Elucidating determinants at the G๐œถ13 N-terminus for binding to the ๐œท๐œธ dimer

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 ๐›ผ, ๐›ฝ, and ๐›พ.

These proteins relay signals from cell surface receptors to various

downstream effector proteins. The ๐›ผ subunits G๐›ผ12 and G๐›ผ13 are

implicated in multiple tumor types as wildtype (WT) and

constitutively active (QL) forms. G๐›ผ13 is overexpressed in

multiple tumor types including invasive breast cancer, prostate

adenocarcinoma, and ovarian cystadenocarcinoma. It is crucial to

identify potential therapeutic sites in G๐›ผ13, 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 G๐›ผ13 required for binding the

๐›ฝ๐›พ dimer, nor whether initial association with ๐›ฝ๐›พ is necessary for

lipid modifications of G๐›ผ13. We have sought to identify amino

acids in G๐›ผ13 required for binding to the ๐›ฝ subunit, and also

determine whether association with ๐›ฝ๐›พ is necessary for signaling

by G๐›ผ13WT compared to the QL form. Therefore, we mutated

amino acids in G๐›ผ13 previously suggested as N-terminal contact

points with the ๐›ฝ subunit. Also, recent work by Wedegaertner and

colleagues identified a single amino acid within a different G

subunit, G๐›ผq, that is crucial for its binding to ๐›ฝ (Aumiller and

Wedegaertner 2023). Based on G๐›ผq and G๐›ผ13 alignment, we also

engineered a single amino acid mutation in G๐›ผ13 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

G๐›ผ13 variants in the sepharose-bound ๐›ฝ subunit samples.

Preliminary results suggest these mutations disrupt both G๐›ผ13 WT

and QL binding to the ๐›ฝ subunit. Currently we are examining these

mutants for tumorigenic growth signaling and ability to interact

with other known binding partners of G๐›ผ13.