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.

Actinin-associated LIM protein: identification of a domain interaction between PDZ and spectrin-like repeat motifs.

PDZ motifs are protein-protein interaction domains that often bind to COOH-terminal peptide sequences. The two PDZ proteins characterized in skeletal muscle, syntrophin and neuronal nitric oxide synthase, occur in the dystrophin complex, suggesting a role for PDZ proteins in muscular dystrophy. Here, we identify actinin-associated LIM protein (ALP), a novel protein in skeletal muscle that contains an NH2-terminal PDZ domain and a COOH-terminal LIM motif. ALP is expressed at high levels only in differentiated skeletal muscle, while an alternatively spliced form occurs at low levels in the heart. ALP is not a component of the dystrophin complex, but occurs in association with alpha-actinin-2 at the Z lines of myofibers. Biochemical and yeast two-hybrid analyses demonstrate that the PDZ domain of ALP binds to the spectrin-like motifs of alpha-actinin-2, defining a new mode for PDZ domain interactions. Fine genetic mapping studies demonstrate that ALP occurs on chromosome 4q35, near the heterochromatic locus that is mutated in fascioscapulohumeral muscular dystrophy.

Dual palmitoylation of PSD-95 mediates its vesiculotubular sorting, postsynaptic targeting, and ion channel clustering.

Postsynaptic density-95 (PSD-95/SAP-90) is a palmitoylated peripheral membrane protein that scaffolds ion channels at excitatory synapses. To elucidate mechanisms for postsynaptic ion channel clustering, we analyzed the cellular trafficking of PSD-95. We find that PSD-95 transiently associates with a perinuclear membranous compartment and traffics with vesiculotubular structures, which migrate in a microtubule-dependent manner. Trafficking of PSD-95 with these vesiculotubular structures requires dual palmitoylation, which is specified by five consecutive hydrophobic residues at the NH(2) terminus. Mutations that disrupt dual palmitoylation of PSD-95 block both ion channel clustering by PSD-95 and its synaptic targeting. Replacing the palmitoylated NH(2) terminus of PSD-95 with alternative palmitoylation motifs at either the NH(2) or COOH termini restores ion channel clustering also induces postsynaptic targeting, respectively. In brain, we find that PSD-95 occurs not only at PSDs but also in association with intracellular smooth tubular structures in dendrites and spines. These data imply that PSD-95 is an itinerant vesicular protein; initial targeting of PSD-95 to an intracellular membrane compartment may participate in postsynaptic ion channel clustering by PSD-95.

Tat protein from human immunodeficiency virus forms a metal-linked dimer.

Tat, the transactivating protein from HIV, forms a metal-linked dimer with metal ions bridging cysteine-rich regions from each monomer. This novel arrangement is distinct from the "zinc finger" domain observed in other eukaryotic regulatory proteins. Ultraviolet absorption spectra show that Tat binds two Zn2+ or two Cd2+ ions per monomer, and electrophoresis of the Tat-metal complexes demonstrates that the protein forms metal-linked dimers. Partial proteolysis and circular dichroism spectra suggest that metal binding has its primary effects in the cysteine-rich region and relatively little effect on the folding of other regions. These results suggest new directions for biological studies and new approaches to drug design.

Autoradiographic imaging of phosphoinositide turnover in the brain.

With [3H]cytidine as a precursor, phosphoinositide turnover can be localized in brain slices by selective autoradiography of the product [3H]cytidine diphosphate diacylglycerol, which is membrane-bound. In the cerebellum, glutamatergic stimulation elicits an increase of phosphoinositide turnover only in Purkinje cells and the molecular layer. In the hippocampus, both glutamatergic and muscarinic cholinergic stimulation increase phosphoinositide turnover, but with distinct localizations. Cholinergic stimulation affects CA1, CA3, CA4, and subiculum, whereas glutamatergic effects are restricted to the subiculum and CA3. Imaging phosphoinositide turnover in brain slices, which are amenable to electrophysiologic studies, will permit a dynamic localized analysis of regulation of this second messenger in response to synaptic stimulation of specific neuronal pathways.

Nitric oxide: a physiologic mediator of penile erection.

Nitric oxide (NO) is a cytotoxic agent of macrophages, a messenger molecule of neurons, and a vasodilator produced by endothelial cells. NO synthase, the synthetic enzyme for NO, was localized to rat penile neurons innervating the corpora cavernosa and to neuronal plexuses in the adventitial layer of penile arteries. Small doses of NO synthase inhibitors abolished electrophysiologically induced penile erections. These results establish NO as a physiologic mediator of erectile function.

