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J Lindstrom

Publications and source records attributed to J Lindstrom.

At least 127 records · Page 7Linked to original sources
PubMed1987

Purification and characterization of a nicotinic acetylcholine receptor from rat brain.

We previously reported the immunoaffinity purification of an acetylcholine receptor from chicken brain that did not bind alpha-bungarotoxin but did bind nicotine and other cholinergic agonists. Antisera and monoclonal antibodies raised against this receptor crossreacted with a receptor from rat brain that had similar pharmacological properties, and also bound to functional acetylcholine receptors in chicken ciliary ganglion cells and rat PC12 cells. Here we report purification of the receptor from rat brain using monoclonal antibody (mAb) 270 raised against receptor from chicken brain. This receptor, similar in size to monomers of receptor from Torpedo electric organ, contained two subunits--apparent Mr, 51,000 and 79,000. The Mr 51,000 subunit was bound by antisera to alpha subunits of receptor from Torpedo electric organ and by mAb 270, which is specific for the Mr 49,000 subunit analogue of receptor from chicken brain. Both subunits were bound by mAb 286, which also binds both subunits of receptors from chicken brain. The alpha-bungarotoxin binding component was purified from the same extracts. It consisted of four subunits of apparent Mr 44,700, 52,300, 56,600, and 65,200. The basic structure of receptors from muscle had evolved to an (alpha)2 beta gamma delta subunit stoichiometry by the time of primitive elasmobranches and is now little changed in mammals. The apparent (alpha)2(beta)2 or (alpha)3(beta)2 structure of the neuronal acetylcholine receptors that we have purified may derive from an early gene duplication event in the evolution of the extended gene family, which now also includes receptors from ganglia and muscle as well as neuronal alpha-bungarotoxin binding sites.

Animals↗
PubMed1987

Location of subunits within the acetylcholine receptor by electron image analysis of tubular crystals from Torpedo marmorata.

The binding sites on the nicotinic acetylcholine receptor of labels specific for the alpha-, beta-, and delta-subunits were determined by electron image analysis, using tubular crystals of receptors grown from the postsynaptic membranes of Torpedo marmorata electric organ. The labels were alpha-bungarotoxin (which attaches to the acetylcholine binding sites on the pair of alpha-subunits), Fab35 (a monoclonal antibody Fab fragment directed against the main immunogenic region of the alpha-subunit), Fab111 (a monoclonal antibody Fab fragment directed against a cytoplasmic site on the beta-subunit), and wheat germ agglutinin (which binds to N-acetylglucosamine residues on the delta-subunit). These labels, bound to receptors in the crystals, were located by comparing labeled with native structures, averaged in each case over more than 5,000 molecules. From the assignments made, we find that the clockwise arrangement of subunits around the receptor, viewed from the synaptic face, is: alpha, beta, alpha, gamma, and delta; that the main immunogenic region is at (or close to) the side of the alpha-subunit; and that the two acetylcholine binding sites are at the synaptic end of the alpha-subunits, 27-28 A from the central axis and approximately 53 A apart. In the crystal lattice, neighboring molecules are paired so that their delta- and alpha-subunits are juxtaposed, an organization that appears to relate closely to the grouping of receptors in vivo.

Animals↗
PubMed1987

Genetic variants of C2 muscle cells that are defective in synthesis of the alpha-subunit of the acetylcholine receptor.

We have analyzed two genetic variants of C2 muscle cells that have reduced levels of binding activity for alpha-bungarotoxin and have found that both synthesize only low levels of the alpha-subunit of the acetylcholine receptor. In both variants the uptake of 22Na in response to carbachol is diminished in proportion to the reduction in toxin-binding activity. In addition, the kinetic and sedimentation properties of the residual toxin-binding activity in both is indistinguishable from that seen in wild-type cells. Immunoblotting experiments on extracts of the variants using subunit-specific antibodies to alpha- and beta-subunits of the acetylcholine receptor demonstrated that the beta-subunit was present, but failed to detect alpha-subunit. In both variants, the amount of alpha-subunit accumulated after a 5-min period of labeling with [35S]methionine was reduced by over 90%, leading to the conclusion that the alpha-subunit is synthesized at greatly reduced rates. Northern blot and S1 nuclease analysis showed no differences between the alpha-subunit mRNA in wild-type and variant cells.

