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R Schoepfer

Publications and source records attributed to R Schoepfer.

48 records · Page 3Linked to original sources
PubMed1990

Structural and functional heterogeneity of nicotinic receptors.

Three gene families of the ligand-gated ion channel gene superfamily encode proteins which bind cholinergic ligands: (1) nicotinic acetylcholine receptors (AChRs) from skeletal muscle, (2) AChRs from neurons, and (3) neuronal alpha-bungarotoxin-binding proteins (alpha BgtBPs). AChRs from muscles and nerves function as ACh-gated cation channels, but alpha BgtBPs do not appear to function in this way. A family of neuronal AChR subtypes has been characterized using monoclonal antibodies and cDNA probes. Neuronal AChRs exhibit sequence homologies with muscle AChRs, but differ in subunit composition, pharmacological and electrophysiological properties, and, in some cases, apparent functional roles. The genes that encode the subunits of the various purified AChR subtypes have been determined in several cases. Histological localization of AChR subunit mRNAs by in situ hybridization and of subunit proteins by immunohistochemistry is being conducted with increasing resolution. The subunit structure of alpha BgtBP is uncertain, but cDNAs have been identified for two subunits. Sequences of these cDNAs reveal that alpha BgtBPs are members of the ligand-gated ion channel gene family, and suggest that they could function as gated cation channels. Biochemical and molecular genetic approaches to studies of neuronal AChRs and related proteins are merging to provide a detailed description of a complex family of AChRs widely dispersed throughout the nervous system, which are probably important to many activities of the nervous system, but whose functional roles are not yet well characterized.

Animals↗
PubMed1990

Brain alpha-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily.

alpha-Bungarotoxin (alpha Bgt) is a potent, high-affinity antagonist for nicotinic acetylcholine receptors (AChRs) from muscle, but not for AChRs from neurons. Both muscle and neuronal AChRs are thought to be formed from multiple homologous subunits aligned around a central cation channel whose opening is regulated by ACh binding. In contrast, the exact structure and function of high-affinity alpha Bgt binding proteins (alpha BgtBPs) found in avian and mammalian neurons remain unknown. Here we show that cDNA clones encoding alpha BgtBP alpha 1 and alpha 2 subunits define alpha BgtBPs as members of a gene family within the ligand-gated ion channel gene superfamily, but distinct from the gene families of AChRs from muscles and nerves. Subunit-specific monoclonal antibodies raised against bacterially expressed alpha BgtBP alpha 1 and alpha 2 subunit fragments reveal the existence of at least two different alpha BgtBP subtypes in embryonic day 18 chicken brains. More than 75% of all alpha BgtBPs have the alpha 1 subunit, but no alpha 2 subunit, and a minor alpha BgtBP subtype (approximately 15%) has both the alpha 1 and alpha 2 subunits.

Amino Acid Sequence↗
PubMed1989

Antisera against an acetylcholine receptor alpha 3 fusion protein bind to ganglionic but not to brain nicotinic acetylcholine receptors.

Neuronal nicotinic acetylcholine receptor (AChR) subtypes have been defined pharmacologically, immunologically, and by DNA cloning, but the correlations between these approaches are incomplete. Vertebrate neuronal AChRs that have been isolated are composed of structural subunits and ACh-binding subunits. A single kind of subunit can be used in more than one AChR subtype. Monoclonal antibody (mAb) 35 binds to structural subunits of subtypes of AChRs from both chicken brain and ganglia. By using antisera to a unique sequence of alpha 3 ACh-binding subunits expressed in bacteria, we show that ganglionic AChRs contain alpha 3 ACh-binding subunits, whereas the brain AChR subtype that binds mAb 35 does not. Subunit-specific antisera raised against recombinant proteins should be a valuable approach for identifying the subunit composition of receptors in multigene, multisubunit families.

Amino Acid Sequence↗
PubMed1989

A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671.