PSD-95 involvement in maturation of excitatory synapses.

PSD-95 is a neuronal PDZ protein that associates with receptors and cytoskeletal elements at synapses, but whose function is uncertain. We found that overexpression of PSD-95 in hippocampal neurons can drive maturation of glutamatergic synapses. PSD-95 expression enhanced postsynaptic clustering and activity of glutamate receptors. Postsynaptic expression of PSD-95 also enhanced maturation of the presynaptic terminal. These effects required synaptic clustering of PSD-95 but did not rely on its guanylate kinase domain. PSD-95 expression also increased the number and size of dendritic spines. These results demonstrate that PSD-95 can orchestrate synaptic development and are suggestive of roles for PSD-95 in synapse stabilization and plasticity.

Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex.

The PDZ protein interaction domain of neuronal nitric oxide synthase (nNOS) can heterodimerize with the PDZ domains of postsynaptic density protein 95 and syntrophin through interactions that are not mediated by recognition of a typical carboxyl-terminal motif. The nNOS-syntrophin PDZ complex structure revealed that the domains interact in an unusual linear head-to-tail arrangement. The nNOS PDZ domain has two opposite interaction surfaces-one face has the canonical peptide binding groove, whereas the other has a beta-hairpin "finger." This nNOS beta finger docks in the syntrophin peptide binding groove, mimicking a peptide ligand, except that a sharp beta turn replaces the normally required carboxyl terminus. This structure explains how PDZ domains can participate in diverse interaction modes to assemble protein networks.

Transient nitric oxide synthase neurons in embryonic cerebral cortical plate, sensory ganglia, and olfactory epithelium.

Neuronal nitric oxide synthase (NOS), visualized immunohistochemically or with NADPH diaphorase histochemistry, is transiently expressed in discrete areas of the developing rat nervous system. In the brain transient NOS expression occurs in the cerebral cortical plate. At E15-E19, the majority of cells in the plate stain, with their processes extending through the corpus striatum to the thalamus. This staining decreases after birth and vanishes by the 15th postnatal day. Neurons in olfactory epithelium also express NOS from E15 till early postnatal life. In embryonic sensory ganglia virtually all neuronal cells are NOS positive, whereas by early adulthood only 1% express NOS. By contrast to these areas of transient NOS expression, in other neuronal sites NOS staining appears after cell bodies cease dividing and cells extend processes, and the staining persists in adult life. The transient expression of neuronal NOS may reflect a role in developmental processes such as programmed cell death.

N-terminal palmitoylation of PSD-95 regulates association with cell membranes and interaction with K+ channel Kv1.4.

Ion channels and associated signal transduction cascades are clustered at excitatory synapses by PSD-95 and related PDZ-containing proteins. Mechanisms that target PSD-95 to synaptic membranes, however, are unknown. Here, PSD-95 is shown to partition as an integral membrane protein in brain homogenates. Metabolic labeling of brain slices or cultured cells demonstrates that PSD-95 is modified by thioester-linked palmitate, a long chain fatty acid that targets proteins to cell membranes. In fact, PSD-95 is a major palmitoylated protein in intact cells, and palmitoylated PSD-95 partitions exclusively with cell membranes. Mutagenesis indicates that palmitoylation of PSD-95 occurs on conserved N-terminal cysteines 3 and 5. Palmitoylation-deficient mutants of PSD-95 do not partition as integral membrane proteins and do not participate in PDZ-ion channel interactions in vivo. This work identifies palmitoylation as a critical regulatory mechanism for receptor interactions with PSD-95.

Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs.

During synaptic development, proteins aggregate at specialized pre- and postsynaptic structures. Mechanisms that mediate protein clustering at these sites remain unknown. To investigate this process, we analyzed synaptic targeting of a postsynaptic density protein, PSD-95, by expressing green fluorescent protein- (GFP-) tagged PSD-95 in cultured hippocampal neurons. We find that postsynaptic clustering relies on three elements of PSD-95: N-terminal palmitoylation, the first two PDZ domains, and a C-terminal targeting motif. In contrast, disruptions of PDZ3, SH3, or guanylate kinase (GK) domains do not affect synaptic targeting. Palmitoylation is sufficient to target the diffusely expressed SAP-97 to synapses, and palmitoylation cannot be replaced with alternative membrane association motifs, suggesting that a specialized synaptic lipid environment mediates postsynaptic clustering. The requirements for PDZ domains and a C-terminal domain of PSD-95 indicate that protein-protein interactions cooperate with lipid interactions in synaptic targeting.

