Characterization of a component in chick ciliary ganglia that cross- reacts with monoclonal antibodies to muscle and electric organ acetylcholine receptor

Abstract

Chick ciliary ganglion neurons have previously been shown to contain a component that shares an antigenic determinant with the "main immunogenic region" of the alpha-subunit in nicotinic acetylcholine receptor from skeletal muscle and electric organ. Ultrastructural studies of antibody binding in the ganglion have shown that the cross-reacting antigen exposed on the surface of the neurons is located predominantly in synaptic membrane. Here we show that the neuronal antigen can be identified in detergent extracts of ciliary and sympathetic ganglia, but not in extracts of heart, liver, spinal cord, retina, or dorsal root ganglia. In the ciliary ganglion the component is present as an integral membrane constituent, and, when detergent solubilized, it sediments as a 10 S species and binds to concanavalin A. The component is distinct from the alpha-bungarotoxin-binding site on the neurons since toxin-binding sites and antibody-binding sites can be precipitated separately in ganglion extracts. The component reaches peak levels per ganglionic protein between embryonic days 8 and 12. These are some of the properties expected for the nicotinic acetylcholine receptor on ciliary ganglion neurons.

Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture

“…determined bv including a 20-fold excess of unlabeled mAb 35 in thelabeling reaction, usually-constituted 20-30% of total binding and was subtracted in all cases to yield specific binding. Previous studies have shown that the saturable mAb 35 binding determined in this way is specific (Smith et al, 1985(Smith et al, , 1986.…”

“…Extracts were pooled and assayed in 50 ~1 aliquots for specific Y-mAb 35 binding, using small DEAE-cellulose columns to separate bound and free mAb 35 as previously described (Smith et al, 1985). The following modifications were made in the assay procedure: Reaction volumes contained the equivalent of 2-3 culture wells; binding was carried out for 30 min; fetal calf serum at 1: 16 dilution replaced the rat serum previously used to decrease nonspecific binding to column material; the reaction was terminated by dilution with 0.60 ml HB; aliquots were assayed in triplicate on individual DEAE-cellulose columns.…”

“…Cross-linking studies with a photoaffinity derivative of Bgt 3.1 demonstrate that Bgt 3.1 and mAb 35 recognize the same neuronal component Berg, 1986b, 1987). The AChR identified by both Bgt 3.1 and mAb 35 is clearly distinct from the membrane component of unknown function on the neurons that binds a-bungarotoxin (Jacob and Berg, 1983;Smith et al, 1983Smith et al, , 1985Jacob et al, 1984).…”

“…One of these is a monoclonal antibody, mAb 35, to the "main immunogenic region" (MIR) of AChR a-subunits from muscle and electric organ. MAb 35 cross-reacts with a component on chick ciliary ganglion neurons that has the ultrastructural location, biochemical properties and cholinergic modulation expected for the neuronal AChR (Jacob et al, 1984;Smith et al, 1985Smith et al, , 1986. Antisera raised against a similar component immunopurified from chick brain with mAb 35 specifically block the ACh response of ciliary ganglion neurons (Stollberg et al, 1986).…”

Pharmacological properties of immuno-isolated neuronal nicotinic receptors

“…mAb 35 binds to synapses on chicken ciliary ganglion neurons, whereas orBGT binds to sites outside of the synapse (Jacob et al, 1984). The component in ciliary ganglia identified by mAb 35 has biochemical properties expected of an AChR, and its amount can be modulated by treatment of ganglion cultures with cholinergic ligands (Smith et al, 1985(Smith et al, , 1986. By immunoaffinity chromatography on mAb 35 coupled to Sepharose, we have purified its binding component from chicken brain (Whiting and Lindstrom, 1986).…”