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Wednesday, April 22 • 9:20am - 9:40am
Effect of reverting switch II amino acids in Gα13 to their ancestral forms.

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Heterotrimeric guanine nucleotide binding proteins (G proteins), upon activation by cell surface G protein coupled receptors (GPCRs) via external stimuli, transmit signals to downstream pathways regulating a variety of cellular responses, such as cell proliferation and cytoskeletal rearrangements. The G12/13 subfamily of G proteins, consisting of Gα12 and Gα13, has been demonstrated to promote uncontrolled growth of cells and play a role in metastatic cancer progression. Evolutionary comparison of proteins in the G12/13 subfamily of G proteins showed a highly conserved Gln/Gln amino acid pair at a precise structural position in G12/13 proteins in species ranging from sea sponge to humans. Surprisingly, mammalian Gα13 has lost these amino acids and reverted to the Glu/Lys pair common to other G protein subfamilies. To study the effects of this reversion on the ability of Gα13 to bind its effector proteins, we engineered a myc-tagged Gα13 containing a Glu/Lys to Gln/Gln mutation and are testing this in binding assays with a number of proteins that have been shown to interact exclusively with Gα13 or with both Gα12 and Gα13. For this purpose, we constructed several new GST-fusions of Gα13 binding proteins, including Hax-1, JLP, and RGS16. The genes encoding Gα13 binding proteins were isolated from a brain cDNA library, amplified by PCR, and inserted into a vector. The plasmids were transformed into bacterial cells for protein synthesis, and the proteins were purified for ongoing interaction experiments.

Wednesday April 22, 2015 9:20am - 9:40am PDT
014 Zeis Hall