Neurotransmitter receptors that mediate their cellular actions through guanine nucleotide regulatory binding proteins (G proteins) comprise a large and diverse gene superfamily and modulate the activity of a large variety of important physiological functions. To better understand the mechanisms of signal transduction medicated by this family of receptors we have utilized molecular biological approaches including gene cloning, permanent receptor gene expression in cultured cells, and site-directed mutagenesis to study the relationship between receptor structure and function and the mechanisms of receptor regulation. CHO cell lines permanently transfected with single G protein receptor subtypes are of great utility in the characterization of novel receptor agonist and antagonists. Furthermore, these cell lines facilitate studies on the study of specific receptor-G protein interactions. The ability to modify the amino acid sequence of a receptor with site-directed mutagenesis has allowed for identification of conserved cysteine residues in muscarinic acetylcholine receptors that are involved in ligand binding and receptor activation by agonists. Also, we have demonstrated that beta-adrenergic and muscarinic acetylcholine receptors expressed in the same cell line are down-regulated in a coordinated fashion by agonists. Since both of these receptor subtypes can be found on peripheral tissues and in the central nervous system, the phenomenon of cross-regulation becomes an important area of study. Characterization of Beta-adrenergic receptors expressed in CHO cells has revealed a differential coupling between Beta2- and Beta3- adrenergic receptor subtypes and stimulatory G proteins that most likely reflects the marked differences in the primary structure of these receptor subtypes.
