Abstract

Heterotrimeric G proteins relay a variety of extracellular signals to downstream intracellular effector proteins. Activation of the G protein α subunit (Gα), a requisite for signal propagation, requires the external action of a guanine nucleotide exchange factor (GEF), typically a G-protein-coupled receptor, which stimulates the Gα subunit to exchange GDP for GTP by providing a stable nucleotide-free transition state. Recently, cytosolic Ric-8 proteins have been demonstrated to possess GEF activity and interact in vitro with different subsets of monomeric Gα subunits. Since a crystal structure of Ric-8 in complex with a GDP-bound or nucleotide-free Gα subunit is not available, our goal has been to characterize this structural interface in the Drosophila model system using a combination of scanning mutagenesis and co-precipitation assays. A comprehensive Gα12 NAAIRS library was used as a proxy to uncover structural determinates on Concertina (Cta), the Drosophila Gα12 homolog, governing binding to the Ric-8 N-terminus. Initial screening of this library has revealed two distinct regions of Gα12 critical for interaction with the N-terminus of Ric-8. Unfortunately, corresponding Cta mutants, assayed by our collaborators, failed to show any relative binding impairment to Ric-8, suggesting that perhaps the residues mutated are not functionally conserved.

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