FIGQY phosphorylation defines discrete populations of L1 cell adhesion molecules at sites of cell-cell contact and in migrating neurons.

Phosphorylation of neurofascin, a member of the L1 family of cell adhesion molecules (L1 CAMs), at the conserved FIGQY-tyrosine abolishes the ankyrin-neurofascin interaction. This study provides the first evidence, in Drosophila melanogaster and vertebrates, for the physiological occurrence of FIGQY phosphorylation in L1 family members. FIGQY tyrosine phosphorylation is localized at specialized cell junctions, including paranodes of sciatic nerve, neuromuscular junctions of adult rats and Drosophila embryos, epidermal muscle attachment sites of Drosophila, and adherens junctions of developing epithelial cells of rat and Drosophila. In addition, FIGQY-phosphorylated L1 CAMs are abundantly expressed in regions of neuronal migration and axon extension, including the embryonic cortex, the neonatal cerebellum and the rostral migratory stream, a region of continued neurogenesis and migration throughout adulthood in the rat. Based on our results, physiological FIGQY-tyrosine phosphorylation of the L1 family likely regulates adhesion molecule-ankyrin interactions establishing ankyrin-free and ankyrin-containing microdomains and participates in an ankyrin-independent intracellular signaling pathway at specialized sites of intercellular contact in epithelial and nervous tissue.

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.

Syntrophin isoforms at the neuromuscular junction: developmental time course and differential localization.

The syntrophins are a family of cytoplasmic adapter proteins that associate with dystrophin family proteins and have putative signaling and structural roles at the neuromuscular junction. We have localized the syntrophin family members within the rodent junction from birth to adulthood. Alpha-syntrophin is the only isoform on the postsynaptic membrane at birth. In the adult, it occurs on the crests of the junctional folds, with utrophin, and in the troughs, with dystrophin. Surprisingly, neuronal nitric oxide synthase (nNOS) does not accompany alpha-syntrophin onto the crests. Beta2-syntrophin, a junction-specific form, is not present at birth and occurs mainly in the troughs in the adult. Beta1-syntrophin is a sarcolemmal form at birth, not concentrated at the junction, and disappears entirely from most fibers by 6 weeks. In positive fibers, junctional beta1-syntrophin occurs exclusively in the troughs. These results suggest that the syntrophin isoforms have distinct functions at the junction and show that the known protein-protein associations of the syntrophins and nNOS in skeletal muscle are not sufficient to explain their localizations.

Differential membrane localization and intermolecular associations of alpha-dystrobrevin isoforms in skeletal muscle.

alpha-Dystrobrevin is both a dystrophin homologue and a component of the dystrophin protein complex. Alternative splicing yields five forms, of which two predominate in skeletal muscle: full-length alpha-dystrobrevin-1 (84 kD), and COOH-terminal truncated alpha-dystrobrevin-2 (65 kD). Using isoform-specific antibodies, we find that alpha-dystrobrevin-2 is localized on the sarcolemma and at the neuromuscular synapse, where, like dystrophin, it is most concentrated in the depths of the postjunctional folds. alpha-Dystrobrevin-2 preferentially copurifies with dystrophin from muscle extracts. In contrast, alpha-dystrobrevin-1 is more highly restricted to the synapse, like the dystrophin homologue utrophin, and preferentially copurifies with utrophin. In yeast two-hybrid experiments and coimmunoprecipitation of in vitro-translated proteins, alpha-dystrobrevin-2 binds dystrophin, whereas alpha-dystrobrevin-1 binds both dystrophin and utrophin. alpha-Dystrobrevin-2 was lost from the nonsynaptic sarcolemma of dystrophin-deficient mdx mice, but was retained on the perisynaptic sarcolemma even in mice lacking both utrophin and dystrophin. In contrast, alpha-dystrobrevin-1 remained synaptically localized in mdx and utrophin-negative muscle, but was absent in double mutants. Thus, the distinct distributions of alpha-dystrobrevin-1 and -2 can be partly explained by specific associations with utrophin and dystrophin, but other factors are also involved. These results show that alternative splicing confers distinct properties of association on the alpha-dystrobrevins.

beta 2-Syntrophin: localization at the neuromuscular junction in skeletal muscle.

