Role of SNTA1 in Rac1 activation, modulation of ROS generation, and migratory potential of human breast cancer cells.

BACKGROUND: Alpha-1-syntrophin (SNTA1) has been implicated in the activation of Rac1. However, the underlying mechanism has not yet been explored. Here, we show that a novel complex, involving SNTA1, P66shc, and Grb2 proteins, is involved in Rac1 activation. METHODS: Co-immunoprecipitation assays were used to show the complex formation, while siRNAs and shRNAs were used to downregulate expression of these proteins. Various Rac1 activation assays and functional assays, such as migration assays, in vitro wound healing assays, cell proliferation assays, and ROS generation assays, were also performed. RESULTS: The results showed a significant increase in activation of Rac1 when SNTA1 and P66shc were overexpressed, whereas depletion of SNTA1 and P66shc expression effectively reduced the levels of active Rac1. The results indicated a significant displacement of Sos1 protein from Grb2 when SNTA1 and P66shc are overexpressed in breast cancer cell lines, resulting in Sos1 predominantly forming a complex with Eps8 and E3b1. In addition, the SNTA1/P66shc-mediated Rac1 activation resulted in an increase in reactive oxygen species (ROS) production and migratory potential in human breast cancer cells. CONCLUSION: Together, our results present a possible mechanism of Rac1 activation involving SNTA1 and emphasise its role in ROS generation, cell migration, and acquisition of malignancy.

Differential association of syntrophin pairs with the dystrophin complex.

The syntrophins are a multigene family of intracellular dystrophin-associated proteins comprising three isoforms, alpha1, beta1, and beta2. Based on their domain organization and association with neuronal nitric oxide synthase, syntrophins are thought to function as modular adapters that recruit signaling proteins to the membrane via association with the dystrophin complex. Using sequences derived from a new mouse beta1-syntrophin cDNA, and previously isolated cDNAs for alpha1- and beta2-syntrophins, we prepared isoform-specific antibodies to study the expression, skeletal muscle localization, and dystrophin family association of all three syntrophins. Most tissues express multiple syntrophin isoforms. In mouse gastrocnemius skeletal muscle, alpha1- and beta1-syntrophin are concentrated at the neuromuscular junction but are also present on the extrasynaptic sarcolemma. beta1-syntrophin is restricted to fast-twitch muscle fibers, the first fibers to degenerate in Duchenne muscular dystrophy. beta2-syntrophin is largely restricted to the neuromuscular junction. The sarcolemmal distribution of alpha1- and beta1-syntrophins suggests association with dystrophin and dystrobrevin, whereas all three syntrophins could potentially associate with utrophin at the neuromuscular junction. Utrophin complexes immunoisolated from skeletal muscle are highly enriched in beta1- and beta2-syntrophins, while dystrophin complexes contain mostly alpha1- and beta1-syntrophins. Dystrobrevin complexes contain dystrophin and alpha1- and beta1-syntrophins. From these results, we propose a model in which a dystrophin-dystrobrevin complex is associated with two syntrophins. Since individual syntrophins do not have intrinsic binding specificity for dystrophin, dystrobrevin, or utrophin, the observed preferential pairing of syntrophins must depend on extrinsic regulatory mechanisms.

In vivo requirement of the alpha-syntrophin PDZ domain for the sarcolemmal localization of nNOS and aquaporin-4.

alpha-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of alpha-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of alpha-syntrophin in vivo by generating transgenic mouse lines expressing full-length alpha-syntrophin or a mutated alpha-syntrophin lacking the PDZ domain (Delta PDZ). The Delta PDZ alpha-syntrophin displaced endogenous alpha- and beta 1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the alpha-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an alpha-syntrophin-null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length alpha-syntrophin, not the Delta PDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound alpha-syntrophin PDZ domain.

Absence of alpha-syntrophin leads to structurally aberrant neuromuscular synapses deficient in utrophin.

The syntrophins are a family of structurally related proteins that contain multiple protein interaction motifs. Syntrophins associate directly with dystrophin, the product of the Duchenne muscular dystrophy locus, and its homologues. We have generated alpha-syntrophin null mice by targeted gene disruption to test the function of this association. The alpha-Syn(-/)- mice show no evidence of myopathy, despite reduced levels of alpha-dystrobrevin-2. Neuronal nitric oxide synthase, a component of the dystrophin protein complex, is absent from the sarcolemma of the alpha-Syn(-/)- mice, even where other syntrophin isoforms are present. alpha-Syn(-/)- neuromuscular junctions have undetectable levels of postsynaptic utrophin and reduced levels of acetylcholine receptor and acetylcholinesterase. The mutant junctions have shallow nerve gutters, abnormal distributions of acetylcholine receptors, and postjunctional folds that are generally less organized and have fewer openings to the synaptic cleft than controls. Thus, alpha-syntrophin has an important role in synapse formation and in the organization of utrophin, acetylcholine receptor, and acetylcholinesterase at the neuromuscular synapse.

