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Structure-function analyses of the kit receptor for the steel factor
1Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Abstract:
Binding of the Steel factor (SLF) to the product of the c-kit proto-oncogene stimulates the receptor's intrinsic tyrosine kinase that phosphorylates a set of cytoplasmic signaling molecules. Germ-line mutations in the genes that encode the receptor or the ligand result in remarkably similar phenotypes that affect melanogenesis, erythropoiesis and gametogenesis in mice. We concentrated on the initial events of the signal transduction pathway that underlies these processes. The extracellular portion of Kit is comprised of five immunoglobulin-(Ig)-like domains. Ligand binding to this domain induces rapid and extensive dimerization of the receptor molecules in a mechanism that involves monovalent binding of the dimeric ligand, followed by an increase in receptors' affinity and gradual stabilization of the dimers. It thus appears that Kit has at least two functions: ligand binding and ligand-induced receptor dimerization, in addition to the kinase activity. Both functions are independent of the transmembrane and cytoplasmic domains, as a recombinant soluble ectodomain retained high affinity to SLF and ligand-dependent dimerization. In order to correlate these functions with specific structures, we employed ligand-competitive monoclonal antibodies, soluble deletion mutants of the ectodomain and chimeric human-mouse Kit proteins. These approaches indicated that the N-terminal three Ig-like domains constitute the binding site, whose core is the second domain. Further experiments suggested that a putative dimerization site is distinct from the binding cleft and may be located on the fourth Ig-like domain.
Insights
Steel factor (SLF) binding to c-Kit receptor triggers tyrosine kinase activity. The N-terminal domains of c-Kit mediate ligand binding and dimerization, crucial for cell signaling.
Area of Science:
- Cellular signaling
- Molecular biology
- Biochemistry
Background:
- The c-Kit receptor tyrosine kinase and its ligand, Steel factor (SLF), are critical for melanogenesis, erythropoiesis, and gametogenesis.
- Mutations in c-Kit or SLF lead to similar developmental defects in mice.
- Understanding the initial signal transduction events mediated by c-Kit is essential.
Purpose of the Study:
- To investigate the initial molecular events in the c-Kit signal transduction pathway.
- To delineate the structural basis for SLF binding and ligand-induced c-Kit dimerization.
- To correlate specific functions of the c-Kit ectodomain with its structural domains.
Main Methods:
- Utilized ligand-competitive monoclonal antibodies to map binding sites.
- Employed soluble deletion mutants of the c-Kit ectodomain.
- Constructed chimeric human-mouse c-Kit proteins to analyze domain functions.
- Investigated the binding affinity and dimerization properties of recombinant soluble c-Kit ectodomain.
Main Results:
- The extracellular portion of c-Kit, comprising five immunoglobulin-like (Ig) domains, mediates SLF binding and receptor dimerization.
- SLF binding to the N-terminal three Ig-like domains, particularly the second domain, is necessary for initiating signaling.
- A distinct dimerization site, potentially located on the fourth Ig-like domain, is responsible for stabilizing receptor dimers.
- A soluble recombinant c-Kit ectodomain retains high affinity for SLF and exhibits ligand-dependent dimerization, independent of transmembrane and cytoplasmic domains.
Conclusions:
- c-Kit possesses distinct functional sites for ligand binding and dimerization within its extracellular ectodomain.
- The N-terminal Ig-like domains are crucial for SLF recognition and initial receptor activation.
- The fourth Ig-like domain likely plays a key role in mediating receptor dimerization upon ligand binding.
- These findings provide structural insights into the mechanism of c-Kit receptor activation and signal initiation.