Neuronal nicotinic acetylcholine receptor beta-subunit is coded for by the cDNA clone alpha 4

Acetylcholine receptors (AChRs) with high affinity for nicotine but no affinity for alpha-bungarotoxin, which have been purified from rat and chicken brains by immuno-affinity chromatography, consist of two types of subunits, alpha and beta. The beta-subunits form the ACh binding sites. Putative nicotinic AChR subunit cDNAs alpha 3 and alpha 4 have been identified by screening cDNA libraries prepared from rat PC12 cells and rat brain with cDNA probes encoding the mouse muscle AChR alpha-subunit. Here we determine the amino-terminal amino acid sequence of the rat brain AChR beta-subunit by protein microsequencing to be the same as amino acid residues 27-43 of the protein which could be coded by alpha 4. Further, we present evidence consistent with a subunit stoichiometry of alpha 3 beta 2 for this neuronal nicotinic AChR.     

Chromosomes move poleward in anaphase along stationary microtubules that coordinately disassemble from their kinetochore ends

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.     

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.     

Surface and intracellular distribution of a putative neuronal nicotinic acetylcholine receptor

Chick ciliary ganglion neurons have a membrane component that shares an antigenic determinant with the main immunogenic region (MIR) of nicotinic acetylcholine receptors from skeletal muscle and electric organ. Previous studies have shown that the component has many of the properties expected for a ganglionic nicotinic acetylcholine receptor, and that its distribution on the neuron surface in vivo is restricted predominantly to synaptic membrane. Here we report the presence of a large intracellular pool of the putative receptor in embryonic neurons and demonstrate that it is associated with organelles known to comprise the biosynthetic and regulatory pathways of integral plasma membrane proteins. Embryonic chick ciliary ganglia were lightly fixed, saponin-permeabilized, incubated with an anti-MIR monoclonal antibody (mAb) followed by horseradish peroxidase-conjugated secondary antibody, reacted for peroxidase activity, and examined by electron microscopy. Deposits of reaction product were associated with synaptic membrane, small portions of the pseudodendrite surface membrane, most of the rough endoplasmic reticulum, small portions of the nuclear envelope, some Golgi complexes, and a few coated pits, coated vesicles, multivesicular bodies, and smooth-membraned vacuoles. No other labeling was present in the neurons. The labeling was specific in that it was not present when the anti-MIR mAb was replaced with either nonimmune serum or mAbs of different specificity. Chick dorsal root ganglion neurons thought to lack nicotinic acetylcholine receptors were not labeled by the anti-MIR mAb. Substantial intracellular populations have also been reported for the muscle acetylcholine receptor and brain voltage-dependent sodium channel alpha-subunit. This may represent a general pattern for multisubunit membrane proteins during development.     

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.     

Serum gp70 production regulated by a gene on murine chromosome 7

We mated 129 mice (prototype strain of G_IX ⁺ with C5713L/6 mice (prototype strain of G_IX ^–) and thereby identified the gene controlling enhancement of serum gp70 production after lipopolysaccharide (LPS) injection. This gene has been tentatively designated Sgp-2. Sgp-2 is linked with the Hbb locus on chromosome 7. The estimated frequency of recombination between Sgp-2 and Hbb in this setting is about 20%.     

Cellular immune response to acetylcholine receptors in murine experimental autoimmune myasthenia gravis: inhibition with monoclonal anti-I-A antibodies

Gene(s) at the I-A subregion of the murine major histocompatibility complex influence susceptibility to experimental autoimmune myasthenia gravis. C57Bl/6 mice immunized with acetylcholine receptors (AChR) in complete Freund's adjuvant demonstrated cellular and humoral immune responses to AChR. They developed muscle weakness characteristic of myasthenia gravis and demonstrated a reduction in the muscle AChR content. The kinetics of AChR-specific lymphocyte proliferation generally correlate with anti-AChR antibody response. AChR-specific lymphocyte proliferation was also observed in C57Bl/6 splenocytes after secondary immunization with AChR. The in vitro cellular reactivity to AChR in experimental autoimmune myasthenia gravis (EAMG) mice (C57Bl/6) was suppressed by monoclonal anti-I-Ab antibodies directed against private (Ia20) or public (Ia8) specificities, suggesting a critical role for these Ia determinants in the cellular immune response to AChR in murine EAMG.     

