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The kit ligand: a cell surface molecule altered in steel mutant fibroblasts
1Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
The c-kit proto-oncogene, the gene at the mouse W developmental locus, is one of a substantial group of genes that appear to encode cell surface receptors but for which the ligands are unknown. We have characterized the kit ligand by a generally applicable approach: the receptor extracellular domain was genetically fused to placental alkaline phosphatase, producing a soluble receptor affinity reagent with an enzyme tag that could be easily and sensitively traced. This fusion protein, APtag-KIT, was used to demonstrate a specific binding interaction (KD = 3 x 10(-8) M) with a ligand on 3T3 fibroblast lines. In situ staining showed labeling over the whole surface of the 3T3 cells, but not extending to adjacent nonexpressing cells. These findings provide direct molecular evidence that the kit ligand can exist as a cell surface protein. Binding was not detected on 3T3 fibroblasts carrying the steel (Sl) mutation, confirming the biological significance of the binding activity and demonstrating that mutations at the Sl locus affect the expression or structure of the kit ligand.
Insights
Researchers identified the kit ligand, a protein interacting with the c-kit receptor. This discovery confirms the kit ligand
Area of Science:
- Molecular Biology
- Developmental Biology
- Cell Signaling
Background:
- The c-kit proto-oncogene encodes a cell surface receptor with an unknown ligand.
- Understanding c-kit ligand is crucial for deciphering its role in development and disease.
- Existing methods lack the sensitivity to identify and characterize unknown ligands.
Purpose of the Study:
- To identify and characterize the unknown ligand for the c-kit receptor.
- To develop a novel method for detecting and studying cell surface receptor ligands.
- To investigate the role of the steel (Sl) locus in kit ligand expression.
Main Methods:
- Genetically fused the extracellular domain of the c-kit receptor to placental alkaline phosphatase (AP).
- Created a soluble receptor affinity reagent, APtag-KIT, for ligand detection.
- Utilized APtag-KIT for binding assays and in situ staining on 3T3 fibroblast cell lines.
Main Results:
- APtag-KIT specifically bound to a ligand on 3T3 fibroblasts with a dissociation constant (KD) of 3 x 10(-8) M.
- In situ staining revealed the ligand is expressed on the cell surface of 3T3 fibroblasts.
- Binding was abolished on 3T3 fibroblasts with the steel (Sl) mutation, indicating Sl locus involvement.
Conclusions:
- Direct molecular evidence confirms the kit ligand exists as a cell surface protein.
- The steel (Sl) locus is essential for the expression or structure of the kit ligand.
- This study provides a novel approach for identifying and characterizing unknown receptor ligands.
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