We are in the process of cloning the genes for major classes of neurotransmitter receptors including cholinergic, adrenergic and GABAergic. cDNA libraries have been obtained or are being constructed from human brain, heart and lung, and porcine, shark, and Drosophila tissue for evolutionary comparison of receptor structures to test the hypothesis developed by this group that neurotransmitter receptors are part of a multigene family. Full length genes are being utilized as probes for mRNA synthesis in developmental models for receptors. To date we have isolated two human brain cDNA clones which cross hybridized to oligonucleotide probes specific for the N-terminal, middle and C-terminal regions of the beta2-adrenergic receptor gene. The nucleotide sequences of these two cDNA clones are being determined by the M13-dideoxy chain termination method. In order to determine the number of copies of beta-adrenergic receptor present in the genome, genomic DNAs from human, porcine, shark, etc., will be examined by Southern blot analysis. Genomic DNA libraries from various species will be constructed to screen for these potentially different genes. The cloned genes will be expressed in different prokaryotic and eucaryotic expression vectors to study the molecular structure of these receptors. Site-directed mutagenesis will be used to study the structure-function relationship of these receptors.
