Using In Vitro Mutagenesis to Characterize Structure-Function Relationships in G Protein-Coupled Receptors

The use of in vitro mutagenesis to characterize structure-function relationships in G protein-coupled receptors has led to the identification of specific amino acid residues that contribute to ligand binding, G protein coupling, and receptor folding. Mutagenesis is commonly used to change or mutate a DNA sequence so that one or more amino acid residues in a given G protein-coupled receptor are changed to different residues. These techniques can also be used to delete or insert one or more amino acids into a receptor and to exchange DNA sequences between homologous receptors. Of the available techniques, site-directed mutagenesis is the most widely employed, and this method can be used to change, insert, or delete specific amino acids residues in a receptor. This chapter describes a reliable PCR-based protocol for this method. We also briefly describe other mutagenesis techniques including random mutagenesis, scanning mutagenesis, deletion mutagenesis, and the construction of receptor chimeras. Important considerations for conducting and interpreting mutagenesis studies are also highlighted.