A novel K+ channel with unique localizations in mammalian brain: molecular cloning and characterization.

Using a cDNA library prepared from circumvallate papillae of rat tongue, we have identified, cloned, and sequenced a novel K+ channel, designated cdrk. The cdrk channel appears to be a member of the Shab subfamily, most closely resembling drk1. Electrophysiologic analysis of expressed cdrk channels reveals delayed rectifier properties similar to those of drk1 channels. Localizations of cdrk mRNA in rat brain and peripheral tissues, assessed by in situ hybridization and Northern blot analysis, differ from any other reported K+ channels. In the brain cdrk mRNA is most concentrated in granule cells of the olfactory bulb and cerebellum. In peripheral tissues, mRNAs for cdrk and drk1 are reciprocally localized, indicating that the K+ channel properties contributed by mammalian Shab homologs may be important in a variety of excitable tissues.

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.

Nitric oxide mediates the formation of synaptic connections in developing and regenerating olfactory receptor neurons.

Nitric oxide (NO) is a diffusible free radical that functions as a second messenger and neurotransmitter. NO synthase (NOS) is highly and transiently expressed in neurons of the developing olfactory epithelium during migration and establishment of primary synapses in the olfactory bulb. NOS is first expressed at E11 in cells of the presumptive nervous layer of the olfactory placode. NOS immunoreactivity persists in the descendants of these cells that differentiate into embryonic olfactory receptor neurons (ORNs). Olfactory NOS expression in the ORN and in its afferents rapidly declines after birth and is undetectable by P7. Following bulbectomy, NOS expression is rapidly induced in the regenerating ORN and is particularly enriched in their outgrowing axons. Immunoblot and Northern blot analyses similarly demonstrate an induction of NOS protein and mRNA expression, respectively, the highest levels of which coincide with peaks of ORN regeneration. These data argue against a role for NO in odorant-sensitive signal transduction, but suggest a prominent function for NO in activity-dependent establishment of connections in both developing and regenerating olfactory neurons.

Nitric oxide synthase protein and mRNA are discretely localized in neuronal populations of the mammalian CNS together with NADPH diaphorase.

Nitric oxide is a free radical that has been recently recognized as a neural messenger molecule. Nitric oxide synthase has now been purified and molecularly cloned from brain. Using specific antibodies and oligonucleotide probes, we have localized brain nitric oxide synthase to discrete neuronal populations in the rat and primate brain. Nitric oxide synthase is exclusively neuronal, and its localization is absolutely coincident with NADPH diaphorase staining in both rat and primate.

PDZ proteins bind, cluster, and synaptically colocalize with Eph receptors and their ephrin ligands.

Localizing cell surface receptors to specific subcellular positions can be critical for their proper functioning, as most notably demonstrated at neuronal synapses. PDZ proteins apparently play critical roles in such protein localizations. Receptor tyrosine kinases have not been previously shown to interact with PDZ proteins in vertebrates. We report that Eph receptors and their membrane-linked ligands all contain PDZ recognition motifs and can bind and be clustered by PDZ proteins. In addition, we find that Eph receptors and ligands colocalize with PDZ proteins at synapses. Thus, PDZ proteins may play critical roles in localizing vertebrate receptor tyrosine kinases and/or their ligands and may be particularly important for Eph function in guidance or patterning or at the synapse.

Cypin: a cytosolic regulator of PSD-95 postsynaptic targeting.

Postsynaptic density 95 (PSD-95/SAP-90) is a membrane associated guanylate kinase (GK) PDZ protein that scaffolds glutamate receptors and associated signaling networks at excitatory synapses. Affinity chromatography identifies cypin as a major PSD-95-binding protein in brain extracts. Cypin is homologous to a family of hydrolytic bacterial enzymes and shares some similarity with collapsin response mediator protein (CRMP), a cytoplasmic mediator of semaphorin III signalling. Cypin is discretely expressed in neurons and is polarized to basal membranes in intestinal epithelial cells. Overexpression of cypin in hippocampal neurons specifically perturbs postsynaptic trafficking of PSD-95 and SAP-102, an effect not produced by overexpression of other PDZ ligands. In fact, PSD-95 can induce postsynaptic clustering of an otherwise diffusely localized K+ channel, Kv1.4. By regulating postsynaptic protein sorting, cypin may influence synaptic development and plasticity.