Animals↗
PubMed1987

Immunohistochemical localization of neuronal nicotinic receptors in the rodent central nervous system.

The distribution of nicotinic acetylcholine receptors (AChR) in the rat and mouse central nervous system has been mapped in detail using monoclonal antibodies to receptors purified from chicken and rat brain. Initial studies in the chicken brain indicate that different neuronal AChRs are contained in axonal projections to the optic lobe in the midbrain from neurons in the lateral spiriform nucleus and from retinal ganglion cells. Monoclonal antibodies to the chicken and rat brain AChRs also label apparently identical regions in all major subdivisions of the central nervous system of rats and mice, and this pattern is very similar to previous reports of 3H-nicotine binding, but quite different from that of alpha-bungarotoxin binding. In several instances, the immunohistochemical evidence has strongly indicated that neuronal AChR undergoes axonal transport. The clearest example of this has been in the visual system, where labeling was observed in the retina, the optic nerve and tract, and in all of the major terminal fields of the optic nerve except the ventral suprachiasmatic nucleus. This was confirmed in unilateral enucleation experiments in the rat, where labeling was greatly reduced in the contralateral optic tract, ventral lateral geniculate nucleus, pretectal nuclei receiving direct visual input, superficial layers of the superior colliculus, and medical terminal nucleus, and was significantly reduced in the dorsal lateral geniculate nucleus. Clear neuronal labeling was also observed in dorsal root ganglion cells and in cranial nerve nuclei containing motoneurons that innervate branchial arch-derived muscles, although the possibility that neuronal AChR undergoes axonal transport in the latter cells was not tested experimentally.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

The effects of inhibiting oligosaccharide trimming by 1-deoxynojirimycin on the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor has a subunit stoichiometry of alpha 2 beta gamma delta; all 5 subunits contain N-linked oligosaccharides. We investigated what role trimming of the oligosaccharides played in the post-translational processing of the subunits and assembly of the receptor by examining the receptor synthesized in the presence of an inhibitor of oligosaccharide trimming, 1-deoxynojirimycin. BC3H-1 cells express one-third fewer receptors when grown in the presence of 1-deoxynojirimycin. The receptor subunits that are expressed have decreased mobility by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating an inhibition of oligosaccharide trimming. In control cells, 40% of the translated alpha subunit acquires the capacity to bind alpha-bungarotoxin with a half-time of 40 min before assembly with the other subunits; the rest is rapidly degraded. In 1-deoxynojirimycin-treated cells approximately the same amount of alpha subunit is translated as in control cells, but that alpha subunit is degraded more rapidly, and only 25% acquires the capacity to bind alpha-bungarotoxin. From these results, we conclude that oligosaccharide processing either may aid in protecting the alpha subunit primary translation product from degradation or may be required for the conformational change or other post-translational modification(s) necessary for formation of the alpha-bungarotoxin binding form of the alpha subunit, which is then protected from proteolytic degradation. The cell surface receptor that is expressed in the presence of 1-deoxynojirimycin, however, is not altered in its affinity for cholinergic ligands. Thus, we conclude that differential N-linked oligosaccharide trimming of the 2 alpha subunits does not appear to play a part in the differences in affinities of the 2 alpha subunits for cholinergic ligands.

1-Deoxynojirimycin↗
PubMed1986

Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding.

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1986

Evidence that the acetylcholine binding site is not formed by the sequence alpha 127-143 of the acetylcholine receptor.

