Irm Hermans-Borgmeyer

Neuronal acetylcholine receptors in Drosophila: the ARD protein is a component of a high-affinity alpha-bungarotoxin binding complex.

The ard gene of Drosophila melanogaster encodes a structural homologue of vertebrate nicotinic acetylcholine receptors (AChR) and is expressed exclusively in nervous tissue. To study the nature of the ARD protein, antibodies were raised against fusion constructs containing two regions of this polypeptide. One segment is putatively extracellular (amino acids 65‐212), the other domain is exposed to the cytoplasm (amino acids 305‐444). The ARD antisera obtained served to investigate the physical relationship between the ARD protein and alpha‐bungarotoxin (alpha‐Btx) binding sites occurring in Drosophila. Two different high‐affinity binding sites for [125I]alpha‐Btx, a highly potent antagonist of vertebrate muscle AChR, were detected in fly head membranes. Equilibrium binding and kinetic studies revealed Kd values of approximately 0.1 nM (site 1) and approximately 4 nM (site 2). The estimated maximal binding (Bmax) was approximately 240 and 1080 fmol/mg protein respectively. Both sites exhibited a nicotinic‐cholinergic pharmacology. Immunoprecipitation experiments with the ARD antisera indicated that the ARD protein is associated with the [125I]alpha‐Btx binding site 1 only. These data support the previously postulated hypothesis that the ARD protein is part of an alpha‐Btx binding neuronal AChR of Drosophila. Furthermore, they indicate heterogeneity in nicotinic‐cholinergic binding sites in the insect nervous system.

Sequence of a Drosophila Ligand‐Gated Ion‐Channel Polypeptide with an Unusual Amino‐Terminal Extracellular Domain

Abstract: We report the isolation of a full‐length clone from a Drosophila melanogaster head cDNA library that encodes a 614‐residue polypeptide that exhibits all of the features of a ligand‐gated chloride‐channel/receptor subunit. This polypeptide, which has been named GRD (denoting that the polypeptide is a GABAA and glycine receptor‐like subunit of Drosophila), displays between 33 and 44% identity to vertebrate GABAA and glycine receptor subunits and 32–37% identity to the GABAA receptor‐like polypeptides from Drosophila and Lymnaea. It is interesting that the large amino‐terminal, presumed extracellular domain of the GRD protein contains an insertion, between the dicysteine loop and the first putative membrane‐spanning domain, of 75 amino acids that is not found in any other ligand‐gated chloride‐channel subunit. Analysis of cDNA and genomic DMA reveals that these residues are encoded by an extension of an exon that is equivalent to exon 6 of vertebrate GABAA and glycine receptor genes. The gene (named Grd) that encodes the Drosophila polypeptide has been mapped, by in situ hybridization, to position 75A on the left arm of chromosome 3.

Neuraxin, a novel putative structural protein of the rat central nervous system that is immunologically related to microtubule-associated protein 5.

During screening of a rat spinal cord lambda gt11 cDNA library with poly‐ and monoclonal antibodies against the postsynaptic glycine receptor a cDNA was isolated which covers an open reading frame encoding a protein of calculated mol. wt 94 kd. Sequence analysis identified a novel type of neuron‐specific protein (named neuraxin) which is characterized by an unusual amino acid composition, 12 central heptadecarepeats and putative protein and/or membrane interaction sites. The gene encoding neuraxin appears to be unique in the haploid rat genome and conserved in higher vertebrates. Northern blot and in situ hybridization revealed neuraxin mRNA to be expressed throughout the rodent central nervous system (CNS). In spinal cord, neuraxin transcripts were abundant in motoneurons which also expressed glycine receptor subunit mRNA. A bacterial fusion protein containing approximately 90% of the neuraxin sequence was found to specifically bind tubulin. Polyclonal neuraxin antibodies cross‐reacted with microtubule‐associated protein 5 (MAP5), and a monoclonal antibody against MAP5 recognized the neuraxin fusion construct. Based on these data we suggest that neuraxin is related to MAP5 and may be implicated in neuronal membrane‐microtubule interactions.

Characterization of an invertebrate nicotinic acetylcholine receptor gene: The ard gene of Drosophila melanogaster

The ard gene encodes a neuronal nicotinic acetylcholine receptor (AChR) protein from Drosophila (ARD protein). Cytogenetically this gene maps at position 64B/C on the left arm of the 3rd chromosome. Five introns interrupt the protein coding region of the gene, and one is found upstream of the translation start site. The ard gene thus contains less introns than vertebrate muscle AChR genes, but, with one exception, the positions of the resident introns are precisely conserved. Implications for the evolution of AChR genes are discussed.

