REPORTER EPITOPES - A NOVEL-APPROACH TO EXAMINE TRANSMEMBRANE TOPOLOGY OF INTEGRAL MEMBRANE-PROTEINS APPLIED TO THE ALPHA-1 SUBUNIT OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR

The development of a novel immunological method called the 'reporter epitope' technique to probe the transmembrane topology of integral membrane proteins is described. Using this method, synthetic oligonucleotides encoding epitopes (reporter epitopes) for well characterized monoclonal antibodies (reporter mAbs) were inserted at various locations within the human acetylcholine receptor (AChR) alpha1 subunit cDNA. The engineered subunits were then expressed along with Torpedo beta1, gamma, and delta subunits in Xenopus oocytes, and the transmembrane location of the site of insertion was determined by the binding of the I-125-labeled reporter mAbs to whole oocytes. Control reporter epitope insertions at alpha347 exhibited the expected cytoplasmic location. Reporter epitopes inserted at alpha429 are located on the extracellular surface. Reporter epitopes that are 16-48 amino acids long do not disrupt assembly or function of hybrid AChRs when inserted near the carboxy terminus (at alpha429) or in the large cytoplasmic domain (at alpha347). However, because two reporter epitopes inserted at alpha157 obliterated subunit assembly and a third reporter epitope when tolerated at this position was inaccessible from the extracellular surface and only marginally accessible after detergent solubilization of the AChRs, a definitive transmembrane location for this region was not possible. Nonetheless, the use of this approach has been successfully demonstrated, and it may be generally applicable to the study of other integral membrane proteins.