Identification of a novel amino acid alpha-tyrosine 93 within the cholinergic ligands-binding sites of the acetylcholine receptor by photoaffinity labeling. Additional evidence for a three-loop model of the cholinergic ligands-binding sites.
The native, membrane-bound, acetylcholine receptor from Torpedo marmorata was photolabeled by the competitive antagonist p-[3H]dimethylaminobenzene-diazonium fluoroborate (DDF) in the presence of the noncompetitive blocker phencyclidine and under energy transfer conditions. The isolated alpha-subunits were treated with cyanogen bromide and fractionation of the resulting fragments yielded three radiolabeled peptides, at the level of which, incorporation of [3H]DDF (i) was equally inhibited by the agonist carbamoylcholine and the competitive antagonist alpha-bungarotoxin and (ii) was insensitive to "scavenging" reagents. Subfragmentation of cyanogen bromide peptide III with omicron-iodosobenzoic acid or trypsin and sequence analysis of the fragments led to the identification of a novel amino acid alpha-Tyr-93 (and possibly Trp-86) as labeled by [3H]DDF in a carbamoylcholine-sensitive manner. alpha-Tyr-93 is conserved in the muscle and neuronal alpha-subunits but not in the other subunits of muscle receptor. This result provides evidence for a site involving at least a third loop of the alpha-subunit amino-terminal hydrophilic domain, in addition to the ones previously identified (Dennis, M., Giraudat, J., Kotzyba-Hibert, F., Goeldner, M., Hirth, C., Chang, J. Y., Lazure, C., Chretien, M., and Changeux, J. P. (1988) Biochemistry 27, 2346-2357). Possible contribution of tyrosine side-chains to the complexation of the quaternary ammonium group of cholinergic ligands is discussed.
Galzi
J L
JL
Département des Biotechnologies, Institut Pasteur, Paris, France.
Revah
F
F
Black
D
D
Goeldner
M
M
Hirth
C
C
Changeux
J P
JP
D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003488:Cyanogen Bromide; D003979:Diazonium Compounds / Q000378:metabolism*; D008024:Ligands; D046911:Macromolecular Substances; D008954:Models, Biological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010777:Photochemistry; D011487:Protein Conformation; D051381:Rats; D011950:Receptors, Cholinergic / Q000302:isolation & purification / Q000378:metabolism*; D012689:Sequence Homology, Nucleic Acid; D014101:Torpedo; D014443:Tyrosine
D000345:Affinity Labels; D003979:Diazonium Compounds; D008024:Ligands; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; C028214:4-dimethylaminobenzenediazonium fluoroborate; D014443:Tyrosine; D003488:Cyanogen Bromide