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

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.

Regulation of neuronal nitric oxide synthase through alternative transcripts.

Nitric oxide (NO) participates in diverse physiological processes ranging from neurotransmission to muscle relaxation. Neuronal-derived NO can be either beneficial or detrimental depending on the cellular context. Neuronal NO synthase (nNOS) must therefore be tightly regulated. One level of regulation involves synthesis of numerous nNOS mRNA transcripts. At least six distinct molecular species of nNOS mRNA are expressed in a tissue and developmentally-regulated manner. Alternative splicing allows the creation of nNOS proteins differing in both enzymatic characteristics and structural features. As one example, we find that there are nNOS mRNAs lacking exon 2. One isoform, nNOS beta, retains full enzymatic activity but lacks a major protein-protein interaction domain (PDZ domain) responsible for targeting nNOS to synaptic membranes. This alternative splicing produces a mislocalized but fully active protein which may be relevant to certain pathologies. As evidence of this, we find that many human brain tumors express an alternatively spliced form of nNOS that co-migrates with nNOS beta, and lacks exon 2. Finally, we also find a 2.5-kb testis-specific nNOS mRNA corresponding to the C-terminal reductase domain of nNOS whose function is unclear.

Binding of the inward rectifier K+ channel Kir 2.3 to PSD-95 is regulated by protein kinase A phosphorylation.

Dynamic regulation of ion channel interactions with the cytoskeleton mediates aspects of synaptic plasticity, yet mechanisms for this process are largely unknown. Here, we report that two inwardly rectifying K+ channels, Kir 2.1 and 2.3, bind to PSD-95, a cytoskeletal protein of postsynaptic densities that clusters NMDA receptors and voltage-dependent K+ channels. Kir 2.3 colocalizes with PSD-95 in neuronal populations in forebrain, and a PSD-95/Kir 2.3 complex occurs in hippocampus. Within the C-terminal tail of Kir 2.3, a serine residue critical for interaction with PSD-95, is also a substrate for phosphorylation by protein kinase A (PKA). Stimulation of PKA in intact cells causes rapid dissociation of the channel from PSD-95. This work identifies a physiological mechanism for regulating ion channel interactions with the postsynaptic density.

Selective loss of sarcolemmal nitric oxide synthase in Becker muscular dystrophy.

Becker muscular dystrophy is an X-linked disease due to mutations of the dystrophin gene. We now show that neuronal-type nitric oxide synthase (nNOS), an identified enzyme in the dystrophin complex, is uniquely absent from skeletal muscle plasma membrane in many human Becker patients and in mouse models of dystrophinopathy. An NH2-terminal domain of nNOS directly interacts with alpha 1-syntrophin but not with other proteins in the dystrophin complex analyzed. However, nNOS does not associate with alpha 1-syntrophin on the sarcolemma in transgenic mdx mice expressing truncated dystrophin proteins. This suggests a ternary interaction of nNOS, alpha 1-syntrophin, and the central domain of dystrophin in vivo, a conclusion supported by developmental studies in muscle. These data indicate that proper assembly of the dystrophin complex is dependent upon the structure of the central rodlike domain and have implications for the design of dystrophin-containing vectors for gene therapy.

Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains.

Neuronal nitric oxide synthase (nNOS) is concentrated at synaptic junctions in brain and motor endplates in skeletal muscle. Here, we show that the N-terminus of nNOS, which contains a PDZ protein motif, interacts with similar motifs in postsynaptic density-95 protein (PSD-95) and a related novel protein, PSD-93.nNOS and PSD-95 are coexpressed in numerous neuronal populations, and a PSD-95/nNOS complex occurs in cerebellum. PDZ domain interactions also mediate binding of nNOS to skeletal muscle syntrophin, a dystrophin-associated protein. nNOS isoforms lacking a PDZ domain, identified in nNOSdelta/delta mutant mice, do not associate with PSD-95 in brain or with skeletal muscle sarcolemma. Interaction of PDZ-containing domains therefore mediates synaptic association of nNOS and may play a more general role in formation of macromolecular signaling complexes.

Expression of nitric oxide synthase in human central nervous system tumors.

The nitric oxide synthases (NOS) are a family of related enzymes which regulate the production of NO, a free radical gas implicated in a wide variety of biological processes. Vasodilation and increased tumor blood flow, increased vascular permeability, modulation of host tumoricidal activity, and free radical injury to tumor cells and adjacent normal tissues are pathophysiological features of malignant tumors that may be mediated by NO. We examined human brain tumors for three NOS isoforms and NADPH diaphrase, a histochemical marker of NOS activity in the brain. We detected increased expression of the brain and endothelial forms of NOS [NOS I and NOS II, respectively (C. Nathan and Q. Xie. Cell, 78: 915-919, 1994)] in astrocytic tumors, and the highest levels of expression was found in higher grade tumors. Each of these two isoforms was found in tumor cells and tumor endothelial cells. The macrophage isoform of NOS (NOS III) was less frequently detected and expressed at a lower level, predominantly in tumor endothelial cells. NADPH diaphorase staining for NOS activity paralleled this pattern of NOS expression. Western blot analysis of tumor tissues for these NOS isoforms confirmed these observations. Our data indicate that malignant central nervous system neoplasms express unexpectedly high levels of NOS and suggest that NO production may be associated with pathophysiological processes important to these tumors.