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 bee n implicated in the assembly, clustering, regulation of function and d egradation rate of the molecule. Torpedo AChR is phosphorylated at eig ht cytoplasmic residues, four of which are on the delta subunit. We ha ve precisely mapped the epitopes of eleven monoclonal antibodies (mAbs ) directed against the Torpedo AChR delta-subunit regions delta 350-39 6 and delta 484-493, which are therefore exposed on the surface of the intact AChR, and now have four highly specific tools for analysing th e role of delta-subunit phosphorylation. More than 160 synthetic pepti des attached to polethylene rods were used for epitope mapping. Four m Abs bound within the region delta 350-380, which contains all of the d elta-subunit phosphorylation sites (Ser361, Ser362, Tyr372 and Ser377) . Specifically, the epitope for mAb 134 (delta 365-375) contains Tyr37 2, 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 consen sus sequences. Using peptide analogues with single residue substitutio ns, Tyr372 was found to be essential for the binding of mAb 134 and Se r377 was found contributing to the binding of mAbs 139 and 166. Finall y, tyrosine phosphorylation of Torpedo AChR selectively inhibited bind ing of mAb 134. These data, and the availability of the defined mAb pr obes, should facilitate the study of the functional role of single ACh R phosphorylation sites.