The syntrophins are a multigene family of proteins which bind C-terminal domains of dystrophin, utrophin and homologs thereof. We report here that antibodies specific for one isoform, beta 2-syntrophin, labeled only the neuromuscular junction (NMJ) in rat skeletal muscle. Anti-alpha 1-syntrophin antibodies gave strong labeling of the sarcolemma and NMJ in normal rat and mouse muscle, and similar but much weaker labeling in dystrophin-minus mdx muscle. beta 2-Syntrophin therefore appears to be specific to the NMJ in normal muscle, as is utrophin, and may be involved in acetylcholine receptor clustering. alpha 1-Syntrophin appears to be associated mainly with dystrophin, as expected, but a small portion must be associated with another protein, possibly homologs of the electric tissue 87K protein.

Association of utrophin and multiple dystrophin short forms with the mammalian M(r) 58,000 dystrophin-associated protein (syntrophin).

Electric tissue syntrophin, originally described as an M(r) 58,000 postsynaptic protein having homologs in mammalian muscle, was previously shown to associate with dystrophin in Triton extracts of Torpedo postsynaptic membranes. It also associates with the Torpedo M(r) 87,000 postsynaptic protein (87K), the core of which is a superdomain homologous to the cysteine-rich (CR) and COOH-terminal (CT) domains of human dystrophin. Using immunoaffinity purifications from various rat tissues and immunoblotting, we find that syntrophin associates with dystrophin, utrophin (the chromosome 6-encoded dystrophin homolog formerly known as dystrophin-related protein), multiple proteins which are cross-reactive with 87K, and two subfamilies of 71K-like proteins (CRCT-containing proteins encoded by the dystrophin gene under the control of an alternative promoter in intron 62). One 71K subfamily retains the dystrophin COOH-terminal sequence; the other has an alternative COOH-terminal sequence caused by deletion of the penultimate exon by alternative splicing. The relative masses of the members of the subfamilies suggest they arise by alternative splicing at other previously described sites within CT. These results establish that syntrophin is a general ligand for the CRCT domain in mammalian dystrophin and its homologs. They also reveal a greater diversity in 71K proteins than has previously been apparent.

Amphibian ryanodine receptor isoforms are related to those of mammalian skeletal or cardiac muscle.

The ryanodine receptor (RyR)-Ca2+ release channels of frog skeletal muscle have been purified as 30S protein complexes comprised of two high molecular weight polypeptides. The upper and lower bands of the frog doublet comigrated on sodium dodecyl sulfate polyacylamide gels with the mammalian skeletal and cardiac RyR polypeptides, respectively. Immunoblot analysis showed that a polyclonal antiserum to the rat skeletal RyR preferentially cross-reacted with the upper band, whereas monoclonal antibodies to the canine cardiac RyR preferentially cross-reacted with the lower band of the frog receptor doublet. Immunoprecipitation studies indicated the presence of two homooligomer 30S RyR complexes comprised of either the lower or upper polypeptide band of the frog doublet, and immunocytochemical staining revealed their colocalization in frog gastrocnemius muscle. After planar lipid bilayer reconstitution of the 30S frog RyR, single-channel currents were observed that exhibited a Na+ and Ca2+ conductance and pharmacological characteristics similar to those of the mammalian skeletal and cardiac Ca2+ release channels. These results suggest that amphibian skeletal muscle expresses two distinct RyR isoforms that share epitopes in common with the mammalian skeletal or cardiac RyR.

The 30 S lobster skeletal muscle Ca2+ release channel (ryanodine receptor) has functional properties distinct from the mammalian channel proteins.

The 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps)-solubilized ryanodine receptor (RyR) of lobster skeletal muscle has been isolated by rate density centrifugation as a 30 S protein complex. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the purified 30 S receptor revealed a single high molecular weight protein band with a mobility intermediate between those of the mammalian skeletal and cardiac M(r) 565,000 RyR polypeptides. Immunoblot analysis showed no or only minimal cross-reactivity with the rabbit skeletal and canine cardiac RyR polypeptides. By immunofluorescence the lobster RyR was localized to the junctions of the A-I bands. Following planar lipid bilayer reconstitution of the purified 30 S lobster RyR, single channel K+ and Ca2+ currents were observed which were modified by ryanodine and optimally activated by millimolar concentrations of cis (cytoplasmic) Ca2+. Vesicle-45Ca2+ flux measurements also indicated an optimal activation of the lobster Ca2+ channel by millimolar Ca2+, whereas 45Ca2+ efflux from mammalian skeletal and cardiac muscle sarcoplasmic reticulum (SR) vesicles is optimally activated by micromolar Ca2+. Further, mammalian muscle SR Ca2+ release activity is modulated by Mg2+ and ATP, whereas neither ligand appreciably affected 45Ca2+ efflux from lobster SR vesicles. These results suggested that lobster and mammalian muscle express immunologically and functionally distinct SR Ca2+ release channel protein complexes.

