Structure and developmental expression of the Dα2 gene encoding a novel nicotinic acetylcholine receptor protein of <i>Drosophila melanogaster</i>

Jonas, Petra; Baumann, Arnd; Merz, Barbara; Gundelfinger, Eckart D.

doi:10.1016/0014-5793(90)81170-s

Cited by 33 publications

(11 citation statements)

References 31 publications

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“…The gene Cha which encodes the ChAT enzyme is expressed at low levels in 6-7 h embryos, and there is a dramatic increase in levels of Cha mRNA at the 13 h stage, followed by a similar but more gradual increase in ChAT enzyme activity (Carbini et at., 1990). The earliest appearance of Ace and ChAT enzymes coincides with the expression of an ~x-like subunit of the multisubunit nicotinic acetylcholine receptor which is first observed after 4-12 h of embryogenesis (Jonas et at., 1990). Other nicotinic receptor subunits are not detected until 10-14 h (Bossy et at., 1988;Wadsworth et at., 1988;Hermans-Borgmeyer et al, 1989;Sawruk et al, 1990aSawruk et al, , 1990b.…”

Section: Discussionmentioning

confidence: 93%

Temporal and spatial expression patterns of two G-protein coupled receptors inDrosophila melanogaster

Hannan

Hall

1996

Invertebrate Neuroscience

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Temporal and spatial expression patterns of a muscarinic acetylcholine receptor (Acr60C) and an octopamine/tyramine receptor (Octyr) were determined in Drosophila melanogaster using quantitative Northern analysis and in situ hybridization to tissue sections. Expression of mRNA encoding both of these G-protein coupled receptors peaks initially in 18 to 21 hour embryos following the formation of the mature larval nervous system. Levels of mRNA then decline during larval stages, rising to a second peak in 3 to 4-day-old pupae after a period of major nervous system reorganization. The muscarinic acetylcholine receptor mRNA is expressed throughout the cortical regions of the central nervous system in adults and embryos. Particularly high levels of expression of Acr60C are observed in cell bodies adjacent to the antennal lobes, suggesting a major role for this muscarinic receptor in the processing of olfactory information. In contrast, the octopamine/tyramine receptor mRNA is distributed diffusely throughout the adult brain, with patches of signal concentrated in the cortex of the dorsal protocerebrum near the mushroom bodies. These patches may represent individual cells expressing Octyr receptors.

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Section: Discussionmentioning

confidence: 93%

Temporal and spatial expression patterns of two G-protein coupled receptors inDrosophila melanogaster

Hannan

Hall

1996

Invertebrate Neuroscience

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“…nACRα96Aa , nACRα96Ab and nACRβ96A , which all map to the 96 A region of the third Drosophila chromosome (Bossy et al . 1988; Jonas et al . 1990; Sawruk et al .…”

Section: Resultsmentioning

confidence: 99%

Nicotinic acetylcholine receptors of Drosophila: three subunits encoded by genomically linked genes can co‐assemble  into the same receptor complex

Chamaon

Smalla

Thomas

et al. 2001

Journal of Neurochemistry

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The second b-like subunit (SBD) is a putative structural subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here we have produced speci®c antibodies against SBD to study, which other nAChR subunits can co-assemble with SBD in receptor complexes of the Drosophila nervous system. Immunohistochemical studies in the adult optic lobe revealed that SBD has a distribution similar to that of the a-subunit ALS in the synaptic neuropil. The subunits ALS, Da2 and SBD can be co-puri®ed by a-bungarotoxin af®nity chromatography. Moreover, anti-SBD antibodies co-precipitate ALS and Da2 and, vice versa, ALS and Da2 antibodies co-immunoprecipitate SBD protein. Two-step immunoaf®nity chromatography with immobilized antibodies against ALS and Da2 revealed the existence of nAChR complexes that include ALS, Da2 and SBD as integral components. Interestingly, the genes encoding these three subunits appear to be directly linked in the Drosophila genome at region 96 A of the third chromosome. In addition, SBD appears to be a component of a different receptor complex, which includes the ARD protein as an additional b-subunit, but neither ALS nor Da2 nor the third a-subunit Da3. These ®ndings suggest a considerable complexity of the Drosophila nicotinic receptor system. Keywords: Drosophila genome, gene cluster, immunoprecipitation, insect nervous system, neurotransmitter receptor, quaternary protein structure. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels assembled from multiple subunits. Prototypic nicotinic receptors are found at the vertebrate neuromuscular junction where they mediate excitatory neurotransmission. These muscle receptors are pentamers of known subunit composition, i.e. a 2 bcd or a 2 bed (reviewed by Galzi et al. 1991;Hucho et al. 1996). A variety of nAChR subunits (a2±a9 and b2±b4) are expressed in the vertebrate nervous system, where they assemble into multiple subtypes of heteromeric and homomeric neuronal receptors (for review see Lindstrom 2000).In the insect CNS nAChRs are widely expressed and mediate fast excitatory synaptic transmission (Sattelle 1980). Most of these receptors are thought to constitute heteromeric complexes (Gundelfinger 1992); however, the subunit composition of native nAChRs in insects is still unknown. From the fruit fly Drosophila melanogaster, six neuronal nAChR subunits have been cloned to date; these are the a-type subunits ALS, Da2/SAD, Da3, Da4 and the b-type subunits ARD and SBD (Gundelfinger and Hess 1992;Gundelfinger and Schulz 2000;Lansdell and Millar 2000). The existence of additional nAChR subunits can be predicted based on the analysis of the Drosophila genome These authors contributed equally to this work. Abbreviations used: aBgt, a-bungarotoxin; ALS, a-like subunit; ARD, acetylcholine receptor protein of Drosophila; Da2, Drosophila a-subunit 2; Da3, Drosophila a-subunit 3; Da4, Drosophila a-subunit 4; mAb, monoclonal antibody; nAChR, nicotinic acetylcholine receptor; pAb, polyclonal antibody; SBD, second b-like ...

