Monday, November 25, 12:50pm โ 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 ๐ผ, ๐ฝ, and ๐พ.
These proteins relay signals from cell surface receptors to various
downstream effector proteins. The ๐ผ subunits G๐ผ12 and G๐ผ13 are
implicated in multiple tumor types as wildtype (WT) and
constitutively active (QL) forms. G๐ผ13 is overexpressed in
multiple tumor types including invasive breast cancer, prostate
adenocarcinoma, and ovarian cystadenocarcinoma. It is crucial to
identify potential therapeutic sites in G๐ผ13, 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 G๐ผ13 required for binding the
๐ฝ๐พ dimer, nor whether initial association with ๐ฝ๐พ is necessary for
lipid modifications of G๐ผ13. We have sought to identify amino
acids in G๐ผ13 required for binding to the ๐ฝ subunit, and also
determine whether association with ๐ฝ๐พ is necessary for signaling
by G๐ผ13WT compared to the QL form. Therefore, we mutated
amino acids in G๐ผ13 previously suggested as N-terminal contact
points with the ๐ฝ subunit. Also, recent work by Wedegaertner and
colleagues identified a single amino acid within a different G
subunit, G๐ผq, that is crucial for its binding to ๐ฝ (Aumiller and
Wedegaertner 2023). Based on G๐ผq and G๐ผ13 alignment, we also
engineered a single amino acid mutation in G๐ผ13 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
G๐ผ13 variants in the sepharose-bound ๐ฝ subunit samples.
Preliminary results suggest these mutations disrupt both G๐ผ13 WT
and QL binding to the ๐ฝ subunit. Currently we are examining these
mutants for tumorigenic growth signaling and ability to interact
with other known binding partners of G๐ผ13.