The objective of these studies is to understand the structure and function of two important membrane signaling receptors: the muscarinic acetylcholine (subtype M1) receptor and the alpha 2B adrenergic receptor. Both are members of a large family of structurally related G protein-coupled receptors. The experimental approach is to use a novel protein expression system with the potential of generating each receptor in the quantities large enough to permit many standard structural and functional analyses that have not yet been possible. The approach entails the construction of chimeras of the genes encoding the receptors and the bacteriorhodopsin gene to direct high level expression in the halophilic archaeon Halobacterium salinarum. Screening of chimeras with different portions of the bacteriorhodopsin gene will define an optimal strategy for receptor over-expression. In addition, constructs will also be expressed in previously employed eukaryotic cell systems as a means to test receptor function in vivo. The method will ensure that the successfully expressed chimeras retain their ability to couple to endogenous downstream signaling components. The studies have a high potential to yield significant new information about the molecular mechanisms of signaling by these membrane proteins.
