Monoclonal antibodies to cytoplasmic domains of the acetylcholine receptor.

Froehner, Stanley C.; Douville, Karen; Klink, S; Culp, William

doi:10.1016/s0021-9258(18)32339-1

Cited by 116 publications

(57 citation statements)

References 45 publications

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“…Antibodies mAb 61 and 124 (Mtartos et al ., 1981 ;Gullick and Lindstrom, 1983), which recognize the a and 0 subunit, respectively, were generously given to us by Dr. Jon Lindstrom (The Salk Institute for Biological Studies, San Diego, CA) ; mAb 14-3-F7, specific for the a subunit, was prepared as described in Dowding and Hall (1987) ; mAb 88B (Froehner et al ., 1983) that recognizes the S subunit was the generous gift of Dr. Stanley C . Froehner (Dartmouth Medical School, Hanover, NH) .…”

Section: Materials and Methods Cdnas And Vectormentioning

confidence: 99%

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells.

Forsayeth

Verrall

et al. 1991

The Journal of Cell Biology

114

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Abstract. We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AM) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A . F. Franco, Jr., P D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron . 5:147-157) . When incomplete combinations of AChR subunits were expressed, surface binding of 'III-a-bungarotoxin was not detected except in the case of aOy which expressed <15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed T RANSMEMBRANE ion channels comprise several families of proteins with a common structural design in which homologous subunits or protein domains surround a central aqueous pore (Unwin, 1989) . The simplest oligomeric channels are homopolymers ; others contain as many as four different polypeptide subunits. Although the structure and function of many ofthese channels is well understood, relatively little is known about how they are assembled . Indeed, the mechanisms of assembly of only a few oligomeric membrane proteins of any type have been extensively investigated (Carlin and Merlie, 1987;Rose and Doms, 1988;Hurtley and Helenius, 1989) .The most completely studied ion channel is the nicotinic actylcholine receptor (AChR)' from vertebrate muscle or from Torpedo electric organ (McCarthy et al., 1986;Claudio, 1989). The AChR is a pentamer with four different subunits whose stoichiometry is a2,Qyb . The subunits have highly homologous sequences and are presumably evolved from a common ancestor that formed a homo-oligomeric channel in which all of the subunits were interchangeable (Raftery et al ., 1980;Noda et al., 1983 ;Numa et al., 1983) . Each of the subunits is made as a single polypeptide chain (Anderson and Blobel, 1981), and the four are assembled into the complete oligomer in the endoplasmic reticulum (Smith et al., 1987;Gu et al., 1989b) . After synthesis of the polypeptide, the a chain undergoes a maturational step be- that in cells expressing pairs of subunits, aS and ay heterodimers were formed, but a# was not . When three subunits were expressed, abo and ayf complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of S or y cDNAs in a mutually competitive manner. High expression of 6 or y subunits also each inhibited formation of a heterodimer with a and the other subunit . These results are consistent with a defined pathway for AChR assembly in which aS and ay heterodimers are formed first, followed by association with the a subunit and with each other to form the complete AM.

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Section: Materials and Methods Cdnas And Vectormentioning

confidence: 99%

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells.

Forsayeth

Verrall

et al. 1991

The Journal of Cell Biology

114

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“…Immunoprecipitations were performed as previously described (Merlie and Sebbane, 1981;Merlie and Lindstrom, 1983) with modifications (Blount and Merlie, 1988). The antibodies used were the c~ subunit-specific monoclonal antibodies mAb61 (Tzartos et al, 1981) andmAb210 (Rathman et al, 1986), a rabbit polyclonal anti-BTX antibody used to precipitate cc subunit that was prebound to BTX (toxin antitoxin) (Merlie and Sebbane, 1981;Merlie and Lindstrom, 1983), the ~ subunitspecific mAb148 (Gullick and Lindstrom, 1983), and the 6 subunit-specific mAb88B (Froehner et al, 1983). After immunoprecipitation, Staphylococcus aureus pellets were resuspended in sample buffer and subjected to SDS-PAGE on a 10% acrylamide, 0.27 % N,N'-bis-methylene acrylamide gel and buffer system previously described (Laemmli, 1970).…”

Section: Immunoprecipitations and Related Methodsmentioning

confidence: 99%

“…However, the 6 subunit is often difficult to detect because it migrates on SDS-PAGE as a heterogeneous band, comigrates with a nonspecifically precipitated protein, and is extremely sensitive to proteolysis. Therefore, the coimmunoprecipitation of ~ with 6 specific mAb88B (Froehner et al, 1983), a more sensitive and reliable assay than coimmunoprecipitation of 6 with ct specific antibodies, was used to routinely detect or/6 association. One caveat to these experiments is that high levels of expression or nonstoichiometric expression of subunits in transfected fibroblasts may lead to nonspecific subunit association.…”

Section: Bungarotoxin Binding and Assembly Of Subunit Mutantsmentioning

confidence: 99%

Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly.

