Generation and use of epitope-tagged receptors

Epitope tagging of a receptor involves introducing a defined amino acid sequence, to which an antibody has already been produced, into the primary amino acid sequence of the receptor. The new sequence can be as short as 10-15 amino acids and the method allows the receptor to be monitored without having to raise an antibody specific to it, and so permits its biochemical characterization and immunolocalization within cells. Other related techniques involve the introduction of functional domains of proteins such as green fluorescence protein or one of its derivatives into receptors to allow their direct visualization. There are a wide range of amino acid tag sequences, and fluorescent proteins, available for use as tags, and choice of tag will depend on several factors including the availability of antisera and cost. The position for the introduction of the tag into the native receptor will depend on precisely what the requirements of the experiments are but it must always be such that the receptor is normally processed, trafficked, and remains functional after tagging. These considerations are discussed fully in this chapter, which also describes examples of strategies for introducing tags, and some general methods for use in the characterization of the tagged proteins.