Monoclonal Antibodies as Site-Specific Probes for the Acetylcholine-receptor delta-Subunit Tyrosine and Serine Phosphorylation Sites

Abstract

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.