9407251 Breitweiser Although hundreds of "seven transmembrane helix" G protein - coupled receptors and a sizable family of heterotrimeric G proteins (consisting of , and subunits) have been identified and cloned, there is as yet a minimal understanding of the effector proteins of these signaling pathways. Most cells contain many types of G protein- coupled receptors, a host of G protein subtypes, and numerous effectors. The ability to respond to a given agonist with activation of the appropriate range of effector enzymes may be controlled by subcellular localization of signaling pathways, variable activation of G protein subtypes and effectors by receptors, by modulation of the levels of expression of components of the various signaling pathways, or most likely, a combination of these and other measures. We have extensively characterized the kinetics of muscarinic receptor-mediated activation of an inwardly rectifying K+ channel in bullfrog atrium via a pertussis toxin-sensitive G protein, Gk1, and have developed a minimal model for regulation of muscarinic K+ current activation and desensitization. With the recent cloning of the muscarinic K+ channel (GIRK1), we now have the means to test the hypotheses derived from studies in intact myocytes by expression of the complete signal transduction pathway (m2 muscarinic receptor, inhibitory G proteins and GIRK1) in a heterologous system. The experiments in this proposal will yield significant new information about the protein-protein interactions involved in G protein-mediated signal transduction. We will examine muscarinic K+ current activation in a heterologous mammalian expression system, which will allow kinetic analysis of G protein-channel interactions with high time resolution, under conditions in which the levels of expression of the various components in the signal transduction pathway are known. We will also make GIRK1 - IRK1 chimeras, by exchanging the conduction pore regions, to examine the r ole of cytoplasmic domains in channel gating. These chimeric molecules will be transiently expressed in the mammalian cell line containing m2 mAChR, to assess the functional consequences of the cytoplasmically-localized domains on channel gating. The techniques implemented during the accomplishment of these two aims will establish the approaches which can be used for a more extensive examination of the role of protein - protein interactions in membrane signaling. %%% Cells are capable of responding to a variety of signals carried by small molecules which bind to specific receptors on their surface membranes. The mechanisms by which these events at the cell surface are translated into appropriate alterations in cell function are the focus of this study. Many distinct receptors have been identified, and a large number of them interact with a family of "signal transducing" proteins, called G proteins, which carry the signal from receptor to the appropriate enzyme which must be activated or inhibited. The ability to respond to a specific small molecule (agonist) with activation of the appropriate enzyme is a complicated process which may be regulated in a variety of ways. We have extensively characterized the kinetics of activation by acetylcholine of an inwardly rectifying K+ channel in bull frog atrium. This ion channel has now been cloned, which permits us to insert this signal transduction pathway into a cell type which does not normally contain it, and to study it under controlled conditions. The experiments in this proposal will yield significant new information about the interactions involved in G protein-mediated signal transduction. We will examine how channel activation is regulated in a well-controlled system in which the amounts of each protein in the signaling pathway are known. We will also examine the role of various regions of the channel molecule in the activation process by producing hybrid channels in which various regions are intercha nged with similar, but distinctly regulated channel types. The techniques implemented during the accomplishment of these two aims will establish the approaches which can be used for a more extensive examination of the role of protein - protein interactions in membrane signaling. ***