Pentapeptide (proctolin) associated with an identified neuron.

Individual neurons can be recognized and identified anatomically, physiologically, and biochemically in the insect central nervous system. Biochemical analyses of extracts prepared from one such identified neuron show it to be associated with a bioactive pentapeptide called proctolin. This peptide may be a neurotransmitter, and a preparation is established in which its physiological action can be studied at the cellular level.

Calcium channels coupled to glutamate release identified by omega-Aga-IVA.

Presynaptic calcium channels are crucial elements of neuronal excitation-secretion coupling. In mammalian brain, they have been difficult to characterize because most presynaptic terminals are too small to probe with electrodes, and available pharmacological tools such as dihydropyridines and omega-conotoxin are largely ineffective. Subsecond measurements of synaptosomal glutamate release have now been used to assess presynaptic calcium channel activity in order to study the action of peptide toxins from the venom of the funnel web spider Agelenopsis aperta, which is known to inhibit dihydropyridine and omega-conotoxin-resistant neuronal calcium currents. A presynaptic calcium channel important in glutamate release is shown to be omega-Aga-IVA sensitive and omega-conotoxin resistant.

Peptide cotransmitter at a neuromuscular junction.

The neuropeptide proctolin (H-Arg-Tyr-Leu-Pro-Thr-OH) is present in the nerve terminals of an identified slow skeletal motoneuron in the cockroach. Proctolin is released onto the target muscle, a coxal depressor, by neuron stimulation and by depolarization with potassium. The physiological action of the motoneuron suggests that proctolin acts as a cotransmitter. Proctolin and neural stimulation produce delayed and sustained contractile effects without muscle depolarization.

Identification of ecdysis-triggering hormone from an epitracheal endocrine system.

Developing insects repeatedly shed their cuticle by means of a stereotyped behavior called ecdysis, thought to be initiated by the brain peptide eclosion hormone. Here an ecdysis-triggering hormone, Mas-ETH, is described from the tobacco hornworm Manduca sexta. Mas-ETH contains 26 amino acids and is produced by a segmentally distributed endocrine system of epitracheal glands (EGs). The EGs undergo a marked reduction in volume, appearance, and immunohistochemical staining during ecdysis, at which time Mas-ETH is found in the hemolymph. Injection of EGs extract or synthetic Mas-ETH into pharate larvae, pupae, or adults initiates preecdysis within 2 to 10 minutes, followed by ecdysis. Sensitivity to injected Mas-ETH appears much earlier before ecdysis and occurs with shorter latency than that reported for eclosion hormone. The isolated central nervous system responds to Mas-ETH, but not to eclosion hormone, with patterned motor bursting corresponding to in vivo preecdysis and ecdysis. Mas-ETH may be an immediate blood-borne trigger for ecdysis through a direct action on the nervous system.

Differential activation of adenosine receptors decreases N-type but potentiates P-type Ca2+ current in hippocampal CA3 neurons.

Adenosine is released in the brain in significant quantities in response to increased cellular activity. Adenosine has been shown either to decrease synaptic transmission or to produce an excitatory response in hippocampal synapses, resulting in increased glutamate release. Previous reports have shown that adenosine or its analogs reduced Ca2+ current in dorsal root ganglion and hippocampal neurons. Here we show that the selective activation of adenosine receptor subtypes has different effects on Ca2+ channels from acutely isolated pyramidal neurons from the CA3 region of guinea pig hippocampus. Activation of A1 receptors inhibited primarily N-type Ca2+ current. In contrast, activation of A2b receptors resulted in significant potentiation of P-type but not N-type Ca2+ current. This potentiation could be inhibited by blocking the cAMP-dependent protein kinase. Because of the ubiquity of adenosine, the differential effects on Ca2+ channels of adenosine receptor subtype activation may have significant implications for neuronal excitability.

P-type calcium channels in rat central and peripheral neurons.

