Abstract
The G12 class of heterotrimeric G protein α subunits is important for several cellular processes that include mitogenesis, cytoskeletal rearrangements, and migration. Previous work in the Meigs lab revealed that an evolutionary G12 homolog from Drosophila, termed Concertina, lacked the ability of mammalian G12 proteins to stimulate a growth signaling pathway in cells. Therefore, several chimeras of Gα12 and Concertina were engineered, and these provided evidence that the C-terminal domain of Gα12 is essential for activating serum response element (SRE) mediated transcription. To dissect this large C-terminal region, overlapping PCR was used to engineer a SRE-coupled chimera that included an additional 45-residue Concertina domain in the middle of its C- terminal region. This modification completely uncoupled the chimeric protein from SRE-mediated transcription as demonstrated by a firefly luciferase reporter gene assay. The N-terminal Concertina domain of this chimera was then replaced with Gα12 sequence, and it was demonstrated that this 45-residue Concertina substitution in the C-terminus was sufficient to uncouple Gα12 from SRE signaling. This new chimera can now be used to more precisely define the determinants of growth signaling activation within Gα12.
How to Cite
Hamilton, T., (2013) “Domain Replacement in Galpha12 by an Evolutionary Homolog Reveals Key Residues in Cell Growth Signaling”, Capstone, The UNC Asheville Journal of Undergraduate Scholarship 26(1).
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