Monoclonal antibodies against the acetylcholine receptor gamma-subunit as site specific probes for receptor tyrosine phosphorylation.

Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) may be involved in AChR desensitization and clustering. Torpedo AChR gamma-subunit is phosphorylated at Tyr365. Using overlapping synthetic peptides, we have precisely mapped the epitopes of five anti-gamma-subunit monoclonal antibodies (mAbs) and found that the epitope(s) for the mAbs 154, 165 and 168 (gamma 365-370) all contain Tyr365. mAb 168 is a known blocker of AChR channel function. Using peptide analogues, Tyr365 was found to be indispensable for mAb165 binding; furthermore its binding was selectively inhibited by in vitro AChR tyrosine phosphorylation. The possible connection between gamma-subunit phosphorylation and regulation of AChR function and the proven usefulness of these mAbs as tools should facilitate functional studies of AChR gamma-subunit phosphorylation.

Monoclonal antibodies as site-specific probes for the acetylcholine-receptor delta-subunit tyrosine and serine phosphorylation sites.

Phosphorylation of the nicotinic acetylcholine receptor (AChR) has been implicated in the assembly, clustering, regulation of function and degradation rate of the molecule. Torpedo AChR is phosphorylated at eight cytoplasmic residues, four of which are on the delta subunit. We have precisely mapped the epitopes of eleven monoclonal antibodies (mAbs) directed against the Torpedo AChR delta-subunit regions delta 350-396 and delta 484-493, which are therefore exposed on the surface of the intact AChR, and now have four highly specific tools for analysing the role of delta-subunit phosphorylation. More than 160 synthetic peptides attached to polyethylene rods were used for epitope mapping. Four mAbs bound within the region delta 350-380, which contains all of the delta-subunit phosphorylation sites (Ser361, Ser362, Tyr372 and Ser377). Specifically, the epitope for mAb 134 (delta 365-375) contains Tyr372, the epitope(s) for mAbs 139 and 166 (delta 376-381) contains Ser377, while the epitope for mAb 146 (delta 350-359) is close to Ser361 and Ser362 and includes parts of the corresponding phosphorylation consensus sequences. Using peptide analogues with single residue substitutions, Tyr372 was found to be essential for the binding of mAb 134 and Ser377 was found contributing to the binding of mAbs 139 and 166. Finally, tyrosine phosphorylation of Torpedo AChR selectively inhibited binding of mAb 134. These data, and the availability of the defined mAb probes, should facilitate the study of the functional role of single AChR phosphorylation sites.