Inhibition of sphincter of Oddi function by the nitric oxide carrier S-nitroso-N-acetylcysteine in rabbits and humans.

Nitric oxide (NO) is an inhibitor of gastrointestinal smooth muscle. Model systems of the gut predict the NO will complex with biological thiol (SH) groups, yielding S-nitrosothiols (RS-NO), which may limit the propensity to form mutagenic nitrosamines. The inhibitory effects of NO and its biologically relevant adducts on sphincter of Oddi (SO) motility have been inferred from animal studies; however, their importance in regulating human SO is not known. The objectives of this study were to (a) provide histologic confirmation of nitric oxide synthase (NOS) in human SO; (b) characterize the pharmacology of S-nitroso-N-acetylcysteine (SNAC), an exemplary S-nitrosothiol, on SO motility in a rabbit model; and (c) study the effects of topical SNAC on SO motility in humans. Immunocytochemical and histochemical identification of NOS was performed in human SO. The pharmacologic response of SNAC was defined in isolated rabbit SO using a standard bioassay. Topical SNAC was then applied to the duodenal papilla in patients undergoing endoscopic retrograde cholangiopancreatography (ERCP) and biliary manometry. NOS was localized to nerve fibers and bundles of the SO in rabbits and humans. SNAC inhibited spontaneous motility (frequency and amplitude) as well as acetylcholine-induced elevations in SO basal pressure in the rabbit model. In patients undergoing ERCP and biliary manometry, topical SNAC inhibited SO contraction freqency, basal pressure, and duodenal motility. NOS is localized to neural elements in human SO, implicating a role for NO in regulating SO function. Supporting this concept, SNAC is an inhibitor of SO and duodenal motility when applied topically to humans during ERCP. Our data suggest a novel clinical approach using local NO donors to control gastrointestinal motility and regulate sphincteric function.

Actinin-associated LIM protein-deficient mice maintain normal development and structure of skeletal muscle.

The actinin-associated LIM protein, ALP, is the prototype of a large family of proteins containing an N-terminal PDZ domain and a C-terminal LIM domain. These PDZ-LIM proteins are components of the muscle cytoskeleton and occur along the Z lines owing to interaction of the PDZ domain with the spectrin-like repeats of alpha-actinin. Because PDZ and LIM domains are typically found in proteins that mediate cellular signaling, PDZ-LIM proteins are suspected to participate in muscle development. Interestingly the ALP gene occurs at 4q35 near the heterochromatic region mutated in facioscapulohumeral muscular dystrophy, indicating a possible role for ALP in this disease. Here, we describe the generation and analysis of mice lacking the ALP gene. Surprisingly, the ALP knockout mice show no gross histological abnormalities and maintain sarcolemmal integrity as determined by serum pyruvate kinase assays. The absence of a dystrophic phenotype in these mice suggests that down-regulation of ALP does not participate in facioscapulohumeral muscular dystrophy. These data suggest that ALP does not participate in muscle development or that an alternative PDZ-LIM protein can compensate for the lack of ALP.

PDZ domain of neuronal nitric oxide synthase recognizes novel C-terminal peptide sequences.

PDZ domains are multifunctional protein-interaction motifs that often bind to the C-terminus of protein targets. Nitric oxide (NO), an endogenous signaling molecule, plays critical roles in nervous, immune, and cardiovascular function. Although there are numerous physiological functions for neuron-derived NO, produced primarily by the neuronal NO synthase (nNOS), excess nNOS activity mediates brain injury in cerebral ischemia and in animal models of Parkinson's disease. Subcellular localization of nNOS activity must therefore be tightly regulated. To determine ligands for the PDZ domain of nNOS, we screened 13 billion distinct peptides and found that the nNOS-PDZ domain binds tightly to peptides ending Asp-X-Val. This differs from the only known (Thr/Ser)-X-Val consensus that interacts with PDZ domains from PSD-95. Preference for Asp at the -2 peptide position is mediated by Tyr-77 of nNOS. A Y77D78 to H77E78 substitution changes the binding specificity from Asp-X-Val to Thr-X-Val. Guided by the Asp-X-Val consensus, candidate nNOS interacting proteins have been identified including glutamate and melatonin receptors. Our results demonstrate that PDZ domains have distinct peptide binding specificity.