title: The fifth immunoglobulin-like domain of the Kit receptor is required for proteolytic cleavage from the cell surface.
abstract: Stem cell factor (SCF) initiates its biological effects by binding to its receptor Kit. Cell surface Kit is proteolytically cleaved to generate soluble Kit. Structure-function analysis of the extracellular region of Kit has implicated the first three immunoglobulin-like domains in SCF binding, and the fourth immunoglobulin-like domain in receptor dimerization. However, the role of the fifth immunoglobulin-like domain is unknown. To test the hypothesis that the fifth immunoglobulin-like domain is important for proteolytic cleavage of Kit from the cell surface, we constructed a mutant form of Kit in which the first four immunoglobulin-like domains are linked to the transmembrane and cytoplasmic domains (designated Kit-Del5). Kit-wild type (Kit-WT) and Kit-Del5 were expressed in the murine mast cell line IC2. Flow cytometry demonstrated that both Kit-WT and Kit-Del5 are displayed on the IC2 cell surface, and immunoblotting confirmed the presence of Kit proteins of the expected molecular weights, 154 kDa and 134 kDa, respectively. Although IC2-Kit-WT cells proteolytically cleave cell surface Kit, generating a 98 kDa soluble form of Kit, IC2-Kit-Del5 cells do not. These findings demonstrate that the fifth immunoglobulin-like domain of Kit is required for proteolytic cleavage of Kit from the cell surface.
authors.affiliation: Division of Hematology, University of Washington, Seattle, WA, USA. vcbroudy@u.washington.edu
authors.forename: V C
authors.initials: VC
authors.lastname: Broudy
authors.forename: N L
authors.initials: NL
authors.lastname: Lin
authors.forename: D F
authors.initials: DF
authors.lastname: Sabath
mesh_terms: D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005434:Flow Cytometry; D017353:Gene Deletion; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry*; D051379:Mice; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019009:Proto-Oncogene Proteins c-kit / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019089:Stem Cell Factor / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D007136:Immunoglobulins; D019089:Stem Cell Factor; D019009:Proto-Oncogene Proteins c-kit; D013755:Tetradecanoylphorbol Acetate