The tyrosine phosphorylation site of the acetylcholine receptor beta subunit is located in a highly immunogenic epitope implicated in channel function: antibody probes for beta subunit phosphorylation and function.
Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) seems to be involved in AChR desensitization and localization on the postsynaptic membrane. This study reveals a probable function of the single known beta subunit phosphorylation site (beta Tyr355) and provides suitable tools for its study. The epitopes for 15 monoclonal antibodies (mAbs) against the cytoplasmic side of the AChR beta subunit were precisely mapped using > 100 synthetic peptides attached on polyethylene rods. Eleven mAbs bound to a very immunogenic cytoplasmic epitope (VICE-beta) on Torpedo beta 352-359, which contains the beta Tyr355, and to the corresponding sequence of human AChR. The contribution of each VICE-beta residue to mAb binding was then studied by peptide analogues having single residue substitutions. Overall, each of the residues beta 354-359, including beta Tyr355, proved critical for mAb binding. Two of our four mAbs known to block the ion channel were found to bind at (mAb148) or close (mAb10) to VICE-beta. Tyrosine phosphorylation of Torpedo AChR by endogenous kinase(s) selectively reduced binding of some VICE-beta mAbs, including the channel blocking mAb148. We conclude that VICE-beta probably plays a key role in AChR function. Elucidation of this role should be facilitated by the identified mAb tools.
Tzartos
S J
SJ
Department of Biochemistry, Hellenic Pasteur Institute, Athens, Greece.
Valcana
C
C
Kouvatsou
R
R
Kokla
A
A
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D000939:Epitopes; D006801:Humans; D007473:Ion Channels / Q000737:chemistry / Q000276:immunology* / Q000502:physiology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000276:immunology; D019000:Phosphotyrosine; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000276:immunology* / Q000502:physiology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014101:Torpedo; D014443:Tyrosine / Q000031:analogs & derivatives / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007473:Ion Channels; D010455:Peptides; D011978:Receptors, Nicotinic; D019000:Phosphotyrosine; D014443:Tyrosine