The sequence alpha 127-143 of the alpha subunit of the acetylcholine receptor has been proposed to contain several important features: (1) the acetylcholine binding site, (2) the only N-glycosylation site of the alpha subunit, at asparagine-alpha 141, and (3) two cysteine residues, at alpha 128 and alpha 142, that may participate in a disulfide bond known to be near the binding site. We tested these hypotheses by using antisera to receptor and its subunits and monoclonal antibodies to the synthetic peptide alpha 127-143 cyclized by a disulfide bond between alpha 128 and alpha 142. Antisera to receptor and its alpha subunit were able to immunoprecipitate the iodinated peptide, and this reaction was inhibited by soluble receptor, but not by membrane-bound receptor. alpha-Bungarotoxin did not inhibit antiserum binding to solubilized receptor. Similarly, cholinergic ligands had little or no effect on binding to immobilized receptors of anti-peptide monoclonal antibodies. In addition, these monoclonal antibodies, when bound to the receptor, did not affect toxin binding kinetics. By contrast, preincubation with concanavalin A did inhibit monoclonal antibody binding. Reduction of the receptor significantly decreased the binding of three of the monoclonal antibodies, but subsequent alkylation with N-ethylmaleimide or the affinity labeling reagent bromoacetylcholine had no additional effect on binding. A dithiothreitol concentration about 100-fold higher that the one needed to reduce the disulfide near the acetylcholine binding site was necessary to inhibit monoclonal antibody binding. We conclude that the sequence alpha 127-143 is not fully exposed on the surface when the receptor is in the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1986

Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping.

The binding domains of 28 monoclonal antibodies (mAbs) against the alpha, beta, and delta subunits of the Torpedo acetylcholine receptor were mapped on the primary sequences of these subunits. Small peptide fragments (2000-20,000 daltons) of the purified subunits were obtained by digestion with staphylococcal V8 protease and papain, separated on a discontinuous polyacrylamide gel electrophoretic system, and electroblotted onto diaminophenyl thioether paper. The blots were probed with the various monoclonal antibodies and also with antibodies against carboxy-terminal decapeptides of the alpha, beta, and delta subunits to identify the carboxy-terminal fragments. From inspection of the binding patterns of the various antibodies to the subunits fragments and the molecular weights of these fragments, and by using the carboxy termini of the subunits as reference points, it was possible to deduce the regions on the primary sequence of each subunit in which the antibodies bound and in some cases to order the binding sites within these sequences. mAb 148, which inhibits receptor function by cross-linking receptor molecules on the cytoplasmic side, was mapped to the sequence beta 368-406. The main immunogenic region of the native receptor, which is of pathological importance in the autoimmune disease myasthenia gravis, was mapped by using mAb 210 to within 80 amino acid residues (alpha 46-127). The overall antigenic structure of alpha subunits was examined. Synthetic peptides have been used to locate determinants responsible for 83% of the antibodies in antisera to denatured alpha subunits and 46% of the antibodies to denatured alpha subunits in antisera to intact receptor. Theoretical models of the transmembrane orientation of the subunit polypeptide chains were tested by determining whether mapped monoclonal antibodies bound to the extracellular or intracellular surface of receptor-rich membranes. Our results confirm previous reports that the carboxy termini of the subunits are exposed on the intracellular surface, as is part of the region between a putative channel-forming domain (M5) and a putative membrane-spanning region (M3). However, contrary to current theoretical models, the region between M5 and the putative membrane-spanning sequence M4 also appears to be on the intracellular surface, implying that M4 and M5 are not membrane-spanning domains.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1986

Transmembrane topography of nicotinic acetylcholine receptor: immunochemical tests contradict theoretical predictions based on hydrophobicity profiles.