Neuronal acetylcholine receptors in drosophila: Mature and immature transcripts of the ard gene in the developing central nervous system

The ARD protein is a Drosophila homolog of vertebrate nicotinic acetylcholine receptor (AChR) polypeptides. Here, an analysis of transcripts of the corresponding ard gene is presented. In situ hybridization experiments revealed ard gene expression in nervous tissue only. During development, ard transcripts are prevalent in late embryos, pupae, and newly eclosed flies. Both the spatial and the temporal pattern of ard gene expression is consistent with the ARD protein being part of a neuronal AChR that is produced in large amounts during major periods of neuronal differentiation. In situ hybridization with an intron-specific probe indicated codistribution of immature and mature ard RNAs in pupae and adult flies. In addition to the mature 3.2 kb RNA species, two large immature transcripts are found in newly eclosed flies but not in embryos, suggesting a developmentally regulated processing of ard RNA.

Biochemical and Molecular Biology Approaches to Central Nicotinic Acetylcholine Receptors

The structural investigation of central nicotinic acetylcholine receptors (nAChRs) has so far relied on the use of probes derived from studies on nAChR in skeletal muscle and Torpedo electric organ. Here we describe experiments in which the potent antagonist of neuromuscular nAChR, α-bungarotoxin (α-Btx), and a Torpedo AChR cDNA probe have been used for characterizing putative neuronal nicotinic receptors in chick and Drosophila, and we discuss the suitability of these approaches.

Characterization of the mRNA and the Gene of a Putative Neuronal Nicotinic Acetylcholine Receptor Protein from Drosophila

In insects, acetylcholine is a major excitatory transmitter of the central nervous system, while neuromuscular transmission is mediated by amino acids (1). A variety of studies have identified an α-bungarotoxin-binding component in the brain of Drosophila, which displays a pharmacology similar to that of the nicotinic acetycholine receptor (AChR) from the vertebrate nervous system (2–5). The concentration of α-toxin-binding sites in Drosophila heads is remarkably higher than in any nervous tissue of vertebrates (6). Recently the α-bungarotoxin-binding protein of the locust central nervous system has been shown to be a functional AChR protein containing four identical (or very similar) polypeptides of Mr = 65,000 which are immunologically related to the nicotinic AChR of Torpedo electroplax (7, 8). Using DNA probes of the Torpedo receptor γ-subunit we have isolated cDNA clones encoding a putative neuronal AChR protein of Drosophila (ARD protein). The appearance of its mRNA during development coincides with the major periods of differentiation of the Drosophila central nervous system (9). Here, the general features of the ARD cDNA/mRNA and the deduced protein are summarized and compared to the structure of the vertebrate AChR subunits. A preliminary characterization of organization of the ARD gene is presented.

Ligand-Gated Ion Channels of Drosophila

Protein subunits of ligand-gated ion channels including excitatory nicotinic acetylcholine receptors (AChR) as well as inhibitory γ-amino butyric acid (GABA) and glycine receptors (GlyR) are encoded by a large super-family of related genes (Schofield et al., 1987; Grenningloh et al., 1987a, b). Using DNA probes of vertebrate receptor subunits we have started to isolate genomic and cDNAs encoding ion channel proteins of the fruitfly Drosophila melanogaster. Two of these genes/cDNAs and the deduced proteins will be described: the ard gene coding for an AChR-like Drosophila protein (ARD protein) and the grd gene encoding a potential ligand-gated chloride channel polypeptide.

Molecular Biology Approaches to the Function and Development of CNS Synapses.

Synapses are the structural elements of the neuronal network through which cellular communication occurs. A prerequisite for understanding synaptic information transfer is the identification of the various components of the synaptic machinery. Recently, different molecular genetic techniques have been developed for the isolation of neuron-specific gene products, such as differential screening, gene transfer, deletion mutant analysis, and screening of expression libraries with monoclonal antibodies or selective ligands. Here, we describe two cloning approaches which have been successfully used in our laboratory. The first illustrates how neural gene products can be isolated and identified by selectively cloning mRNAs which appear in the avian optic lobe during the major period of synaptogenesis.

Central nicotinic acetylcholine receptors in the chicken and Drosophila CNS: Biochemical and molecular biology approaches

Putative neuronal nicotinic acetylcholine receptors (nAChRs) were investigated using biochemical and molecular biology approaches. In the chick visual system, a nicotinic cholinergic binding site was localized on a polypeptide of Mr 57,000 using the potent antagonist, alpha-bungarotoxin (alpha-Btx). Ion flux experiments indicate that this membrane protein is different from the toxin-insensitive nAChR present in vertebrate neurons. Crosshybridization with a Torpedo nAChR cDNA probe allowed isolation of a cholinergic receptor cDNA (ARD) from the Drosophila CNS. Analysis of the corresponding gene, its transcripts and the ARD protein are presented here.