Our purpose is to examine the structural and functional evolution of neurotransmitter receptor proteins. Using muscarinic cholinergic and beta adrenergic receptors as the initial model systems we have isolated and characterized these receptors 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 as well as from lower species including insects (Drosophila). Data from muscarinic receptor studies demonstrate that out of all of the parameters studied (SDS gel molecular weight, isoelectric point, monoclonal antibody cross reactivity, agonist affinity, antagonist affinity, stereospecificity of ligand binding, GTP shifts of agonist affinity, and receptor density) only receptor density appears to have changed over 900 million years of evolution gradually increasing in higher species. In contrast, beta receptor structure has appeared to change more dramatically over the past 400 million years. Muscarinic receptors are being purified from human brain, rat brain and heart, pig heart and shark brain and heart in order to compare primary structures in detail. The evolutionary relationship between muscarinic cholinergic receptors and other neurotransmitter receptors is also being studied. Protein sequence data and defined gene probes will be employed to follow the evolution of adrenergic, cholinergic and GABAergic receptors. Neurotransmitter biosynthetic/degradative enzymes have been examined using receptor-specific monoclonal antibodies. In addition, primary sequence data for these proteins has been compared with that of neurotransmitter receptor proteins for any evolutionary or structural relationships. Gene cloning of adrenergic and cholinergic receptors is underway from a wide variety of species to study specific receptor domains involved in such functions as ligand binding and coupling to other membrane effectors.
