Negative Regulation of the Folded gastrulation Signaling Pathway by the Non-visual -arrestin Kurtz. Emily J. Simon, Alyssa J. Manning, Stephen L. Rogers. Dept Biology, Univ North Carolina-Chapel Hill, Chapel Hill, NC.

   Epithelial morphogenesis is an important developmental process that underlies gastrulation and formation of tissues and organ systems in Drosophila as well as human development. We are using Drosophila as a model to study epithelial apical constriction during mesoderm invagination and imaginal wing disc development. This process is regulated by the Folded gastrulation (Fog) pathway. Recently our lab identified Mist, a G-protein coupled transmembrane receptor, which is activated by the ligand Fog and which downstream triggers cell contraction. Currently how this pathway is inactivated or regulated is not understood. We hypothesize that Mist inactivation is mediated by a -arrestin. In our model, GPRK2 phosphorylates Mist on its C-terminal cytoplasmic domain causing recruitment of a -arrestin/Drosophila Kurtz, which contributes to termination of the contraction signal. Through immunoprecipitation we have shown that Mist interacts with Kurtz. We are using a novel cell culture assay to study this pathway. Drosophila S2R+ cells undergo a dramatic acto-myosin based contraction upon application of exogenous Fog protein. Measuring the percentage of S2R+ cells contracted over time following Fog application shows that the percent contracted plateaus after about 5 minutes. Consequent washout of Fog results in nearly complete relaxation of cells within 30 minutes. Data thus far show that cells overexpressing Kurtz exhibit a reduction in the percentage of contracted cells as compared to wild type cells after Fog treatment for a fixed time period. Conversely, Kurtz knockdown via RNAi results in an increased percentage of contracted cells. These experiments support our model for Mist desensitization and suggest that Kurtz is active during Fog pathway attenuation. We will use our cell culture assay to examine percentage of cellular contraction and relaxation after other perturbations to the pathway, such as alteration of Kurtz and GPRK2 levels. Thus our data demonstrates that Kurtz is a key component in the regulation of this cell contraction pathway via interaction with the Mist receptor.