Blount

Merlie

1990

The Journal of Cell Biology

119

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Abstract. The structural elements required for normal maturation and assembly of the nicotinic acetylcholine receptor a subunit were investigated by expression of mutated subunits in transfected fibroblasts. Normally, the wild-type ct subunit acquires high affinity ol bungarotoxin binding in a time-dependent manner; however, mutation of the 128 and/or 142 cysteines to either serine or alanine, as well as deletion of the entire 14 amino acids in this region abolished all detectable high affinity binding. Nonglycosylated subunits that had a serine to glycine mutation in the consensus sequence also did not efficiently attain high affinity binding to toxin. In contrast, mutation of the proline at position 136 to glycine or alanine, or a double mutation of the cysteines at position 192 and 193 to serines had no effect on the acquisition of high affinity toxin binding. These data suggest that a disulfide bridge between cysteines 128 and 142 and oligosaccharide addition at asparagine 141 are required for the normal maturation of ot subunit as assayed by high affinity toxin binding. The unassembled wild-type ot subunit expressed in fibroblasts is normally degraded with a tlr2 of 2 h; upon assembly with the 6 subunit, the degradation rate slows significantly (tt/2 > 13 h). All mutated c~ subunits retained the capacity to assemble with a 6 subunit coexpressed in fibroblasts; however, mutated t~ subunits that were not glycosylated or did not acquire high affinity toxin binding were rapidly degraded (tlr2 = 20 rain to 2 h) regardless of whether or not they assembled with the ~ subunit. Assembly and rapid degradation of nonglycosylated acetylcholine receptor (AChR) subunits and subunit complexes were also observed in tunicamycin-treated BC3H-1 cells, a mouse musclelike cell line that normally expresses functional AChR. Hence, rapid degradation may be one form of regulation assuring that only correctly processed and assembled subunits accumulate, and ultimately make functional receptors in AChR-expressing cells.

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“…Monoclonal antibodies. The production, characterization, and purification of monoclonal antibodies (mabs) to the Torpedo AChR (Froehner et al, 1983), 43K protein (Froehner, 1984), and 58K protein (Froehner et al, 1987) have been described. Anti-actin mab was purchased from Amersham.…”

Section: Methodsmentioning

confidence: 99%

“…For SDS gel electrophoresis, an aliquot of the Triton extract was diluted to 1 mM in N-ethylmaleimide, and an equal volume of 2x sample electrophoresis bufler containing 50 mM dithiothreitol was added. Both gel electrophoresis in SDS and immunoblotting were performed as previously described (Froehner et al, 1983;Froehner, 1984) using the mab IgG at a concentration of 50 no or ascites fluid at a l/100 dilution. For immunoblotting of actin, tissue homogenates were solubilized directly with an equal volume of double-strength sample electrophoresis buffer.…”

Section: Methodsmentioning

confidence: 99%

Developmental expression of the 43K and 58K postsynaptic membrane proteins and nicotinic acetylcholine receptors in Torpedo electrocytes

et al. 1990

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The expression of the postsynaptic 43-kDa and 58-kDa proteins and actin during development of the Torpedo marmorata electric organ was compared to that of nicotinic acetylcholine receptors (AChRs). Western blot analysis demonstrates that AChRs and proteins of 43 kDa (43K protein) and 58 kDa (58K protein) are all present prior to synaptogenesis. Subsequently, levels of all 3 synaptic proteins increase dramatically during differentiation and innervation of electrocytes. In contrast, actin is present in relatively high concentrations at early times and decreases thereafter. The equimolar ratio of AChRs and the 43K protein found in the adult electric organ is established early in development. Furthermore, the AChR and 43K protein share a common postsynaptic localization in electrocytes following synapse formation. Aggregates of the AChR that form at the ventral pole of the oval-shaped electrocytes prior to innervation, however, show no detectable immunofluorescence staining with anti-43K monoclonal antibodies. Therefore, in some cases, aggregation of AChRs occurs without the 43K protein.

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Monoclonal antibodies to cytoplasmic domains of the acetylcholine receptor.

Cited by 116 publications

References 45 publications

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells.

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells.

Mutational analysis of muscle nicotinic acetylcholine receptor subunit assembly.

Developmental expression of the 43K and 58K postsynaptic membrane proteins and nicotinic acetylcholine receptors in Torpedo electrocytes

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