Structure and functional analysis of G protein-coupled receptors and potential diagnostic ligands.

G protein-coupled receptors are a diverse class of proteins that mediate signal transduction across the plasma membrane. More than 200 receptors in this extended gene family have been cloned, and comparison of the deduced amino-acid sequences indicates that these proteins have marked homology and share a common membrane topology consisting of seven transmembrane helices. Although there is considerable variability in the physiologic ligands responsible for receptor activation, all receptors in this group interact with trimeric, guanine nucleotide-binding proteins to initiate signaling cascades in the cell cytosol. To investigate the structural motifs responsible for ligand binding, we have established a model system to express heterologously human G protein-coupled receptors in a mammalian cell line. This experimental system allows each receptor subtype to be studied in isolation and provides a direct means to link receptor activation to a particular second messenger cascade. Furthermore, the efficacy and specificity of new pharmaceuticals can now be evaluated readily with cloned human receptors, eliminating the need for animal tissues. We have used this expression system in conjunction with an experimental strategy of site-directed mutagenesis to identify amino-acid residues that have a functional role in ligand binding. Because of the strong homology that exists within this family of receptor proteins, the results of this work are applicable to other systems and, therefore, can help to establish a more complete understanding of ligand-receptor interactions. This combined molecular and biochemical approach to the study of G protein-coupled receptors can pave the way for the development of isoform-specific ligands that may be used for radionuclide imaging and therapy.