We propose to study the biochemical mechanisms that underlie the timing and integration of G protein-mediated signals in cells. G protein signaling modules control diverse cellular functions in response to equally diverse inputs. Consequently, G protein signaling is involved in many disease processes, and is the target of a huge number of drugs. G protein modules consist of a conserved group of interacting proteins: receptors, G protein Ga and Gbg subunits, GTPase activating proteins (GAP) and effector proteins. A mammalian cell typically expresses about 30 G protein-coupled receptors, half-dozen G proteins and GAPs and a dozen effectors. Mechanism of action is conserved, but varies quantitatively to allow different cells to respond to extracellular signals with a wide variety of kinetic patterns, intracellular outputs and modes of signal integration. Output from a G protein module quantitatively reflects a balance of receptor-catalyzed G protein activation and GAP-promoted deactivation. The rates of signal initiation and termination thus seem linked to the level of output, but cells can control response kinetics and response levels independently. We developed a quantitative framework for analyzing how receptors and GAPs interact to solve this problem. We will test mechanisms of receptor-GAP interaction and evaluate how and when each contributes to the temporal control of signaling. They include the ability of GAPs to stabilize the association of receptors and G proteins and to directly potentiate receptor function. We recently discovered that Gaq and Gbg, each of which stimulates phospholipase C-bs (PLC- bs) in response to different receptors, together stimulate the PLC-b3 isoform with strong synergism, 10 times the sum of the activities evoked by each subunit individually. Gaq-Gbg synergism is observed in diverse animal cells. We showed that Gaq-Gbg synergism can be explained by a classical two-state allosteric model, and we propose to test the physical basis of that interaction. Further, this model predicts that any two-state enzyme that is stimulated by two different ligands will display significant synergism only if its basal activity is very low, <0.1% of maximum. We will test this prediction by evaluating constitutively active PLC-b3 mutants for loss of synergism and constitutively deactivated PLC- b2 mutants, which should acquire synergistic regulation. We will also use this prediction to search for novel regulatory enzymes that can act as coincidence detectors for two or more ligands.
The proposed research aims to expand our understanding of how cells use simple chemical reactions to amplify and integrate information. The relevance, or applied value, of studying G protein signaling mechanisms lies in the ubiquity of G protein modules among all animals, plants and fungi;the variety and importance of the physiology that they control;the numerous diseases in which they are involved;and the huge number of drugs that act on them. The goal of the proposed research is to develop quantitative understanding of the mechanisms that control cellular information processing by G protein pathways. Such understanding will both help us analyze their malfunction in disease and inform their therapeutic manipulation.
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