The purpose of this project is to examine the structural and functional evolution of neurotransmitter receptor proteins. Using muscarinic cholinergic receptors as the initial model system we have isolated and characterized the muscarinic acetylcholine receptor from the brains and hearts of a wide variety of species. Brain and heart tissue have been obtained from all vertebrate classes including mammals, birds, reptiles, amphibians, and fish. In addition, lower species including insects (drosophila) have provided receptors. The studies underway indicate that all of the studied parameters; SDS gel molecular weight, isoelectric points, monoclonal antibody cross reactivity, agonist affinity, antagonist affinity, stereospecificity, GTP shifts, and receptor density, only the last parameter, receptor density, appears to have changed by increasing gradually over 900 million years of evolution. Muscarinic receptors are being purified from human brain, rat brain and heart, pig heart and shark brain and heart in order to compare in detail primary structures. The evolutionary relationship between muscarinic cholinergic receptors and other neurotransmitter receptors is also being studied. Protein sequencing and gene probes will be used to follow the evolution of adrenergic, cholinergic, dopaminergic, serotonergic, GABAergic and opiate receptor proteins. Neurotransmitter biosynthetic/degradative enzymes, e.g. tyrosine hydroxylase, CAT, acetylcholinesterase will be examined with monoclonal antibodies and primary sequence data for any evolutionary/structural relationship with the neurotransmitter receptor proteins.