Association of the Mr 58,000 postsynaptic protein of electric tissue with Torpedo dystrophin and the Mr 87,000 postsynaptic protein.

Dystrophin was purified by immunoaffinity chromatography from detergent-solubilized Torpedo electric organ postsynaptic membranes using monoclonal antibodies. A major doublet of proteins at Mr 58,000 and minor proteins at Mr 87,000, Mr 45,000, and Mr 30,000 reproducibly copurified with dystrophin. The Mr 58,000 and Mr 87,000 proteins were identical to previously described peripheral membrane proteins (Mr 58,000 protein and 87,000 protein) whose muscle homologs are associated with the sarcolemma (Froehner, S. C., Murnane, A. A., Tobler, M., Peng, H. B., and Sealock, R. (1987) J. Cell Biol. 104, 1633-1646; Carr, C., Fischbach, G. D., and Cohen, J. B. (1989) J. Cell Biol. 109, 1753-1764). The copurification of dystrophin and Mr 58,000 protein was shown to be specific, since dystrophin was also captured with a monoclonal antibody against the Mr 58,000 protein but not by several control antibodies. The Mr 87,000 protein was a major component (along with the Mr 58,000 protein) in material purified on anti-58,000 columns, suggesting that the Mr 58,000 protein forms a distinct complex with the Mr 87,000 protein, as well as with dystrophin. Immunofluorescence staining of skeletal and cardiac muscle from the dystrophin-minus mdx mouse with the anti-58,000 antibody was confined to the sarcolemma as in normal muscle but was much reduced in intensity, even though immunoblotting demonstrated that the contents of Mr 58,000 protein in normal and mdx muscle were comparable. Thus, the Mr 58,000 protein appears to associate inefficiently with the sarcolemmal membrane in the absence of dystrophin. This deficiency may contribute to the membrane abnormalities that lead to muscle necrosis in dystrophic muscle.

Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle.

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.

Localization of paxillin, a focal adhesion protein, to smooth muscle dense plaques, and the myotendinous and neuromuscular junctions of skeletal muscle.

In this report we have demonstrated that paxillin, a cytoskeletal protein which is present in focal adhesions, localizes in vivo to regions of cell-extracellular matrix interaction which are believed to be analogous to focal adhesions. Specifically, it is enriched in the dense plaques of chicken gizzard smooth muscle tissue and in the myotendinous junctions formed in Xenopus laevis tadpole tail skeletal muscle. In addition, paxillin was identified at the rat diaphragm neuromuscular junction. The distribution of paxillin is thus comparable to that of other focal adhesion proteins, for example, talin and vinculin, in these structures.

Commercial preparations of colloidal gold-antibody complexes frequently contain free active antibody.

Using a simple fluorescence test, we show that commercially prepared colloidal gold complexes with goat second antibodies often contain free active antibody. Because such antibodies will compete with antibody-colloidal gold particles for antigen binding sites, labeling intensity at the ultrastructural level must necessarily be submaximal to an unknown degree with such preparations. A survey of five preparations suggests that the problem may be widespread. We recommend that a test of the sort described be incorporated routinely into protocols with all colloidal gold products.

Dystrophin as a focal adhesion protein. Collocalization with talin and the Mr 48,000 sarcolemmal protein in cultured Xenopus muscle.

Monoclonal antibodies against dystrophin and the postsynaptic 58 kDa protein from Torpedo electric organ were used to localize homologs of these proteins in cultured skeletal muscle (Xenopus laevis). The Xenopus homolog is an Mr 48,000 protein and, like dystrophin, is a sarcolemmal protein. Both proteins localized precisely to talin-positive sites, hence with each other, on the substrate-apposed sarcolemma. Therefore, the first sites of appearance of dystrophin on cultured muscle cells are focal adhesions, i.e. specific sites of cytoskeleton/extracellular matrix interaction. These data also add to evidence that dystrophin and the 58 kDa act together.