The peptide toxin omega-Aga-IVA blocked P-type Ca2+ channel current in rat Purkinje neurons (KD approximately 2 nM) but had no effect on identified T-type, L-type, or N-type currents in a variety of central and peripheral neurons. omega-Aga-IVA blocked a substantial fraction of high threshold Ca2+ channel current in neurons from the hippocampal CA1 region (mean 26%), visual cortex (32%), spinal cord (45%), and dorsal root ganglia (23%), but less in hippocampal CA3 neurons (14%) and none in sympathetic neurons. In all cases, omega-Aga-IVA block could be reversed by a brief train of strong depolarizations. There was no overlap between current blocked by omega-Aga-IVA and the fractions blocked by dihydropyridines and omega-conotoxin GVIA, but not all current resistant to dihydropyridines and omega-conotoxin was blocked by omega-Aga-IVA. The results suggest that omega-Aga-IVA is highly selective for P-type channels and that many central neurons and some peripheral neurons possess substantial P-type current.

Distinctive biophysical and pharmacological properties of class A (BI) calcium channel alpha 1 subunits.

Transcripts for the class A Ca2+ channel alpha 1 subunit (also known as BI) are present at high levels in many parts of the mammalian CNS and are widely assumed to encode the P-type Ca2+ channel. To characterize the biophysical and pharmacological properties of alpha 1A channels, macroscopic and single-channel recordings were made in Xenopus oocytes injected with alpha 1A cRNA. alpha 1-specific properties were identified by making systematic comparisons with the more familiar class C alpha 1 subunit under the condition of a standard ancillary subunit (alpha 2/delta + beta) makeup. alpha 1A currents activate and inactivate more rapidly and display steeper voltage dependence of gating than alpha 1C currents. Unlike alpha 1C, alpha 1A channels are largely insensitive to dihydropyridines and FPL 64176, but respond to the cone snail peptide omega-CTx-MVIIC(SNX-230), a potent and fairly selective inhibitor. In comparison with P-type Ca2+ channels in rat cerebellar Purkinje cells, alpha 1A channels in oocytes are approximately 10(2)-fold less sensitive to omega-Aga-IVA and approximately 10-fold more sensitive to omega-CTx-MVIIC. alpha 1A channels are not inhibited by Bay K 8644 and inactivate much more rapidly than P-type Ca2+ channels. Thus, alpha 1A is capable of generating a Ca2+ channel phenotype quite different from P-type current.

Steroid induction of a peptide hormone gene leads to orchestration of a defined behavioral sequence.

At the end of each molt, insects shed the old cuticle by performing preecdysis and ecdysis behaviors. Regulation of these centrally patterned movements involves peptide signaling between endocrine Inka cells and the CNS. In Inka cells, we have identified the cDNA and gene encoding preecdysis-triggering hormone (PETH) and ecdysis-triggering hormone (ETH), which activate these behaviors. Prior to behavioral onset, rising ecdysteroid levels induce expression of the ecdysone receptor (EcR) and ETH gene in Inka cells and evoke CNS sensitivity to PETH and ETH. Subsequent ecdysteroid decline is required for peptide release, which initiates three motor patterns in specific order: PETH triggers preecdysis I, while ETH activates preecdysis II and ecdysis. The Inka cell provides a model for linking steroid regulation of peptide hormone expression and release with activation of a defined behavioral sequence.

Interactions between entorhinal axons and target hippocampal neurons: a role for glutamate in the development of hippocampal circuitry.

A coculture system consisting of input axons from entorhinal cortex explants and target hippocampal pyramidal neurons was used to demonstrate that glutamate, released spontaneously from afferent axons, can influence both dendritic geometry of target neurons and formation of presumptive synaptic sites. Dendritic outgrowth was reduced in hippocampal neurons growing on entorhinal axons when compared with neurons growing off the axons. Presumptive presynaptic sites were observed in association with hippocampal neuron dendrites and somas. HPLC analysis showed that glutamate was released from the explants in an activity- and Ca2(+)-dependent manner. The general glutamate receptor antagonist D-glutamylglycine significantly increased dendritic outgrowth in pyramidal neurons associated with entorhinal axons and reduced presumptive presynaptic sites. Tetrodotoxin and reduction of extracellular Ca2+ also promoted dendritic outgrowth and reduced the formation of presumptive synaptic sites. The results suggest that the neurotransmitter glutamate may play important roles in the development of hippocampal circuitry.

Two forms of mouse syntrophin, a 58 kd dystrophin-associated protein, differ in primary structure and tissue distribution.

Syntrophin, a 58 kd extrinsic membrane protein, is concentrated at postsynaptic sites at the neuromuscular junction and may be involved in clustering acetylcholine receptors. In muscle and nonmuscle tissues, syntrophin is associated with dystrophin, utrophin, and two homologs of the dystrophin carboxy-terminal region. We have isolated three cDNAs encoding Torpedo and mouse syntrophins. The Torpedo cDNA encodes a full-length protein, and on Northern blots recognizes a 3.5 kb mRNA. The two mouse syntrophin cDNAs are products of separate genes but encode proteins that share 50% identity. Syntrophin-1 mRNA (2.2 kb) is expressed at highest levels in skeletal muscle. Syntrophin-2 mRNAs (2.2, 5.0, and 10 kb) are expressed in all mouse tissues examined. These patterns of expression suggest that syntrophin-1 and syntrophin-2 may associate with different members of the dystrophin family.

