Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins.

Evidence was presented earlier that a host-cell receptor for the highly neurotropic rabies virus might be the acetylcholine receptor. The amino acid sequence of the glycoprotein of rabies virus was compared by computer analysis with that of snake venom curaremimetic neurotoxins, potent ligands of the acetylcholine receptor. A statistically significant sequence relation was found between a segment of the rabies glycoprotein and the entire sequence of long neurotoxins. The greatest identity occurs with residues considered most important in neurotoxicity, including those interacting with the acetylcholine binding site of the acetylcholine receptor. Because of the similarity between the glycoprotein and the receptor-binding region of the neurotoxins, this region of the viral glycoprotein may function as a recognition site for the acetylcholine receptor. Direct binding of the rabies virus glycoprotein to the acetylcholine receptor could contribute to the neurotropism of this virus.

Monoclonal antibodies directed against a synthetic peptide corresponding to the alpha-bungarotoxin binding region of the acetylcholine receptor.

Murine monoclonal antibodies have been produced against a 32 amino acid synthetic peptide corresponding to residues 173-204 on the alpha-subunit of the nicotinic acetylcholine receptor from Torpedo californica. All of the monoclonal antibodies were of the IgM subtype and most cross-reacted with the purified native receptor. None of the antibodies were effective in blocking alpha-bungarotoxin binding to the receptor nor, conversely, did alpha-bungarotoxin interfere with antibody binding. However, two monoclonal antibodies, previously shown to bind near the ligand binding site on the native receptor, did compete partially (50%) with the binding of one of the IgM monoclonal antibodies.

alpha-Bungarotoxin binding to two acetylcholine receptor alpha-peptides and their methylmercury-modified analogs: intrinsic phosphorescence and optically detected magnetic resonance studies.

Phosphorescence and optically detected magnetic resonance (ODMR) have been used to characterize two synthetic peptides, alpha 181-198 and alpha 185-196, of the major binding determinant of the alpha-acetylcholine receptor (AChR) of Torpedo californica and its interaction with alpha-bungarotoxin (BgTX) using Trp as an intrinsic probe. BgTX conformational changes are suggested upon complexation with the peptides. Methylmercury-modified peptides show conformational heterogeneity which brings some of the modified Cys residues into proximity of peptide Trp(s). These modified peptides, when bound to BgTX, undergo structural changes which remove the tagged Cys from its close contact with the Trp residue(s) of the peptide.

Phosphorescence and ODMR study of the binding interactions of acetylcholine receptor alpha-subunit peptides with alpha-cobratoxin.

Optical detection of magnetic resonance (ODMR) and phosphorescence spectroscopy have been applied to synthetic peptides derived from the alpha-subunit of the nicotinic acetylcholine receptor of Torpedo californica and their complexes with alpha-cobratoxin (CBTX). The CBTX Trp phosphorescence is strongly quenched by the proximal disulfide linkage, while the emission wavelengths and ODMR frequencies of the 18-mer alpha 181-198 indicate a more hydrophobic Trp environment than in the 12-mer alpha 185-196. Binding to CBTX produces a subtle increase in the hydrophobicity of the Trp environment for the peptides, in qualitative agreement with a recently proposed binding model, in which a receptor Trp residue interacts strongly with a hydrophobic cleft of the toxin.

Demonstration of a tandem pair of complement protein modules in GABA(B) receptor 1a.

We have subcloned and expressed the N-terminal portion of the recently sequenced metabotropic GABA receptor, GABA(B)R1a. This region of the receptor contains a complement protein-like amino acid sequence. The purified 140-residue recombinant protein fragment was soluble and stable. Mass spectrometry indicated formation of four disulfide bonds, as expected if two complement protein modules (CPs, also known as SCRs, Sushi domains) are formed. The circular dichroism spectrum was unusual and characteristic of CPs. Differential scanning calorimetry demonstrated a melting point (64 degrees C), and total enthalpy commensurate with two fully folded domains. We thus conclude that the 1a subtype of the GABA(B) receptor, but not the 1b subtype, contains a pair of CPs and we present a three-dimensional model of this region.

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

A panel of monoclonal antibodies with known specificity for the well-characterized nicotinic acetylcholine receptor from the electroplax of Torpedo californica, many of which cross-react with the mammalian muscle acetylcholine receptor, were examined for cross-reactivity in the fly, Drosophila melanogaster. Monoclonal antibodies with specificities for different epitopes on the transmembrane receptor complex from Torpedo cross-react with different regional subsets of neural tissue in Drosophila. Axonal tracts, neuropil, mechano-sensory bristle elements and photoreceptors, each are detected by separate monoclonal antibody classes corresponding to different epitope domains. A preliminary characterization of an antigenic determinant in Drosophila heads recognized by one of the cross-reacting monoclonal antibodies is presented. Monoclonal antibodies such as these may be useful in identifying molecules of homologous structure or function, possibly including a neuronal acetylcholine receptor.