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“…PCR primer deduced from published sequences of Drosophila and Schistocerca (Hermans-Borgmeyer et al 1986;Jonas et al 1990;Sawruk et al 1990;Marshall et al 1990;Gundelfinger & Hess 1992), were: h1-subunits forward A1: 5%-GGC ATC(T) GAC CTG CG(A)T(C;G) GAG TAC A-3% and l1: 5%-GAC(G) TAC TAC ATC TCG(A) GTG(C) g-3%; backward: A2: 5%ATC AGG TTG(A) ACC GTG TAG AAG(C) AG-3%; l2: 5%-CAG TGA T(C)GT T(G)GG G(C)GG AAT GAT T(C)TC-3% and M6: 5%-ACC(G) AGC AA(T;C)C ATG GT AAC(G) AG-3%. h2-subunit: forward: M5: 5%-GAA GTT CGG C(A)TC C(G)TG GAC CTA CGA-3%; ll1: 5%-GAC ATA TCG ACG AGG CGC GAG G-3% and M9: 5%-TCG ACC TCT CCG AGT TTT ACA CC-3%; backward: A2 and M6.…”

Section: Amplification and Sequencing Of Nicotine Binding Sites Of H-mentioning

confidence: 99%

Alkaloid tolerance in Manduca sexta and phylogenetically related sphingids (Lepidoptera: Sphingidae)

Wink

Theile

2002

Chemoecology

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Nicotine tolerance is well known for Manduca sexta. It also occurs in several other sphingids of the subfamilies Macroglossinae and Sphinginae. Only members of the subfamily Smerinthinae appear to be more susceptible to nicotine intoxication. Phylogenetic trees have been reconstructed from mitochondrial 16S rDNA and nuclear DNA to map nicotine tolerance.The nicotine binding site of both h-subunits of nicotinic acetylcholine receptors (nAChR) have been amplified and sequenced. No apparent amino acid substitution can be seen in the putative nicotine binding site of the h-subunits of nAChR from nicotine tolerant and nicotine sensitive sphingids. Thus, a simple targetsite modification can be ruled out as a cause for nicotine tolerance. This finding agrees with feeding experiments: larvae of M. sexta and other sphingids of the Macroglossinae and Sphinginae not only tolerated nicotine, but also many other alkaloids that affect neuroreceptors other than acetylcholine receptors (nAChR, mAChR).Only 10 to 20% of nicotine injected into larvae of nicotine-tolerant taxa could be recovered later as free nicotine, nicotine N-oxide or cotinine, i.e., 80 to 90% must have been converted to polar conjugates or degradation products which are not detectable with the methods applied. Usually more than 98% of the recoverable alkaloids were found in the faeces. Excretion reached a maximum 6 h after injection in tolerant taxa. Larvae of Manduca sexta, which were reared on a nicotine-rich diet, showed higher nicotine degradation and faster nicotine elimination than naïve larvae. Application of the cytochrome P450 inhibitor SKF 525A (proadifen) reduced the formation of nicotine N-oxide and the rate of alkaloid degradation. Thus, an inducible detoxification mechanism, coupled with a rapid and inducible excretion, appear to be a strategy in Sphingidae that helps them to live on host plants rich in otherwise toxic secondary metabolites.

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Structure and developmental expression of the Dα2 gene encoding a novel nicotinic acetylcholine receptor protein of Drosophila melanogaster

Cited by 33 publications

References 31 publications

Temporal and spatial expression patterns of two G-protein coupled receptors inDrosophila melanogaster

Temporal and spatial expression patterns of two G-protein coupled receptors inDrosophila melanogaster

Nicotinic acetylcholine receptors of Drosophila: three subunits encoded by genomically linked genes can co‐assemble  into the same receptor complex

Alkaloid tolerance in Manduca sexta and phylogenetically related sphingids (Lepidoptera: Sphingidae)

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Structure and developmental expression of the Dα2 gene encoding a novel nicotinic acetylcholine receptor protein of Drosophila melanogaster

Cited by 33 publications

References 31 publications

Temporal and spatial expression patterns of two G-protein coupled receptors inDrosophila melanogaster

Temporal and spatial expression patterns of two G-protein coupled receptors inDrosophila melanogaster

Nicotinic acetylcholine receptors of Drosophila: three subunits encoded by genomically linked genes can co‐assemble into the same receptor complex

Alkaloid tolerance in Manduca sexta and phylogenetically related sphingids (Lepidoptera: Sphingidae)

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Nicotinic acetylcholine receptors of Drosophila: three subunits encoded by genomically linked genes can co‐assemble  into the same receptor complex