The purpose of this project is to achieve stable, continuous expression of neurotransmitter receptor genes in cultured cell lines lacking the receptors. This experimental approach provides a means for 1) definitive identification of cloned neurotransmitter receptor genes using pharmacological and biochemical assays; 2) comparison of the pharmacological and biochemical properties of the same receptor expressed in different cell lines as well as between related receptor proteins; 3) large scale production of receptor proteins; and 4) analysis of the relationship of receptor structure and function using the technique of site-directed mutagenesis without phenotypic selection. To facilitate these goals, we are also constructing new plasmid expression vectors that routinely allow for high density receptor expression. To date, we have produced several cell lines in murine B-82 cells and Chinese hamster ovary (CHO) cells that are expressing human and rat beta 2-adrenergic, human beta 1 adenergic, human alpha2 adrenergic, rat M1-M4 muscarinic cholinergic, and Drosophila octopamine receptors at densities in the range of 100 fmol to 10 pmol of receptor/mg membrane protein. These receptors display all of the expected pharmacological and biochemical properties for each respective receptor subtype. Using site-directed mutagenesis. we have also examined the role of highly conserved aspartate and cysteine residues in beta2-adrenergic and muscarinic acetylcholine receptor function. The aspartate and cysteine residues, located in the putative second and third transmembrane domains of the receptors appear to be involved in agonist binding and agonist induced activation of quanine nucleotide regulatory proteins. Extracellular cysteine residues also appear to be involved in agonist and antagonist binding to the receptors, perhaps as important structural elements of the receptor proteins. These data illustrated that the conservation of receptor structure among these related receptors may reflect conservation of receptor mechanisms.