Neurotoxins: overview of an emerging research technology.

Neurotoxins have highly specific actions on molecular targets, and thus offer an effective means of characterizing the growing number of identified ion channels and receptors in the nervous system. This article and the Neurotoxins Supplement accompanying this issue of TINS provide a convenient reference source to facilitate the use of toxins as selective, diagnostic ligands in research. However, while many toxins exert potent actions on target receptors, it must be emphasized that their effects can be complex, and certain general pitfalls often become apparent. Some examples will be given illustrating these complexities and their impact on experimental interpretation. In addition, the potential for the purposeful creation of new 'designer' toxins using molecular cloning will also be addressed.

Differential effect of alpha-syntrophin knockout on aquaporin-4 and Kir4.1 expression in retinal macroglial cells in mice.

Aquaporin-4 water channels and the inwardly rectifying potassium channels Kir4.1 are coexpressed in a highly polarized manner at the perivascular and subvitreal endfeet of retinal Müller cells and astrocytes. The present study was aimed at resolving the anchoring mechanisms responsible for the coexpression of these molecules. Both aquaporin-4 and Kir4.1 contain PDZ-domain binding motifs at their C-termini and it was recently shown that mice with targeted disruption of the dystrophin gene display altered distribution of aquaporin-4 and Kir4.1 in the retina. To test our hypothesis that alpha-syntrophin (a PDZ-domain containing protein of the dystrophin associated protein complex) is involved in aquaporin-4 and Kir4.1 anchoring in retinal cells, we studied the expression pattern of these molecules in alpha-syntrophin null mice. Judged by quantitative immunogold cytochemistry, deletion of the alpha-syntrophin gene causes a partial loss (by 70%) of aquaporin-4 labeling at astrocyte and Müller cell endfeet but no decrease in Kir4.1 labeling at these sites. These findings suggest that alpha-syntrophin is not involved in the anchoring of Kir4.1 and only partly responsible for the anchoring of aquaporin-4 in retinal endfeet membranes. Furthermore we show that wild type and alpha-syntrophin null mice exhibit strong beta1 syntrophin labeling at perivascular and subvitreal Müller cell endfeet, raising the possibility that beta1 syntrophin might be involved in the anchoring of Kir4.1 and the alpha-syntrophin independent pool of aquaporin-4.

beta-Integrin-collagen interaction reduces chondrocyte apoptosis.

We have observed that the spent culture media in suspended chondrocyte cultures is essential for the survival of the cells, since complete change of the spent media induces severe programmed cell death (apoptosis). Moreover, we showed that extracellular matrix (ECM) molecules in the culture media provide vital chondrocyte-matrix interactions; when media are changed, cells are deprived of matrix molecules and undergo apoptosis. In this paper we report that interaction with collagen, a ubiquitous extracellular matrix molecule, is essential for chondrocyte survival. Such an interaction causes chondrocyte aggregation and reduces the level of chondrocyte apoptosis. Hyaluronan, an abundant ECM molecule, can influence the effects of collagen by preventing chondrocyte aggregation. Degradation of hyaluronan with hyaluronidase results in chondrocyte aggregation, and this reduces the level of chondrocyte apoptosis. Experiments with an antibody to integrin beta1 suggest that the collagen-chondrocyte interactions are mediated through integrin beta1, and these interactions may protect chondrocytes from apoptosis. We hypothesize that hyaluronan binds aggrecan and link protein, forming stable ternary complexes, which interact with the chondrocyte surface, perhaps via CD44, and thus maintains a stable chondrocyte-matrix network.

Molecular cloning and biological activity of ecdysis-triggering hormones in Drosophila melanogaster.

Ecdysis-triggering hormones (ETH) initiate a defined behavioral sequence leading to shedding of the insect cuticle. We have identified eth, a gene encoding peptides with ETH-like structure and biological activity in Drosophila melanogaster. The open reading frame contains three putative peptides based on canonical endopeptidase cleavage and amidation sites. Two of the predicted peptides (DrmETH1 and DrmETH2) prepared by chemical synthesis induce premature eclosion upon injection into pharate adults. The promoter region of the gene contains a direct repeat ecdysteroid response element. Identification of eth in Drosophila provides opportunities for genetic manipulation of endocrine and behavioral events underlying a stereotypic behavior.