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 for use in the production of monoclonal and polyclonal antibodies; 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 construction 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 beta2-adrenergic, human beta1 adrenergic, human alpha2- adrenergic, rat M1-M4 muscarinic cholinergic and bovine GABA A 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 subtype. Using site-directed mutagenesis, we have also examined the role of highly conserved aspartate and cysteine residues in human beta2- adrenergic receptor function. The aspartate and cysteine residues, located in the putative second and third transmembrane domains of the receptor, appear to be involved in agonist binding and agonist induced activation of the stimulatory guanine nucleotide regulatory protein, Gs. extracellular cysteine residues also appear to be involved in agonist and antagonist binding to the receptor, perhaps as important structural elements of the receptor protein. In addition, we have also utilized site-directed mutagenesis to examine the role of the corresponding aspartate and cysteine residues in rat M1 muscarinic cholinergic and human alpha2- adrenergic receptors. These conserved amino acids appear to play critical roles in receptor function in these members of the neurotransmitter receptor family.
