This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Kitl is a member of a growing class of growth factors that are biologically active as either soluble (S) factors or membrane-bound (MB) factors. KitlSl mutants are the starting point in an approach that combines genetic analysis with knowledge-based computer modeling to gain a better understanding of Kitl function. Based on our preliminary studies, we have obtained evidence that the MB-isoforms of the KitlSl mutants are not glycosylated normally and do not undergo proper intracellular processing. We hypothesize that the KitlSl mutant proteins have severe misfolding defects and are retained intracellularly such that little or no protein reaches the cell surface. We will take complementary approaches to examine this hypothesis. First, we will test whether S- as well as MB- isoforms of KitlSl mutants are defective for intracellular processing, binding to Kit and/or activation of Kit. Second, we will construct and select for new Kitl mutations and test whether these mutations restore Kitl function. The new Kitl mutants will test whether the defects in the existing KitlSl mutants result from the particular aa substitution or the absence of the original residue. We will also perform a selection for second-site mutations in KitlSl mutants that restore in vitro function. Third, we are using molecular modeling studies, based on the crystal structure of the human KITL ortholog, to predict the effect of each of the Kitl sequence alterations on structure. Further understanding of sequence and structural requirements for Kitl function will contribute greatly to understanding how this signaling molecule functions. These studies should also contribute to understanding the function of other important cytokines, which are structurally similar to Kitl.
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