In our preceding paper [Ratnam, M., Sargent, P. B., Sarin, V., Fox, J. L., Le Nguyen, D., Rivier, J., Criado, M., & Lindstrom, J. (1986) Biochemistry (preceding paper in this issue)], we presented results from peptide mapping studies of purified subunits of the Torpedo acetylcholine receptor which suggested that the sequence beta 429-441 is on the cytoplasmic surface of the receptor. Since this finding contradicts earlier theoretical models of the transmembrane structure of the receptor, which placed this sequence of the beta subunit on the extracellular surface, we investigated the location of the corresponding sequence (389-408) and adjacent sequences of the alpha subunit by a more direct approach. We synthesized peptides including the sequences alpha 330-346, alpha 349-364, alpha 360-378, alpha 379-385, and alpha 389-408 and shorter parts of these peptides. These peptides corresponded to a highly immunogenic region, and by using 125I-labeled peptides as antigens, we were able to detect in our library of monoclonal antibodies to alpha subunits between two and six which bound specifically to each of these peptides, except alpha 389-408. We obtained antibodies specific for alpha 389-408 both from antisera against the denatured alpha subunit and from antisera made against the peptide. These antibodies were specific to alpha 389-396. In binding assays, antibodies specific for all of these five peptides bound to receptor-rich membrane vesicles only after permeabilization of the vesicles to permit access of the antibodies to the cytoplasmic surface of the receptors, suggesting that the receptor sequences which bound these antibodies were located on the intracellular side of the membrane. Electron microscopy using colloidal gold to visualize the bound antibodies was used to conclusively demonstrate that all of these sequences are exposed on the cytoplasmic surface of the receptor. These results, along with our previous demonstration that the C-terminal 10 amino acids of each subunit are exposed on the cytoplasmic surface, show that the hydrophobic domain M4 (alpha 409-426), previously predicted from hydropathy profiles to be transmembranous, does not, in fact, cross the membrane. Further, these results show that the putative amphipathic transmembrane domain M5 (alpha 364-399) also does not cross the membrane. Our results thus indicate that the transmembrane topology of a membrane protein cannot be deduced strictly from the hydropathy profile of its primary amino acid sequence. We present a model for the transmembrane orientation of receptor subunit polypeptide chains which is consistent with current data.

Animals↗
PubMed1986

Biosynthesis of the Torpedo californica acetylcholine receptor alpha subunit in yeast.

Yeast cells were transformed with a plasmid containing complementary DNA encoding the alpha subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the alpha subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane. The alpha subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.

Animals↗
PubMed1986

Characteristics of monoclonal antibodies to denatured Torpedo and to native calf acetylcholine receptors: species, subunit and region specificity.

Seventy-five monoclonal antibodies (mAbs) to sodium dodecyl sulfate-denatured Torpedo californica (66 mAbs) and intact fetal calf (9 mAbs) acetylcholine receptor (AChR) were produced. These mAbs were characterized for subunit, region and species specificity, for Ig class and subclass, for protein A binding and for antigen-crosslinking capacity. Fourteen were identified as anti-alpha, 35 were anti-beta, 8 were anti-gamma and 15 were anti-delta. None of the 11 anti-alpha derived from denatured AChR bound to the main immunogenic region (MIR) as judged by antibody competition assays. This contrasts with previous results using mAbs against native AChr, the majority of which bind to the MIR. Thirty-eight mAbs crossreacted with some or all of the tested AChRs from fish electric organs and mammalian muscles in addition to the immunogen. Eight anti-alpha, anti-beta and 1 anti-delta mAbs showed good to excellent autoantibody activity. Analysis by sucrose gradient centrifugation of some AChR-mAb complexes revealed that some mAbs form intermolecular and others form intramolecular crosslinkings of the AChR. The described mAbs have proven valuable tools in AChR and myasthenia gravis research.

Animals↗
PubMed1986

Pharmacological properties of immuno-isolated neuronal nicotinic receptors.