Effects of tunicamycin on the expression of beta-adrenergic receptors in human astrocytoma cells during growth and recovery from agonist-induced down-regulation.

Tunicamycin, which inhibits formation of asparagine-linked glycoproteins, caused a concentration-dependent blockade of beta-adrenergic receptor (beta-AR) accumulation in 1321N1 human astrocytoma cells during growth in culture. A concentration of tunicamycin (0.1 microgram/ml) that inhibited receptor accumulation and [3H]mannose or [3H]glucosamine incorporation into glycoproteins by 90% had only a small effect (10%) on [3H]leucine incorporation into protein, and reduced the rate of cell growth. Incubation in drug-free medium subsequent to treatment of 1321N1 cells with tunicamycin for 48 hr resulted in recovery of beta-AR to control levels within an additional 48 hr. Exposure of cultures to isoproterenol (0.1 microM, 12 hr) caused an 80-90% loss of beta-AR in both pre- and postconfluent cultures; beta-AR recovered to control levels upon removal of isoproterenol. Although both tunicamycin and the protein synthesis inhibitor cycloheximide blocked beta-AR accumulation during growth of 1321N1 cells, neither agent inhibited the appearance of beta-AR during recovery from the down-regulated state in preconfluent cultures. However, cycloheximide, but not tunicamycin, blocked recovery of beta-AR after isoproterenol-induced loss of receptors in postconfluent cultures. In a previous report (Mol. Pharmacol. 26:424-429, 1984), we provided direct evidence that recovery of beta-AR from down-regulation in postconfluent cultures requires de novo synthesis of receptor protein. Thus, the results with tunicamycin are consistent with the idea that recovery of beta-AR in postconfluent cultures requires the synthesis of new beta-AR molecules, but as aglycoproteins that exhibit radioligand-binding characteristics similar to those of native glycoprotein beta-AR.

Effects of drug-induced changes in brain monoamines on aggression and motor behavior in mice.

Mice maintained on a basal casein diet supplemented with 4% L-tyrosine potentiated L-DOPA effects on aggression. At low doses (12.5-25 mg/kg) L-DOPA increased aggression whereas at high doses (50-100 mg/kg) it decreased aggression. 5-HTP (50-200 mg/kg) produced a dose-dependent decrease in aggression and motor activity which was antagonized by pretreatment with dietary L-tyrosine (4%) or L-DOPA (50 mg/kg). L-DOPA induced reductions in motor activity were, in turn, antagonized by 5-HTP. Increases in motor activity following d-amphetamine (3 mg/kg) were sharply reduced by 5-HTP (50-100 mg/kg), but 5-HTP potentiated reductions in aggression following d-amphetamine. The concentration in brain of tyrosine, DOPA, dopamine (DA), noradrenaline (NA), DOPAC, HVA, tryptophan, serotonin (5-HT), and 5-HIAA were obtained following drug and diet treatments. The changes observed, particularly in DA and 5-HT metabolites, provide further evidence for an inhibitory role of brain 5-HT systems in the mediation of the behavioral effects of d-amphetamine and the catecholamine precursors, L-tyrosine and L-DOPA.

Footshock treatment activates catecholamine synthesis in slices of mouse brain regions.

Synthesis of catecholamines was measured in slices of frontal cortex, hypothalamus, striatum, hippocampus and brainstem by the accumulation of [3H]norepinephrine (NE) and [3H]dopamine (DA) following incubation with [3H]tyrosine. Following acute footshock (60 shocks, 0.3 mA 30 min), consistent increases in [3H]DA accumulation were seen in frontal cortex slices, but no significant effect was seen in striatal slices. The accumulation of [3H]NE was not altered consistently in frontal cortex, hypothalamus, hippocampus or brainstem. Brain slices from mice adrenalectomized 24-48 h before footshock showed similar results. However, in hypophysectomized mice, footshock did not increase the [3H]DA accumulation in slices of frontal cortex. Administration of dexamethasone before footshock prevented the footshock-induced increase in frontal cortex [3H]DA accumulation, but footshock then significantly increased [3H]NE accumulation in the hypothalamus and brainstem. Chronic footshock (5 days) had little effect on frontal cortex [3H]catecholamine accumulation but produced a consistent elevation of [3H]NE accumulation in slices from the hypothalamus. In confirmation that the slice data reflected in vivo metabolism, both dihydroxyphenylacetic acid and homovanillic acid were significantly elevated in the frontal cortex but not the striatum of mice receiving acute footshock. Since previous studies have shown that ACTH administered intracerebroventricularly also accelerated [3H]DA accumulation in frontal cortex slices, these results are consistent with the involvement of ACTH in the effects of footshock on frontal cortex DA. The effects of chronic footshock are consistent with the activation of hypothalamic tyrosine hydroxylase by corticosterone.