The human neuromedulloblastoma cell line TE671 is shown by single-channel recordings to express nicotinic acetylcholine receptors (AChRs) that are blocked by alpha-bungarotoxin (alpha Bgt). These AChRs do not react with antisera to the alpha Bgt-binding protein of brain or with monoclonal antibodies (mAbs) to brain nicotinic AChRs that do not bind alpha Bgt. TE671 AChRs do react with autoantibodies to muscle AChRs from myasthenia gravis patients and with mAbs to muscle AChRs, including mAbs specific for extrajunctional AChRs. AChRs. AChRs purified from TE671 cells are composed of 4 kinds of subunits corresponding to those of muscle AChR. Sequences of cDNAs for the ACh-binding alpha subunit and the delta subunit of this AChR further identify it as muscle AChR. Expression of TE671 AChR can be up-regulated by nicotine and dexamethasone, and down-regulated by forskolin.

Amino Acid Sequence↗
PubMed1989

The gene of the alpha-subunit of the acetylcholine receptor: molecular organisation and transcription in myasthenia-associated thymomas.

DNA and RNA were isolated from 5 thymomas of Myasthenia Gravis (MG) patients, from normal tissues, and from the TE671 cell line (which expresses a muscle type acetylcholine receptor, AChR). The cDNA of the alpha-subunit of the AChR, a 159 bp BglII/BstEII fragment encoding the main immunogenic region (MIR) and a 88 bp EcoRV/TaqI fragment encoding the mAb155 binding site (a cytoplasmic epitope of AChR) served as probes. Hybridizations were performed under both high and low stringent conditions. Southern blot analysis of genomic DNA, restricted with EcoRI, HindIII and BamHI/HindIII, showed a normal pattern of restriction fragments in all tissues investigated. In particular, in thymomas there was no deletion of exon 4 which encodes the MIR. Dot and Northern blot analysis of total RNA and mRNA revealed transcription of the alpha-subunit AChR gene in TE671 cells and skeletal muscle but not in other tissues including thymomas. These results confirm former reports that there are no intact AChR in thymomas of MG patients. In addition we show here that there is also no truncated, MIR-deficient AChR or a protein with extensive molecular homology with the AChR in thymomas. These investigations support our idea that an AChR-unrelated protein might play a role in the pathogenesis of thymoma-associated MG [Marx et al., this volume].

Autoimmune Diseases↗
PubMed1988

The human medulloblastoma cell line TE671 expresses a muscle-like acetylcholine receptor. Cloning of the alpha-subunit cDNA.

Nicotinic acetylcholine receptors (AChRs) from muscle bind alpha-bungarotoxin (alpha Bgt) and are composed of four kinds of subunits, whereas AChRs from mammalian brains do not bind alpha Bgt and are composed of two kinds of subunits. alpha Bgt-binding proteins whose function is unknown are also found in brain. All these proteins belong to the same gene family. The human medulloblastoma cell line TE671 expresses a functional AChR which binds alpha Bgt. Surprisingly, the AChR of this neuron-derived cell line has electrophysiological, immunological and biochemical properties different from neuronal AChRs and very similar to muscle AChRs. The TE671 AChR binds alpha Bgt, but is different from alpha Bgt-binding proteins in brain. Here we show that TE671 expresses the alpha-subunit mRNA coding for the muscle AChR, thereby proving that TE671 expresses a muscle-type AChR that is not expressed in adult brain. The isolated cDNA clones should prove useful for expression of large amounts of human muscle-type AChR alpha-subunit protein for studies of the autoimmune response to muscle AChRs in human myasthenia gravis.

Amino Acid Sequence↗
PubMed1988

cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor.

