MUTATIONS REVEAL G PROTEIN ACTIVATION MECHANISM

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Heterotrimeric G proteins relay signals from cell-surface receptors to intracellular effectors. Recently solved crystal structures of the alpha subunit of transducin (Galphat) in both GDP- and GTP-bound forms make Galphat an ideal model for studying the mechanism of G protein signaling. The goals of this project are to define the structural basis of receptor activation of G protein, by perturbing release of GDP through site- directed mutagenesis, and at the same time to select recombinant Galphat molecules to serve as tools for studying the mechanism for terminating the action of G proteins. To study G protein activation by the receptor, retinal rhodopsin, mutations will be introduced in receptor coupling and GDP binding regions of Galphat, based on high resolution structures of this protein. Galphat mutations that accelerate GDP release will constitutively activate the protein and mimic receptor catalyzed activation. Analysis of these mutations will identify structural elements responsible for coupling receptors to G proteins. These GDP-releasing mutations will also be used to probe mechanisms that regulate termination of the activity of Galphat. Because steady-state GTP hydrolysis by such mutant proteins is not limited by the rate of GDP release, their GTPase activities can be used to detect GTPase Activating Proteins (GAPs) in the absence of activated rhodopsin. The role of the Galphat effector, cGMP phosphodiesterase (PDE), and other potential GAPs will be investigated.