Neurotensin induces catalepsy in mice.

Intracerebroventricular (i.c.v.) injection of neurotensin (NT) induced catalepsy in mice at doses greater than or equal to 0.02 microgram. The cataleptic effect progressively increased, reaching a maximum at approx. 2 hr after injection. In contrast, the hypothermic effect of neurotensin reached a maximum 1 hr after the injection, and was declining at 2 hr. Not all mice that showed hypothermia also showed catalepsy, and some mice showed catalepsy without hypothermia. Catalepsy induced by intracerebroventricular injection of neurotensin was not significantly correlated with the hypothermia. Furthermore, oxotremorine induced hypothermia without catalepsy. Thus, several lines of evidence indicate that the catalepsy induced by neurotensin is not the consequence of the neurotensin induced hypothermia. Thyrotropin releasing hormone (TRH), injected either intracerebroventricularly with neurotensin, or intraperitoneally before neurotensin abolished the hypothermia but only diminished the catalepsy scores. The cataleptic effect of neurotensin is consistent with its other neuroleptic-like activities.

ACTH 1-24 and lysine vasopressin selectively activate dopamine synthesis in frontal cortex.

The accumulation of [3H] catecholamines from [3H] tyrosine in frontal cortical, septal, striatal and hippocampal slices was examined following intracerebroventricular (i.c.v.) injections of ACTH 1-24, lysine vasopressin (LVP) and saline. Both ACTH 1-24 and LVP (1 microgram) selectively increased the accumulation of [3H] dopamine (DA) in frontal cortical slices, but did not affect that of [3H] norepinephrine (NE). LVP but not ACTH 1-24 also inhibited the accumulation of [3H] DA in striatal slices. ACTH 1-24 did not alter the accumulation of [3H] NE in hippocampal slices, nor did LVP alter the accumulation of either catecholamine (CA) in septal slices. In vitro incubations with ACTH analogs of LVP failed to alter the rate of accumulation of [3H] CAs in striatal, substantia nigral and frontal cortical slices, except for an inhibitory effect at high doses. This effect is believed to be an artifact of precursor dilution caused by release of tyrosine following degradation of the peptides. Neither peptide modified the increased [3H] CA accumulation stimulated by 26 mM K+, nor did ACTH 1-24 modify the inhibition of [3H] CA accumulation caused by 3 X 10 -6 M haloperidol or 3 X 10 -7 M apomorphine. Selective activation of the mesocortical DA system has also been reported ot occur in response to footshock, suggesting the possibility that endogenous ACTH and/or LVP might mediate the stress-induced activation of mesocortical DA synthesis. Alternatively, i.c.v. injections of these peptides may themselves be stressful and thus indirectly elicit the response.

Effect of fusaric acid on aggression, motor activity, and brain monoamines in mice.

The effects of fusaric acid (FA), a dopamine-beta-hydroxylase (D beta H) inhibitor, were determined on aggression, motor activity, and brain monoamines at doses of 3.2 to 60 mg/kg following administration of dietary supplements of L-tyrosine or balanced protein to male albino mice. Compared to saline injected control animals, both aggression and motor activity were reduced by the highest doses of FA. Somewhat more reduction in aggression was observed in animals administered dietary supplements of casein compared to those given the tyrosine supplement. Treatment with FA at doses of 30 to 60 mg/kg decreased brain norepinephrine and dopamine, and decreased brain tyrosine in animals fed the tyrosine supplement. In contrast, FA increased 5-hydroxytryptamine, and caused marked increases in 5-hydroxyindoleacetic acid at the highest doses. The data suggest that the neurochemical effects of FA may not be the same in rats and mice.