The role of tyrosine at the ligand-binding site of the nicotinic acetylcholine receptor.

Identification of the critical residues in a receptor's ligand-binding site provides valuable structural information important for understanding the basis for ligand recognition. The design of specific ligands targeted for receptor action will depend to a great extent on detailed structural knowledge of this kind. Although the nicotinic acetylcholine receptor (nAChR) is perhaps the best characterized of all receptors, the detailed configuration of the ligand-binding site remains unknown. Structural comparisons of nicotinic agonists and antagonists have long predicted a negative subsite on the receptor to interact with the positively charged alkyl-ammonium moiety common to nearly all nicotinic agents. We have used intrinsic fluorescence spectroscopic analyses together with binding studies of selectively modified peptide fragments of the nAChR to suggest that one or two invariant tyrosine residues at positions 190 and 198 on the alpha-subunit provide the critical negative subsite required for ligand binding. Tyrosines may similarly be part of the negative subsite of muscarinic receptors and other neurotransmitter receptors that bind cationic ligands.

A binding site peptide fragment of the nicotinic acetylcholine receptor. Sequence-specific assignment of 1H-NMR resonances in the dodecamer alpha 185-196.

The total sequence-specific 1H assignment for the alpha 185-peptide was accomplished by analysis of COSY spectra along with spin-decoupling and confirmatory NOE difference experiments. Some ambiguities in the assignments were successfully addressed utilizing additional peptides with selective amino acid substitutions. The chemical shifts of several of the C alpha H resonances, along with evidence for a slowly exchanging amide at Thr-191 suggest that the alpha 185-peptide may contain a certain amount of non-random coil structure. The role of any such ordered structure in the mechanism of binding to alpha-bungarotoxin remains to be determined. The assignment of the peptide 1H resonances will facilitate the analysis and identification of chemical shift perturbations observed upon formation of the complex between alpha-bungarotoxin and the alpha 185-peptide [7].

Binding of rabies virus to purified Torpedo acetylcholine receptor.

The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by alpha-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.

Alpha-bungarotoxin binding to a high molecular weight component from lower vertebrate brain identified on dodecyl sulfate protein-blots.

The binding of [125I]iodo-alpha-bungarotoxin [( 125]alpha-BuTX) to the dissociated alpha-subunit of Torpedo acetylcholine receptor (AChR) can be readily demonstrated in a modified 'protein-blot' analysis utilizing electrophoretically transferred, dissociated subunits immobilized onto positively charged nylon membranes which are then incubated directly with [125I]alpha-BuTX. We report here the use of the protein-blotting technique to detect the alpha-BuTX binding site present in the central nervous system of lower vertebrates and to characterize some of the physicochemical properties of the toxin binding site. High molecular weight (Mr greater than or equal to 200,000 and greater than or equal to 120,000) alpha-BuTX-binding components can be readily demonstrated in avian and fish brain extracts upon protein-blotting with [125I]alpha-BuTX following lithium dodecyl sulfate PAGE. Neither extensive reduction with dithiothreitol nor prior reduction followed by alkylation with iodoacetamide alter the mobility of the CNS-derived BuTX-binding sites. In contrast to our findings with Torpedo AChR or muscle AChR derived from a number of different species, no binding is observed in the molecular weight range of the alpha-subunit (Mr = 40,000) nor is any binding at any molecular weight observed in similar fractions prepared from adult, mammalian (rat, guinea pig) brain using this technique. These results demonstrate the existence in lower vertebrate brain of a BuTX binding site comparable in size to the AChR oligomeric complex of electric organ and muscle. They also suggest, however, striking structural differences between muscle AChR and the central neuronal BuTX-binding complex as well as a considerable difference between the neuronal BuTX-binding sites derived from lower and higher vertebrate brain.

Characteristics of the association of nerve growth factor with primary cultures of rat sympathetic neurons.