Recently we immunoaffinity-purified an ACh receptor from chicken brain using a monoclonal antibody raised against receptors from fish electric organ (Whiting and Lindstrom, 1986). This neuronal receptor could be affinity-labeled with 3H-bromoacetylcholine, and antisera to it specifically blocked ACh-induced depolarization of chicken ciliary ganglion cells. Here we show that this neuronal ACh receptor binds 3H-nicotine with high affinity (KD = 6.61 +/- 0.13 nM). 3H-Nicotine binding was blocked by various nicotinic cholinergic ligands but not by alpha-bungarotoxin or the muscarinic antagonist atropine. Binding was also blocked by affinity labeling the receptor with bromoacetylcholine (after reduction by dithiothreitol). Additionally, we were able to use rat antisera raised against the chicken brain receptor to isolate a component from detergent extracts of rat brain that also bound 3H-nicotine with high affinity (KD = 1.5 nM). The pharmacology of this putative ACh receptor from rat brain was almost identical to the receptor from chicken brain, and its regional distribution was in good agreement with that of 3H-nicotine binding to rodent brain membranes reported by other workers. Thus, by analogy to the receptor we have purified and characterized from chicken brain, this nicotine-binding component from rat brain is probably a functional mammalian neuronal nicotinic ACh receptor.

Animals↗
PubMed1985

Structural localization of the sequence alpha 235-242 of the nicotinic acetylcholine receptor.

Two monoclonal antibodies (mAb 254 and 255) were obtained against a synthetic peptide corresponding to the sequence 235-242 of the alpha-subunit of Torpedo acetylcholine receptor. These mAbs could bind to receptor in native membrane vesicles only when these vesicles were permeabilized, suggesting that the sequence alpha 235-242 is exposed on the cytoplasmic surface of the receptor. Further evidence for the cytoplasmic localization of this sequence was partial competition for binding between these mAbs and mAbs previously demonstrated to bind to the cytoplasmic part of the receptor. A model is proposed which accounts for all the experimental data obtained thus far on the transmembrane orientation of the subunit polypeptide chains.

Amino Acid Sequence↗
PubMed1985

Muscle acetylcholine receptor loss in murine experimental autoimmune myasthenia gravis: correlated with cellular, humoral and clinical responses.

An extensive analysis of the relationship between immunological parameters and clinical responses and biochemical loss of muscle acetylcholine receptors (AChR) was performed in murine experimental autoimmune myasthenia gravis. The onset of clinical muscle weakness correlated strongly with the onset of significant muscle AChR loss. Mice with clinical muscle weakness had greater amount of muscle AChR loss. There was no correlation between the concentration of anti-AChR antibodies and the presence of clinical muscle weakness or amount of muscle AChR loss. However, the kinetics of autoantibody response correlated well with that of muscle AChR loss.

Autoantibodies↗
PubMed1985

Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits.

Two monoclonal antibodies (mAbs 236 and 237) against a synthetic peptide composed of the same amino acid residues as the sequence 152-167 of the alpha subunit of the acetylcholine receptor were obtained, and their crossreaction with the synthetic peptide, alpha subunit, and solubilized receptor was demonstrated. Crossreaction with the synthetic peptide alpha 159-169 was less by a factor of 10(4), suggesting that the mAbs bind primarily to the sequence alpha 152-159. Cholinergic ligands did not inhibit mAb binding. No crossreaction was observed with the receptor in native membranes, but the mAbs could bind to receptor reconstituted into liposomes in which 50% of the receptors have their cytoplasmic surface oriented outside. When native membranes were permeabilized with saponin, mAbs directed against cytoplasmic determinants of the receptor could bind to them, but mAbs 236 and 237 could not. However, after treatments that removed peripheral proteins from the cytoplasmic surface, binding of both mAbs was observed. Further evidence for the cytoplasmic localization of this sequence was provided by observation of partial competition for binding between mAbs 236 and 237 and mAbs previously demonstrated to bind to the cytoplasmic surface of the receptor. To account for these findings, a model for the organization of the polypeptide chains in receptor subunits is proposed that has a total of seven transmembrane domains in each subunit, two of which are amphipathic and one of which is not alpha-helical.

Amino Acid Sequence↗