Nicotinamide adenine dinucleotide-dependent formate dehydrogenase from Rhodopseudomonas palustris

Summary Formate dehydrogenase in extracts of the facultative phototroph, Rhodopseudomonas palustris was shown to be soluble and NAD-linked. The flavin nucleotides, FMN and FAD, stimulated the rate of NAD reduction about fourfold. Reduction of artificial electron acceptors such as DCPIP and cytochrome c was also stimulated by FMN and FAD. The pH optimum for the reduction of NAD was pH 8.0, in contrast to pH 6.8 for cytochrome c and DCPIP reduction. The apparent K _m for formate as measured by NAD reduction was 2.6×10^-4 M. Although the addition of thiosulfate or yeast extract to the formate medium increased both the growth rate and yield of Rhps. palustris, they had little effect on the activity of formate dehydrogenase.     

Perinatal thymocyte antigen expression and postnatal immune development altered by gestational exposure to tetrachlorodibenzo-p-dioxin (TCDD).

In utero exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was found to alter expression of murine thymocyte fetal cell-surface markers. Pregnant mice were treated (via gavage) with 0, 1.5, or 3.0 micrograms TCDD/kg/day in corn oil on gestational days (gd) 6-14. Offspring were examined on gd 18 and postnatally on d6, d14, and d21, and at 7, 8, and 10 weeks of age. Severe thymic atrophy and cellular depletion were found both pre- and postnatally in TCDD-exposed mice. Immunocytochemical localization of the Thy 1.2 antigen on gd 18 thymocytes revealed no TCDD-related changes in cellular distribution. Flow cytometric analysis, however, indicated that the TCDD treatment resulted in a significant decrease in the percentage of CD4+8+ fetal thymocytes, as well as significantly increased CD4-8- and CD4-8+ thymocytes. The increased CD4-8+ population after TCDD was not from induction of Ts cells. At 7-8 weeks postnatally, no differences existed between control and treatment groups in mitogen responses and antibody plaque response. However, altered thymocyte antigen expression was found to correlate with altered postnatal immune function, as evidenced by decreased cytotoxic T lymphocyte response at 8 weeks of age. Taken together, these results indicate that immunosuppression following prenatal exposure to TCDD can be readily detected by qualitative and quantitative changes in the cell surface phenotype of fetal thymocytes. Furthermore, the observed altered distribution suggests that TCDD inhibits normal thymocyte maturational processes.     

A soybean cell wall protein is affected by seed color genotype.

The dominant I gene inhibits accumulation of anthocyanin pigments in epidermal cells of the soybean seed coat. We compared saline-soluble proteins extracted from developing seed coats and identified a 35-kilodalton protein that was abundant in Richland (genotype I/I, yellow) and much reduced in an isogenic mutant line T157 (genotype i/i, imperfect black seed coats). We purified the 35-kilodalton protein by a novel procedure using chromatography on insoluble polyvinylpolypyrrolidone. The 35-kilodalton protein was composed primarily of proline, hydroxyproline, valine, tyrosine, and lysine. Three criteria (N-terminal amino acid sequence, amino acid composition, and sequence of a cDNA) proved that the seed coat 35-kilodalton protein was PRP1, a member of a proline-rich gene family expressed in hypocotyls and other soybean tissues. The levels of soluble PRP1 polypeptides and PRP1 mRNA were reduced in young seed coats with the recessive i/i genotype. These data demonstrated an unexpected and novel correlation between an anthocyanin gene and the quantitative levels of a specific, developmentally regulated cell wall protein. In contrast, PRP2, a closely related cell wall protein, was synthesized later in seed coat development and was not affected by the genotype of the I locus.