ABSTRACT
The role of the beta3 and beta4 subunits of the nicotinic acetylcholine receptor in brain is still unclear. We investigated nicotinic receptor structure with antibodies directed against unique regions of the beta3 and beta4 subunits of the rat nicotinic acetylcholine receptor. Anti-beta4 detected a single band of 66 kDa in most regions of the brain that was strongest in striatum and cerebellum. The 60 kDa beta3 subunit was detected primarily in striatum and cerebellum, and faintly in hippocampus. Immunoprecipitation experiments established that the two subunits were coassembled in the cerebellum along with the beta2 subunit. Antibodies against the alpha4, beta2, beta3, and beta4 subunits immunoprecipitated approximately 75% of the bungarotoxin-insensitive nicotinic receptor from cerebellar extracts as determined by nicotine-dependent acetylcholine binding. Transfection of COS cells with cDNAs for these four subunits induced expression of a high affinity nicotinic receptor. Omission of only a single subunit from the transfection affected either the Bmax or the apparent KD of the receptor. Our data suggest that the beta3 subunit functions as a structural entity that links a relatively unstable alpha4beta2 heterodimer to a more stable alpha4beta4 heterodimer. The agonist-binding site formed by alpha4beta2 has a much greater affinity than does that formed by alpha4beta4. In this respect, nicotinic receptors that contain the beta3 subunit are structurally homologous to the muscle nicotinic receptor.
Subject(s)
Nerve Tissue Proteins/chemistry , Protein Conformation , Receptors, Nicotinic/chemistry , Acetylcholine/metabolism , Animals , Antibodies, Monoclonal/immunology , Bungarotoxins/pharmacology , COS Cells , Cerebellum/chemistry , Cholinergic Antagonists/pharmacology , DNA, Complementary/genetics , Muscle Proteins/chemistry , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/immunology , Nerve Tissue Proteins/metabolism , Polymerase Chain Reaction , Protein Structure, Tertiary , Rats , Receptors, Nicotinic/drug effects , Receptors, Nicotinic/immunology , Receptors, Nicotinic/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Synaptosomes/chemistryABSTRACT
The study of the expression of alpha-galactosyl epitopes on various mammalian cells is of particular interest, since as much as 1% of circulating IgG antibodies in humans interact with this carbohydrate residue. This natural antibody, designated "anti-Gal," was previously found to bind to terminal Gal alpha 1----3Gal beta 1----4GlcNAc-R on biochemically defined glycolipids (Galili, U., Macher, B. A., Buehler, J., and Shohet, S. B. (1985) J. Exp. Med. 162, 573-582; Galili, U., Buehler, J., Shohet, S. B., and Macher, B. A. (1987) J. Exp. Med. 165, 693-704). The expression of anti-Gal binding epitopes on nucleated cells from various mammalian species was studied by immunostaining with this antibody. The binding of anti-Gal to various cells was correlated with the binding of the lectin Bandeiraea (Griffonia) simplicifolia IB4 (BS lectin). The BS lectin also interacts with alpha-galactosyl residues and particularly with high affinity with Gal alpha 1----3Gal beta 1----4GlcNAc residues. We observed a striking evolutionary pattern in the expression of these epitopes on mammalian nucleated cells. Fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, and lymphoid cells of nonprimate mammals, prosimians, and New World monkeys readily bound both anti-Gal and BS lectin. However, no such binding was detectable on cells of Old World monkeys, apes, and humans. Measurment of the binding of radiolabeled BS lectin to the various nucleated cells suggests that cells binding anti-Gal express 10(6) to 3.5 x 10(7) alpha-galactosyl epitopes, most of which, based on the anti-Gal specificity, seem to have the structure of Gal alpha 1----3Gal beta 1----4GlcNAc-R. The absence of these epitopes from human cells results from diminished activity of the enzyme alpha 1----3 galactosyltransferase, which catalyzes the following reaction. Gal beta 1----4GlcNAc-R + UDP-Gal(alpha 1----3-galactosyltransferase)----Gal alpha 1----3Gal beta 1----4GlcNAc-R + UDP This enzyme, which participates in the glycosylation of cell membrane glycoconjugates in nonprimate mammals, prosimians, and New World monkeys, appears to have been suppressed in Old World primates as a result of evolutionary events which occurred 20-30 million years ago. It is argued that an anomalous activity of this enzyme in man may result in initiation of autoimmune diseases because of the de novo expression of Gal alpha 1----3Gal beta 1----4GlcNAc-R epitopes recognized by anti-Gal.