Long-term primary cultures of rat sympathetic neurons require NGF for survival and development. The kinetics of the interaction of 125I-NGF with sympathetic neuron cultures suggests the presence of diffusional barriers preventing a determination of true dissociation and association rate constants. Although the observed rate constants do not accurately reflect the microscopic interaction of NGF with receptor, the ratio of the observed rate constants does provide a good estimate of the KD. This value (1 X 10(-9)M) agrees with earlier steady state measurements of the KD. The association of 125I-NGF with neuronal cultures is temperature-dependent with internalization and retrograde transport occurring at 37 degrees C. The retrograde transport of 125I-NGF in compartmentalized neuronal cultures is concentration dependent and saturates at about 100 ng/ml (4 X 10(-9)M). The amount of 125I-NGF accumulated by retrograde transport appears to be increased subsequent to a period of NGF-starvation. The increase in uptake does not appear to be due to an increase in NGF receptor number since the number of binding sites is not greatly increased upon NGF starvation.

Structural characterization of alpha-bungarotoxin-binding proteins from Aplysia californica.

Structural features of alpha-bungarotoxin-binding proteins from the marine mollusc Aplysia californica have been examined as a first step toward delineating their potential role in cholinergic neurotransmission. Protein blotting with 125I-alpha-bungarotoxin was used to identify binding proteins in membranes prepared from Aplysia muscle and nervous tissue. Binding proteins from both tissues exhibited similar physical characteristics, which distinguish them from the prototypical alpha-bungarotoxin-binding protein, the nicotinic acetylcholine receptor obtained from Torpedo californica electric organ membranes. Aplysia binding activities migrate with an apparent molecular weight of 250 kDa on sodium dodecyl sulfate (SDS) gels in the presence of reducing agents. Binding of alpha-bungarotoxin to blots of Aplysia membranes is abolished by exposure of samples to heat or to low pH but is unaffected by reduction-alkylation treatment. In contrast, the alpha-bungarotoxin-binding subunit of the acetylcholine receptor from Torpedo membranes migrates on SDS gels at 40 kDa. It retains binding activity following exposure to heat or to low pH, but binding is substantially diminished by reduction-alkylation treatments. Another distinguishing characteristic of the Aplysia binding activities is revealed by examining recovery of membrane alpha-bungarotoxin-binding on protein blots; the high recovery of Aplysia binding contrasts sharply with the low recovery of Torpedo binding activity. The high apparent molecular weight of the Aplysia alpha-bungarotoxin-binding activities, their most distinguishing feature, is similar to an alpha-bungarotoxin-binding activity recently identified in lower vertebrate brain. Covalent cross-linking with 125I-alpha-bungarotoxin demonstrates, however, that the mobility of both Aplysia binding activities is due to a multimeric protein that is unusually resistant to dissociation in SDS. The covalently radiolabeled Aplysia alpha-bungarotoxin-binding activity migrates at approximately 260 kDa on SDS gels when solubilized at room temperature. When it was boiled before electrophoresis, the mobility of the radiolabeled protein shifts to approximately 70 kDa. Resistance to dissociation in the absence of boiling may explain both the high recovery of activity on blots and the insensitivity to reductive alkylation. Conversely, dissociation of the multimeric complex upon boiling may explain the observed loss of binding activity. Our results demonstrate structural similarities and differences between Aplysia alpha-bungarotoxin-binding proteins and the Torpedo acetylcholine receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

The metabotropic GABA receptor: molecular insights and their functional consequences.

Recent years have seen rapid and significant advances in our understanding of the G-protein-coupled gamma-amino butyric acid, B-type (GABA(B)) receptor, which could be a therapeutic target in conditions as diverse as epilepsy and hypertension. This progress originated with the ground-breaking work of Bernhard Bettler's team at Novartis who cloned the DNA encoding a GABA(B) receptor in 1997. Currently, the receptor is thought to be an unusual, possibly unique, example of a heterodimer composed of homologous, seven-transmembrane-domain (7TMD) subunits (named GABA(B) R1 and GABA(B) R2), neither of which is fully functional when expressed alone. The large N-terminal domain of the GABA(B) R1 subunit projects extracellularly and contains a ligand binding site. The similarity of the amino acid sequence of this region to some bacterial periplasmic amino acid-binding proteins of known structure has enabled structural and functional modelling of the N-terminal domain, and the identification of residues whose substitution modulates agonist/antagonist binding affinities. The intracellular C-terminal domains of the R1 and R2 subunits appear to constitute an important means of contact between the two subunits. Alternative splice variants, a common and functionally important feature of 7TMD proteins, have been demonstrated for the R1 subunit. Notably GABA(B) R1a differs from GABA(B) R1b by the possession of an N-terminal extension containing two complement protein modules (also called SCRs, or sushi domains) of unknown function. The levels at which each of the respective variants is expressed are not equal to one another, with variations occurring over the course of development and throughout the central nervous system. It is not yet clear, however, whether one variant is predominantly presynaptically located and the other postsynaptically located. The existence of as yet unidentified splice variants, additional receptor subtypes and alternative quaternary composition has not been ruled out as a source of receptor heterogeneity.

