Monday, November 25, 12:50pm – 1:10pm, Zeis 123, via Zoom
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Forbes Fowler
Dr. Ted Meigs
Heterotrimeric guanine nucleotide-binding proteins (G proteins)
are cell signaling conduits composed of the subunits š¼, š½, and š¾.
These proteins relay signals from cell surface receptors to various
downstream effector proteins. The š¼ subunits Gš¼12 and Gš¼13 are
implicated in multiple tumor types as wildtype (WT) and
constitutively active (QL) forms. Gš¼13 is overexpressed in
multiple tumor types including invasive breast cancer, prostate
adenocarcinoma, and ovarian cystadenocarcinoma. It is crucial to
identify potential therapeutic sites in Gš¼13, and considerable work
has examined its mechanism of binding to downstream effectors as
well as its post-translational modifications. No studies have
precisely defined the amino acids of Gš¼13 required for binding the
š½š¾ dimer, nor whether initial association with š½š¾ is necessary for
lipid modifications of Gš¼13. We have sought to identify amino
acids in Gš¼13 required for binding to the š½ subunit, and also
determine whether association with š½š¾ is necessary for signaling
by Gš¼13WT compared to the QL form. Therefore, we mutated
amino acids in Gš¼13 previously suggested as N-terminal contact
points with the š½ subunit. Also, recent work by Wedegaertner and
colleagues identified a single amino acid within a different G
subunit, Gš¼q, that is crucial for its binding to š½ (Aumiller and
Wedegaertner 2023). Based on Gš¼q and Gš¼13 alignment, we also
engineered a single amino acid mutation in Gš¼13 WT and QL. All
mutants were expressed in HEK293 cells, and lysates were used in
a glutathione-S-transferase (GST) pulldown assay. Precipitates
were subjected to immunoblotting to determine the presence of
Gš¼13 variants in the sepharose-bound š½ subunit samples.
Preliminary results suggest these mutations disrupt both Gš¼13 WT
and QL binding to the š½ subunit. Currently we are examining these
mutants for tumorigenic growth signaling and ability to interact
with other known binding partners of Gš¼13.