Mapping of exposed surfaces of the nicotinic acetylcholine receptor by identification of iodinated tyrosine residues.
Here we report on the use of iodination of the membrane-bound nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue in order to define surface-exposed portions of the receptor molecule. Membrane-bound nAChR was 125I-iodinated using the oxidation agent Iodo-Gen. The iodinated subunits were separated by preparative gel electrophoresis, desalted, and cleaved with trypsin. The resulting peptides were separated by reverse-phase HPLC and the radioactive peptides were identified by mass spectrometry and protein sequencing. For the delta-subunit, we identified five iodinated peptides containing the tyrosine residues deltaTyr17, deltaTyr74, deltaTyr365, deltaTyr372, and deltaTyr428. The surface exposition of these amino acids is in agreement with the four-transmembrane-segment model (4TM model) of the nAChR, but the assignment to the intra- or extracellular surface is doubtful. According to this model, the N-terminal portion of the receptor subunits including the iodinated residues deltaTyr17 and deltaTyr74 is extracellular and deltaTyr372 as a site of tyrosine phosphorylation is located on the cytoplasmic side. But since this latter residue is among the first to be iodinated using an immobilized iodination agent, its true position with respect to the membrane bilayer is not clear.
Mund
M
M
Institut für Biochemie, Freie Universität Berlin, Germany.
Weise
C
C
Franke
P
P
Hucho
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004557:Electric Organ / Q000378:metabolism; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D007470:Monoiodotyrosine; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry*; D014101:Torpedo; D014357:Trypsin; D014508:Urea / Q000031:analogs & derivatives
D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; C012385:1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril; D014508:Urea; D014357:Trypsin; D007470:Monoiodotyrosine