Phospholipid composition and membrane function in phosphatidylserine decarboxylase mutants of Escherichia coli.

Temperature-sensitive conditional lethal mutants in phosphatidylserine decarboxylase (psd) accumulate large amounts of phosphatidylserine under nonpermissive conditions (42 degrees C) prior to cell death. In addition, the ratio of cardiolipin to phosphatidylglycerol is increased. At an intermediate temperature (37 degrees C), high levels of phosphatidylserine can be maintained with little effect on cell growth or viability. Under these conditions, both the rate of induction and the function of the lactose transport system are normal. At 42 degrees C addition of Mg2+ or Ca2+ to mutant cultures produces a partial phenotypic suppression. Growth is prolonged and the filaments normally present at 42 degrees C do not form. Upon transfer to the nonpermissive temperature, there is a considerable lag before accumulation of phosphatidylserine begins and the growth rate is affected. Based on the kinetics of heat inactivation of phosphatidylserine decarboxylase activity in extracts, in intact nongrowing cells, and in growing cells, it appears that the enzyme newly synthesized at 42 degrees C is more thermolabile in vivo than enzyme molecules previously inserted into the membrane at the lower temperature. Thus, the older, stable enzymatic activity must be diluted during growth before physiological effects are observed.

Intrinsic fluorescence of binding-site fragments of the nicotinic acetylcholine receptor: perturbations produced upon binding alpha-bungarotoxin.

Synthetic peptides corresponding to sequences contained within residues 173-204 of the alpha-subunit in the nicotinic acetylcholine receptor (nAChR) of Torpedo californica bind the competitive antagonist alpha-bungarotoxin (BGTX) with relative high affinity. Since the synthetic peptide fragments of the receptor and BGTX each contain a small number of aromatic residues, intrinsic fluorescence studies were used to investigate their interaction. We examined a number of receptor-derived peptide fragments of increasing length (4-32 amino acids). Changes in the lambda max and quantum yield with increasing polypeptide chain length suggest an increase in the hydrophobicity of the tryptophan environment. When selective excitation and subtraction were used to reveal the tyrosine fluorescence of the peptides, a significant red shift in emission was observed and was found to be due to an excited-state tyrosinate. The binding of BGTX to the receptor-derived peptide fragments resulted in a large increase in fluorescence. In addition, at equilibrium, the lambda max of tryptophan fluorescence was shifted to shorter wavelengths. The. fluorescence enhancement, which was saturable with either peptide or BGTX, was used to determine the dissociation constants for the complexes. At pH 7.4, the apparent Kd for a dodecameric peptide (alpha 185-196), consisting of residues 185-196 in the alpha-subunit of the nAChR from Torpedo californica, was 1.4 microM. The Kd for an 18-mer (alpha 181-198), consisting of residues 181-198 of the Torpedo alpha-subunit, was 0.3 microM. No binding or enhanced fluorescence was observed with an irrelevant synthetic peptide of comparable composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural studies on the intracellular fate of 125I-nerve growth factor in cultured rat sympathetic neurons.

Primary cell cultures of sympathetic neurons from rat were exposed to 125I-nerve growth factor (NGF) and the fate of the NGF in the cell was followed using electron microscopic autoradiography. The intracellular localization of NGF was determined in the cell bodies and in the proximal neurites of neurons that had been grown in three-chamber dishes, following 5 or 24 hr of retrograde transport of NGF from the distal portions of the neurites. Label in the proximal neurites was predominantly associated with lysosomes and multivesicular bodies (MVBs), and at 5 hr elongated tubular elements were especially heavily labeled. Most of the label in the cell bodies was concentrated in lysosomes and MVBs. Lysosomes accounted for the largest fraction (45-60%) of the grains in the cell body, with a labeling density (LD = % grains/% area) of 3-5, while MVBs accounted for 5-10% of the grains with an LD of 5-20. We observed no evidence of nuclear labeling after 5 or 24 hr of retrograde transport. Mass cultures of neurons were incubated for 22 hr with NGF in the presence of the lysosomal inhibitors chloroquine (CQ, 0.05 mM) or methylamine (MA, 10 mM). In both agents the lysosomes were swollen with membranous material but still sequestered NGF, especially in CQ where the lysosomes were associated with almost 65% of the grains and had an LD of 6. CQ and MA had different effects on the morphology of the MVBs: in CQ they were few in number and compact while in MA they were numerous and appeared swollen and vacuolated. We observed no evidence for the nuclear accumulation of NGF even in the presence of the lysosomotropic agents.