at either the primary (1°) or secondary (2°) stage of MAP1A cDNA clone purification are shown in the top circles. Probing with³²P-labeled domains of PSD-95 reveals that the GK domain of PSD-95 selectively interacts with MAP1A, whereas the PDZ domains of PSD-95 interact with APC. C, Domain structure of MAP1A illustrates that the GK interacting clones overlap in a small region that represents the GK binding site. The locations of the light chain 2 (LC2), self-similarity 1 (SS1), and self-similarity 2 (SS2) domains are indicated.

Fig. 3.

The GK domain potently binds to MAP1A and to proteins of the PSD. A, Immunoprecipitation of crude brain extracts (100 μg) with a monoclonal antibody to MAP1A (Sigma), followed by overlay assay with [³²P]guanylate kinase domain demonstrates that the 350 kDa guanylate kinase-interacting protein is MAP1A. This 350 kDa protein also is enriched specifically in a purified brain microtubule preparation (10 μg). The subcellular fractionation shows the enrichment of the 100–140 kDa GK-interacting protein bands in Triton X-100 soluble synaptic membranes (10 μg) and the postsynaptic density (10 μg). Immunoprecipitation of SDS-solubilized PSD fraction with an antiserum to PSD-95, followed by overlay analysis, shows specific association of the 100–140 kDa bands with PSD-95. These proteins were not precipitated with preimmune serum. B, Saturation binding analysis shows that MAP1A binds with high affinity to the GK domain.

Fig. 4.

MAP1A associates with PSD-93 in vivo. A, MAP1A from brain extracts is retained selectively by GST fusion proteins of the GK domains of PSD-93, PSD-95, and SAP-97 but does not bind to GST fusions containing the SH3 or PDZ domains of PSD-95 or to a GK domain mutant (GK-mut) lacking the final 26 amino acids of PSD-95. nNOS specifically associates with the PDZ domains of PSD-95. “Pull-down” assays with GST fusion proteins were performed from brain extracts as described (Brenman et al., 1996a), and the resulting eluates immunoblotted sequentially for MAP1A and nNOS (Transduction Laboratories, Lexington, KY). The input is equal to 10% of the extract loaded onto the GST fusion protein columns. B, Indirect immunofluorescence (200× magnification) reveals extensive colocalization of PSD-93 and MAP1A in rat brain. MAP1A (green; a, g) and PSD-93 (red; b, h) colocalize in somatodendritic profiles in Purkinje neurons in cerebellum (a, b) and the CA3 region of hippocampus (g, h). PSD-93 (red; e) does not colocalize extensively with MAP2 (green; d) in cerebellum. Composite images show the colocalization of signals (yellow; c, f, i).C, Antiserum to PSD-93 specifically immunoprecipitates MAP1A from brain extracts. Immunoprecipitation followed by immunoblotting reveals that MAP1A is associated with PSD-93 in Triton X-100-solubilized rat cerebellar extracts. Conversely, MAP2 and calcium/calmodulin-dependent protein kinase II (CamK II) do not coimmunoprecipitate with PSD-93.

Fig. 5.

GK binding activity of full-length MAGUK is stimulated by a PDZ ligand. A, Overlay assay was used to compare the binding activity of full-length PSD-95 with that of the isolated GK domain of PSD-95. Autoradiography shows that the isolated GK domain interacts strongly with MAP1A and the 100–140 kDa bands from brain extracts, whereas the full-length MAGUK shows much weaker binding activity (∼5%). A 9-amino-acid peptide corresponding to the tail of NR2B, KLSSIESDV (NR2B), dramatically increases the binding activity of full-length MAGUK (approximately eightfold), whereas a similar mutated peptide, KLSSIEADA (NR2B**), is ineffective. After autoradiography the interacting protein bands were excised and counted in liquid scintillant. B, NR2B peptide stimulates GK binding activity of full-length MAGUK to purified MAP1A fusion protein. NR2B peptides with either a single mutation KLSSIESDA (NR2B*) or a double mutation (NR2B**) do not activate GK binding activity. GK binding activity was quantitated by dot blot assay, monitoring the binding of full-length PSD-95 to a purified MAP1A fusion protein, as described in Materials and Methods. C, The affinity of NR2B peptide for PDZ domains in solution is in the micromolar range. PSD-95-GST fusion proteins were used to “pull down” K⁺channel K_v1.4 from Triton X-100-solubilized brain extract. NR2B peptides were added to the pulldown to block the association of the fusion proteins with K_v1.4 in vitro. Mutant peptide had no effect.

Fig. 6.

PDZ domains 2 and 3 autoinhibit GK domain.A, Schematic showing design of histidine-tagged fusion proteins of PSD-95. “Far Western” analysis demonstrates that PDZ2 and PDZ3 domains inhibit the binding of the GK domain to a purified MAP1A-GST fusion protein. A peptide corresponding to the tail of NR2B, but not a mutated peptide NR2B*, stimulates the binding activity of the full-length MAGUK to purified MAP1A. The NR2B peptide did not affect the binding of the GK construct lacking PDZ domains (ΔPDZ1–3). None of the MAGUK protein constructs bound to GST alone. CB, Coomassie blue. B, C-terminal peptides (9-mers; at 200 μm) corresponding to NR2B, K⁺ channel K_v1.4, APC, or CRIPT all stimulate the binding of full-length MAGUK to MAP1A, as measured by far Western assay. C, Quantitative binding assay shows that distinct PDZ domain ligands (200 μm) stimulate the GK binding activity of full-length MAGUK from 5- to 10-fold. The greatest activation is observed consistently with CRIPT, a PDZ3 ligand. GK binding activity was quantitated by dot blot assay, monitoring the binding of full-length PSD-95 to a purified MAP1A fusion protein.D, A C-terminal peptide corresponding to NR2B stimulates the binding of three distinct MAGUKs, PSD-93, PSD-95, and SAP-97, to MAP1A.