G.P.3.08 Missense mutations in the dystrophin ZZ domain: Effects beyond beta dystroglycan binding?

The DMD gene product, dystrophin, binds to beta dystroglycan (βDG) at the sarcolemmal membrane via interactions in the dystrophin cysteine rich (CR) region. This region is comprised of the WW domain, which plays a primary role in βDG binding; two Ca2+ binding EF hand motifs; and the ZZ domain, a zinc finger domain defined by four cysteine residues. Reported Duchenne muscular dystrophy (DMD) associated ZZ domain mutations include three missense (C3313F, C3340Y, and D3335H) and one single amino acid deletion (E3367, just C-terminal to the canonical ZZ domain). Despite severe DMD phenotypes, muscle biopsies from each of these patients have shown correct sarcolemmal localization of dystrophin, albeit in reduced amounts. The ZZ domain has been shown to increase WW-mediated βDG binding efficiency, and to interact with calmodulin. Previously published studies examining the effect of ZZ missense mutations on interactions between the CR region and βDG have revealed differing results. In vitro overlay assays showed that only the C3340Y mutation abrogates βDG binding. In contrast, co-immunoprecipitation studies of transfected minidystrophin constructs containing mutations at each of the ZZ cysteine residues demonstrated complete ablation of βDG binding with each mutation, along with mislocalization of the minidystrophin to the nucleus. In order to clarify the role of the CR region in βDG binding, we expressed the entire CR region and part the adjacent C-terminal domain in 293FT cells. Binding assays show that this construct retains the ability to bind βDG and calmodulin even in the presence of any of the four ZZ mutations. Expression of the ZZ-mutant truncated dystrophin proteins in mdx myotubes shows no evidence of nuclear localization. These results suggest that there may be functions of the ZZ domain that involve more subtle effects on βDG binding or interactions with unknown protein binding partners. Current studies testing this hypothesis are underway.