Monoclonal antibodies with defined specificities for Torpedo nicotinic acetylcholine receptor cross-react with Drosophila neural tissue

Chase, Bruce A.; Holliday, Janet; Reese, James H.; Chun, Linda L. Y.; Hawrot, Edward

doi:10.1016/0306-4522(87)90051-0

Cited by 30 publications

(8 citation statements)

References 52 publications

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“…DMeltrin induced only low levels of Ser cleavage. The activity of the Mmd Meltrin homolog was not examined, because its expression is restricted to the central nervous system (Chase et al, 1987).…”

Section: Several Drosophila Adams Can Cleave Deltamentioning

confidence: 99%

Unidirectional Notch signaling depends on continuous cleavage of Delta

et al. 2005

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Unidirectional signaling from cells expressing Delta (Dl) to cells expressing Notch is a key feature of many developmental processes. We demonstrate that the Drosophila ADAM metalloprotease Kuzbanian-like(Kul) plays a key role in promoting this asymmetry. Kul cleaves Dl efficiently both in cell culture and in flies, and has previously been shown not to be necessary for Notch processing during signaling. In the absence of Kul in the developing wing, the level of Dl in cells that normally receive the signal is elevated, and subsequent alterations in the directionality of Notch signaling lead to prominent phenotypic defects. Proteolytic cleavage of Dl by Kul represents a general mechanism for refining and maintaining the asymmetric distribution of Dl, in cases where transcriptional repression of Dlexpression does not suffice to eliminate Dl protein.

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Section: Several Drosophila Adams Can Cleave Deltamentioning

confidence: 99%

Unidirectional Notch signaling depends on continuous cleavage of Delta

et al. 2005

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“…Using MAb 35 chicken ciliary ganglion (Jacob et al 1986), rat hypothalamus (Mason 1985) and human skeletal muscle (Smitet al 1987;Jennekens et al 1992) have been investigated. MAb 35.74 -raised in mice using affinity-purified nAChR from Torpedo electric organ membranes (Deutch et al 1987) -completely blocks MAb 35-binding to purified Torpedo nAChR, the epitope recognized by mAb 35.74 therefore most likely lying close to or coincid- (1) Tzartos et al 1981; (2) Jacob et al 1986; (3) Mason 1985; (4) Smit et al 1987;(5) Jennekens et al 1992;(6) Chase et al 1987;(7) Deutch etal. 1987;(8) Whiting and Lindstrom 1986;(9) Swanson etal.…”

Section: Monoclonal Antibodies Have Been Raised Against the Differentmentioning

confidence: 99%

Immunohistochemistry of cholinergic receptors

Schröder

1992

Anat Embryol

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Acetylcholine and its receptors are involved in a variety of important signal transduction processes. As shown here paradigmatically for the human neuromuscular junction and the cerebral cortex, acetylcholine receptors can be visualized immunohistochemically at the cellular and subcellular level under physiological and pathological conditions. At normal motor endplates nicotinic cholinoceptors are localized at the surface of the postsynaptic junctional folds. In myasthenic syndromes investigation of muscle biopsies enables the diagnosis of receptor deficiencies at the ultrastructural level. In normal cerebral cortex pyramidal neurons are equipped with both nicotinic and muscarinic acetylcholine receptors localized to postsynaptic densities. In neuropsychiatric diseases cholinoceptor expression can be monitored at the cellular level by quantitative assessment of immunolabeled cortical neurons.

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“…The gels were transferred onto polyvinylidene difluoride membranes and blocked with phosphate-buffered saline containing 0.1% Tween and 3% bovine serum albumin (Sigma). Primary antibody incubations were performed in the same buffer using a 1:500 dilution of antibody 43.37 (120 g/ml stock) (25). Blots were then incubated with anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (working dilution 1:2500; Transduction Laboratories).…”

Section: Sds-polyacrylamide Gel Electrophoresis and Western Blot Analmentioning

confidence: 99%

Biotinylation of Substituted Cysteines in the Nicotinic Acetylcholine Receptor Reveals Distinct Binding Modes for α-Bungarotoxin and Erabutoxin a

Spura¹,

Riel²,

Freedman³

et al. 2000

Journal of Biological Chemistry

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, little is known about the accessibility of other residues within this region. By determining second-order rate constants for the reaction of cysteine mutants at ␣184 -␣197 with the thiol-specific biotin derivative (؉)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine, we now show that only very subtle differences in reactivity (ϳ10-fold) are detectable, arguing that the entire region is solvent-exposed. Importantly, biotinylation in the presence of saturating concentrations of the long neurotoxin ␣-Bgtx is significantly retarded for positions ␣W187C, ␣F189C, and reduced wild-type receptors (␣Cys 192 and ␣Cys 193 ), further emphasizing their major contribution to the ␣-Bgtx binding site. Interestingly, although biotinylation of position ␣V188C is not affected by the presence of ␣-Bgtx, erabutoxin a, which is a member of the short neurotoxin family, inhibits biotinylation at position ␣V188C, but not at ␣W187C or ␣F189C. Taken together, these results indicate that short and long neurotoxins establish interactions with distinct amino acids on the nicotinic acetylcholine receptor. The nicotinic acetylcholine receptor (AChR)1 is the major prototype for neurotransmitter-gated ion channels and is found at high concentrations in the postsynaptic membranes of muscle cells, where it mediates the rapid propagation of electrical signals at the neuromuscular junction. It is a pentameric protein composed of four subunit types in a molar ratio 2␣:␤:␥:␦ (see Ref. 1 for review).An important first step in assessing the structure and function of such ion channels at a molecular level is to determine their transmembrane topology. To address this issue, a variety of techniques have been developed (see Ref. 2 for review). The most commonly used methods are the epitope protection assay (3-5), in which an epitope that is recognized by a specific antibody is fused to the protein of interest, and N-linked glycosylation tagging, wherein N-linked glycosylation sites can be engineered into the protein under investigation and glycosylation can then be evaluated (6 -8).The above methods, however, are only useful to assess overall topology of membrane proteins. For the nAChR, there is general consensus on the overall topology, although final proof will have to await high resolution structural data; each subunit possesses a large, extracellular amino-terminal domain, which is followed by four transmembrane-spanning regions and a short extracellular carboxyl terminus (9). In contrast, the key structural issue of whether individual residues are solventexposed has not been resolved. To address this issue, Gallivan et al. (10) have recently employed the in vivo nonsense suppression technique to incorporate derivatives of the unnatural amino acid biocytin into the nAChR heterologously expressed in Xenopus oocytes. By evaluating the binding of 125 I-streptavidin to biotinylated receptors, they studied the surface exposure of individual residues comprising the main immunogenic region (spanning positions 67-76; Ref. 11) and showed that positi...

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Monoclonal antibodies with defined specificities for Torpedo nicotinic acetylcholine receptor cross-react with Drosophila neural tissue

Cited by 30 publications

References 52 publications

Unidirectional Notch signaling depends on continuous cleavage of Delta

Unidirectional Notch signaling depends on continuous cleavage of Delta

Immunohistochemistry of cholinergic receptors

Biotinylation of Substituted Cysteines in the Nicotinic Acetylcholine Receptor Reveals Distinct Binding Modes for α-Bungarotoxin and Erabutoxin a

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