Nucleotide and deduced amino acid sequences of Torpedo californica acetylcholine receptor gamma subunit.

Abstract

The nucleotide sequence has been determined of a cDNA clone that codes for the 60,000-dalton y subunit of Torpedo californica acetylcholine receptor. The length of the cDNA clone is 2,010 base pairs. The 5' and 3' untranslated regions have respective lengths of 31 and 461 base pairs. Data suggest that the putative polyadenylylation consensus sequence A-A-T-A-A-A may not be required for polyadenylylation of the mRNA corresponding to the cDNA clone described in this study. From the DNA sequence data, the amino acid sequence of the y subunit was deduced. The subunit is composed of 489 amino acids giving a molecular mass of 56,600 daltons. The deduced amino acid sequence data also indicate the presence of a 17-amino acid extension or signal peptide on this subunit. From these data, structural predictions for the y subunit are made such as potential membrane-spanning regions, possible asparagine-linked glycosylation sites, and the assignment of regions of the protein to the extracellular, internal, and cytoplasmic domains of the lipid bilayer.The nicotinic acetylcholine receptor (AcChoR) is a glycoprotein located in the postsynaptic membrane of the vertebrate neuromuscular junction. When the neurotransmitter acetylcholine binds to the receptor, a channel permeable to small cations opens. The resulting ion flow leads to depolarization and contraction of the muscle cell. AcChoR from the electric organ of Torpedo californica has been most extensively studied because it is a major component of the membrane in this tissue and can be purified in milligram amounts (for recent reviews, see refs. 1 and 2). The AcChoR from T. californica is an oligomeric complex with a molecular mass of --250,000 daltons. The complex is composed of four different polypeptide chains of approximately 40,000 (a), 50,000 (,B), 60,000 (y), and 65,000 (8) daltons (3, 4) with a stoichiometry of 2:1:1:1, respectively (5, 6). The subunits have a number of common features: each subunit is glycosylated (4, 7), the subunits have similar amino acid compositions (8) and show 35-50% sequence homology in the NH2-terminal regions (6), and each subunit spans the lipid bilayer (9). A number of experiments have demonstrated that a large part of the AcChoR extends into the synaptic cleft or extracellular side of the membrane (reviewed in ref. 10). The purified 250,000-dalton oligomer contains the binding sites for acetylcholine and the ligand-gated ion channel (11,12). Acetylcholine analogs are known to bind to the a-polypeptide chains (3); as yet, no specific functions have been assigned to the other polypeptide chains.This paper reports the complete nucleotide sequence of a cDNA clone (4D8) coding for the y subunit of AcChoR from the electric organ of T. californica. The deduced amino acid sequence of the y subunit and its putative signal peptide are also presented. Several structural predictions based on the amino acid sequence are discussed and incorporated into a model giving a possible partitioning of the y-polypeptide chain into the extracellular, ...

Mutations in M2 alter the selectivity of the mouse nicotinic acetylcholine receptor for organic and alkali metal cations.

“…Aligned amino acid sequences of the mouse (LaPolla, Mixter-Mayne, and Davidson, 1985;Isenberg, Mudd, Shah, and Merlie, 1986;Yu, Lapolla, and Davidson, 1986;Gardner, 1990), Torpedo (Noda, Takahashi, Tanabe, Toyosato, Furutani, Hirose, Asai, Inayama, Miyata, and Numa, 1982;Claudio, Ballivet, Patrick, and Heinemann, 1983;Noda, Takahashi, Tanabe, Toyosato, Kikyotani, Hirose, Asai, Takashima, Inayama, Miyata, and Numa, 1983), and Xenopus M2 regions (Baldwin, Yoshihara, Blackmer, Kintner, and Burden, 1988). Amino acids are given by the single letter code.…”

Primary structure of delta subunit precursor of calf muscle acetylcholine receptor deduced from cDNA sequence

“…Work from this laboratory has elucidated the primary structures of all four subunits of the Torpedo californica acetylcholine receptor by cloning and sequencing cDNAs encoding the respective subunit precursors [4 -61; cDNA sequences for the y subunit precursor of the T. californica receptor [7] and the M subunit precursor of the Torpedo marmorata receptor [8, 91 have also been reported by other groups. The conspicuous amino acid sequence homology found among the four subunits of the T. calijornica receptor suggests that the genes encoding them have been generated from a single common ancestor by gene duplications.…”

Monoclonal antibodies specific to the beta and gamma subunits of the Torpedo acetylcholine receptor inhibit single-channel activity

“…Changeux et al, 1984). All four ACh receptor subunits are transmembrane polypeptides (Froehner, 198 1;Strader and Rafferty, 1980;Strader et al, 1979;Tarrab-Hazdai et al, 1978;Wennogle and Changeux, 1980) and the complete amino acid sequences of all four subunits have been deduced from the nucleotide sequences of cDNA clones (Claudio et al, 1983;Devillers-Thiery et al, 1983;Noda et al, 1982Noda et al, , 1983a. Reconstitution of the purified ACh receptor in lipid vesicles (Anholt et al, 1981(Anholt et al, , 1982Changeux et al, 1979;Gonzalez-Ros et al, 1980;Huganir et al, 1979;Lindstrom et al, 1980;Wu and Raftery, 1979) and in lipid bilayers (Boheim et al, 1981;Labarca et al, 1984a, b;Nelson et al, 1980;Schindler et al, 1984;Tank et al, 1983) demonstrated that the a,&6 structure is sufjicient to express the transduction of ligand binding into the opening of a cation-specific channel.…”