Wnt-Frizzled interaction involved in chick limb development *
In combination with LRP-5 and LRP-6, ten members of the Frizzled（Fz）family are known to function as Wnt receptors. However the relationship of the receptors to ligands is poorly understood, partly because of the difficulty in obtaining active Wnt proteins for a direct binding assay. Several Wnt members, including chicken Wnt-3a, Wnt-4, Wnt-5a, Wnt-6, Wnt-7a, Wnt-10a, and Wnt-11, which are expressed with specific patterns in the chick limb bud are expressed in DF-1 cells after HA-tagging to the C-terminal end. On the other hands chicken Fz-4 and Fz-10 are expressed after FLAG-tagging to the intracellular region of the receptors. Expression constructs for WntHA and FzFLAG are transfected in DF-1 cells, either independently or in combination, and the Western blot assay was performed to detect receptor binding of WntHA before and after immunoprecipitation of the receptor with anti-FLAG antibody. When the conditioned medium of DF-1 expressing WntHA was added to cells expressing FzFLAG, Wnt binding was detected in the following combinations:Wnt-3a, Wnt-5a, and Wnt-7a for Fz-10, and Wnt-5a for Fz-4. Weak binding was also detected in Wnt-6 and Wnt-10a for Fz-10. A smaller amount of Wnt-7a showed efficient binding to Fz-10, compared with Wnt-5a, implicating highest affinity to be to Wnt-7a. Since Wnt-7a is temporally and spatially co-expressed in the limb bud with Fz-10, but not with Fz-4, these results provide evidence indicating functional interaction between Wnt and Fz members, eventually leading to morphogenetic events during limb development.(Accepted on April 12，2006)