Nicotinic acetylcholine receptors (AChRs) immunoaffinity-purified from brains are composed of only two kinds of subunits rather than the four kinds present in muscle-type AChRs. Here we report the N-terminal protein sequences of the structural subunits of AChRs from rat and chicken brains and the cloning of full-length cDNAs for the chicken brain AChR structural subunit. Previously, the N-terminal amino acid sequence of the ACh-binding subunit of AChR immunoaffinity-purified from rat brain was shown to correspond to the cDNA alpha 4. Thus, cDNA sequences are now known for both of the subunits that form one AChR subtype in vivo.

Amino Acid Sequence↗
PubMed1987

Molecular studies of the neuronal nicotinic acetylcholine receptor family.

Nicotinic acetylcholine receptors on neurons are part of a gene family that includes nicotinic acetylcholine receptors on skeletal muscles and neuronal alpha bungarotoxin-binding proteins that in many species, unlike receptors, do not have an acetylcholine-regulated cation channel. This gene superfamily of ligand-gated receptors also includes receptors for glycine and gamma-aminobutyric acid. Rapid progress on neuronal nicotinic receptors has recently been possible using monoclonal antibodies as probes for receptor proteins and cDNAs as probes for receptor genes. These studies are the primary focus of this review, although other aspects of these receptors are also considered. In birds and mammals, there are subtypes of neuronal nicotinic receptors. All of these receptors differ from nicotinic receptors of muscle pharmacologically (none bind alpha bungarotoxin, and some have very high affinity for nicotine), structurally (having only two types of subunits rather than four), and, in some cases, in functional role (some are located presynaptically). However, there are amino acid sequence homologies between the subunits of these receptors that suggest the location of important functional domains. Sequence homologies also suggest that the subunits of the proteins of this family all evolved from a common ancestral protein subunit. The ligand-gated ion channel characteristic of this superfamily is formed from multiple copies of homologous subunits. Conserved domains responsible for strong stereospecific association of the subunits are probably a fundamental organizing principle of the superfamily. Whereas the structure of muscle-type nicotinic receptors appears to have been established by the time of elasmobranchs and has evolved quite conservatively since then, the evolution of neuronal-type nicotinic receptors appears to be in more rapid flux. Certainly, the studies of these receptors are in rapid flux, with the availability of monoclonal antibody probes for localizing, purifying, and characterizing the proteins, and cDNA probes for determining sequences, localizing mRNAs, expressing functional receptors, and studying genetic regulation. The role of nicotinic receptors in neuromuscular transmission is well understood, but the role of nicotinic receptors in brain function is not. The current deluge of data using antibodies and cDNAs is beginning to come together nicely to describe the structure of these receptors. Soon, these techniques may combine with others to better reveal the functional roles of neuronal nicotinic receptors.

Animals↗
PubMedDate not supplied

Functional acetylcholine receptor in PC12 cells reacts with a monoclonal antibody to brain nicotinic receptors.

The use of a variety of probes for neuronal nicotinic acetylcholine receptors has indicated that there are sites on neurons that bind the acetylcholine receptor antagonist alpha-bungarotoxin, but do not regulate cation channels in response to the binding of acetylcholine. Sites with high binding affinity for nicotine and for alpha-bungarotoxin also show different distributions in brain. A monoclonal antibody (mAb35) raised against acetylcholine receptors from Electrophorus electric organ has been used to purify receptors from chick brain. These receptors do not bind alpha-bungarotoxin but have a high affinity for nicotine and antibodies raised against them block the function of acetylcholine receptors in ciliary ganglia. We have raised a monoclonal antibody (mAb270) against acetylcholine receptors from chicken brain (P.J.W., R. Liu, S. Shimasaki, F. Esch, B. Morley & J.M.L., manuscript in preparation), which has been used to purify a similar receptor from rat brain. Here we show that this antibody identifies a functional nicotinic acetylcholine receptor in rat-neuron-like PC12 cells which is probably not encoded by the cDNA clone (lambda PCA48) previously suggested as a candidate for the acetylcholine receptor gene. Additionally, we show that mAb270 binds to the same areas of rat brain as nicotine.

Adrenal Gland Neoplasms↗