I propose to discover the GTPase-activating protein (GAP) for the heterotrimeric G proteins Golf and Gs that allows rapid responses to odorants by olfactory sensory neurons and similarly fast adenylyl cyclase responses in some central neurons. G protein GAPs accelerate hydrolysis of bound GTP and thereby accelerate the turn-off of G protein signaling upon termination of the receptor stimulus. Since we discovered the first GAP for a trimeric G protein in 1992, GAPs for the Gi, Gq and G12 families have been well studied, but no GAP for a Gs family member, either Golf or Gs itself, has been identified. In olfactory sensory neurons, olfactory receptors activate Golf, a close homolog of Gs, which then activates adenylyl cyclase. Cyclic AMP activates a CNG cation channel to initiate a cascade of channel opening that leads to synaptic transmission. Both electrophysiologic analysis of this pathway in olfactory neurons and quench-flow measurements of adenylyl cyclase in olfactory cilia indicate that Golf and the cyclase are deactivated in <0.2 sec after removal of odorant. Such speed is needed both for physiologically fast responses to odorants and for distinction among multiple odorants, but it is $40- fold faster than the 10-sec deactivation rate of isolated Golf. Such a fast turn-off rate demands a GAP activity. Regulation of the Golf GAP will impact both the kinetics and dynamics of olfactory signaling. Like most primary sensory neurons, olfactory sensory neurons overexpress the major components of their sensory signaling pathway, both proteins and their mRNAs. It is thus likely that the Golf GAP mRNA is also relatively abundant. We will take advantage of these properties of olfactory neurons to clone the Golf GAP cDNA and initiate its study. We will use a sensitive and selective in vitro assay for GAP activity to select GAP cDNA from an from olfactory epithelium expression library. We will also use a selective co-precipitation (TAP-Tag) approach to isolate and identify the expressed GAP protein in cells transfected with the library. The Golf GAP(s) will be evaluated in cellular backgrounds for its ability to modulate adenylyl cyclase III activity and kinetics via Golf, and for any biological regulation of its activity. Identifying a Golf GAP will allow us to understand how its activity is controlled in olfactory neurons and, working with olfactory electrophysiologists, determine its role in slow and fast responses to odorants. The Golf GAP may also be the Gs GAP that is predicted to be active in neurons and heart, or it may lead us to a distinct Gs GAP as a paralog. This work will finally illuminate how cAMP pathways are controlled in cells where adenylyl cyclase signaling is fast.
I propose to identify and study the GTPase-activating protein (GAP) for the olfactory neuron G protein Golf. The Golf GAP is a still-missing component of the initial odorant-sensing signaling pathway. It allows the first step in olfaction to be as fast as it is. In olfactory sensory neurons, the rate of activation and deactivation of Golf limits the rate of detection of, and response to, odorants. The Golf GAP cooperates with odorant receptors to control these rates. However, the identify and precise function of the Golf GAP is unknown even though biochemical and electrophysiological data argue strongly for its existence. I have developed assays and a strategy that makes discovery of the GAP highly feasible over the 2-year R21 time frame. Learning the GAP's function will improve our understanding of signal timing and integration in olfaction. Because Golf is a close homolog of Gs, which mediates hormone and neurotransmitter signaling throughout the body, discovery of the Golf GAP will be of far broader impact on diverse signaling pathways either because Golf and Gs will share a GAP or because the Golf GAP will lead to discovery of a distinct GAP for Gs.
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