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Abstract
Axin is a critical component of the β-catenin destruction complex and is also necessary for Wnt signaling initiation at the level of co-receptor activation. Axin contains an RGS domain, which is similar to that of proteins that accelerate the GTPase activity of heterotrimeric Gα/Gna proteins and thereby limit the duration of active G-protein signaling. Although G-proteins are increasingly recognized as essential components of Wnt signaling, it has been unclear whether this domain of Axin might function in G-protein regulation. This study was performed to test the hypothesis that Axin RGS-Gna interactions would be required to attenuate Wnt signaling. We tested these ideas using an axin1 genetic mutant (masterblind) and antisense oligo knockdowns in developing zebrafish and Xenopus embryos. We generated a point mutation that is predicted to reduce Axin-Gna interaction and tested for the ability of the mutant forms to rescue Axin loss-of-function function. This Axin point mutation was deficient in binding to Gna proteins in vitro, and was unable to relocalize to the plasma membrane upon Gna overexpression. We found that the Axin point mutant construct failed to rescue normal anteroposterior neural patterning in masterblind mutant zebrafish, suggesting a requirement for G-protein interactions in this context. We also found that the same mutant was able to rescue deficiencies in maternal axin1 loss-of-function in Xenopus. These data suggest that maternal and zygotic Wnt signaling may differ in the extent of Axin regulation of G-protein signaling. We further report that expression of a membrane-localized Axin construct is sufficient to inhibit Wnt/β-catenin signaling and to promote Axin protein turnover.
Highlights
Wnt signaling has major roles throughout development, stem cell maintenance and regeneration
Identification of a putative essential Gna-interacting residue in the Axin-RGS domain To address the potential role for Axin RGS activity in Wnt signaling, we sought to mutate single amino acid residues in the RGS domain that would disrupt RGS-associated GAP activity but would not affect interactions with adenomatous polyposis coli (APC), and not affect b-catenin degradation
The presence of the RGS domain in Axin has suggested that interaction with heterotrimeric G-proteins through this domain could be an important step in regulating the activity of the bcatenin degradation complex
Summary
Wnt signaling has major roles throughout development, stem cell maintenance and regeneration. Different Wnt ligand/receptor complexes can regulate distinct signaling networks that either activate the transcription factor b-catenin, or stimulate various bcatenin-independent pathways Binding of Wnt ligands to a receptor complex, composed of Frizzled receptors (Fzd) and one of the LDL receptor-related proteins Lrp5/6, activates the Dishevelled (Dvl) protein to inhibit GSK3b and degradation complex activity. This results in unphosphorylated and stabilized b-catenin, which can enter the nucleus and complex with nuclear T-cell factor/lymphoid enhancer factor (TCF/LEF)related transcription factors to regulate downstream target gene expression. Axin1/masterblind (mbl) mutant zebrafish show a loss of eyes and telencephalon and other anterior defects resulting from constitutive activation of zygotic Wnt/bcatenin signaling [8,9]
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