Role of the RGS (regulator of G protein signaling) domain of axin in vertebrate axis formation

Abstract

The protein complex of Axin, APC and GSK‐3 acts as a primary negative regulator of the Wnt signaling pathway. Axin, a key player in this complex, contains four critical domains: a β‐catenin‐ and GSK‐binding domains, an RGS domain and a DIX domain. Our study focuses on the role of the Axin‐RGS domain. Typically, RGS domains contain a conserved asparagine (Asn), critical for the ability to bind heterotrimeric G‐proteins. Interestingly, we find that the Axin‐RGS domain encodes a glutamine (Gln) at the equivalent position, suggesting a novel function for Axin‐RGS. Because Wnt signaling is thought to involve activation of G‐protein coupled receptors, we tested whether the Axin‐RGS domain functions in this context. We generated point mutations that converts the original Gln to Asn, predicted to increase affinity for Gα, and that converts Gln to Ala, predicted to abolish binding activity. We used an in vitro binding assay to evaluate the ability of these mutants to bind Gα. We also tested for the ability of the mutant forms to rescue Axin function in masterblind zebrafish mutant embryos and in antisense knockdown of Axin in zebrafish and frog (Xenopus laevis). We found that the mutant constructs showed a differential ability to restore normal development in fish and frog embryos. Our results suggest that the interaction of Axin with G‐proteins may play a key role in the regulation of Wnt signaling during axial development.