Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor

Abstract

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4 interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.

Direct contacts between extracellular membrane-proximal domains are required for VEGF receptor activation and cell signaling

“…3D). The structure of D7 dimer is very similar to the homotypic D4 contacts seen in KIT extracellular dimer structure (PDB code: 2E9W) (8). In addition D7 of VEGFR2 exhibits strong polarization of electrostatic field with an overall negatively charged surface with the exception of a positively charged center strip right along the D7-D7 interface (Fig.…”

“…An evolutionarily conserved sequence motif (L∕IxRΦxxxD∕ExG) responsible for mediating homotypic contacts between Ig-like domains was identified by structure-based sequence alignment of D4 of KIT, PDGFRα, PDGFRβ, and colony stimulating factor 1 receptor (CSF1R) (8,10). To determine which domain of VEGFR2 may be functionally related to D4 of KIT, we searched the sequences of D4 and D7 of VEGFRs for this conserved motif.…”

“…The asymmetric unit contains one molecule with solvent content of 64.1%. The structure was solved by molecular replacement with MolRep using models based on the structures of Ig domains from MUSK (2IEP) (18), telokin (1TLK) (17), and D4 of KIT (2E9W) (8) as search models. The structure refinement and model-building cycles were performed using RefMac (27) and COOT (28).…”

“…However, the ectodomains of the PDGF-receptor (PDGFR) family of RTKs (type III) are composed of five Ig-like repeats of which D1, D2, and D3 function as ligand-binding regions of PDGFR and other members of the family (i.e., KIT, CSF1R, and Flt3). Structural and biochemical experiments have shown that SCF binding to the extracellular region induces KIT dimerization, a step followed by homotypic contacts between the two membrane-proximal Ig-like domains D4 and D5 of neighboring KIT molecules (8). Biochemical studies of wild-type (WT) and oncogenic KIT mutants have shown that the homotypic D4 and D5 contacts play a critical role in positioning the cytoplasmic regions of KIT dimers at a distance and orientation that facilitate transautophosphorylation, kinase activation, and cell signaling.…”

CSF-1 Receptor Signaling in Myeloid Cells

“…2B) Elegheert et al 2011;Ma et al 2012;Felix et al 2013). D4 shares a dimerization domain sequence fingerprint that has been identified in other closely related RTK III receptors, Kit and PDGFR (Yuzawa et al 2007;Yang et al 2008), and the presence of domains D4 and D5 in the CSF-1:CSF-1R D1 -D5 significantly decreases the K d of interaction Elegheert et al 2011;Ma et al 2012) owing to CSF-1R homotypic interactions. However, the K d for the CSF-1:CSF-1R D1 -D5 interaction at 37˚C ( 20 nM, human and mouse [Elegheert et al 2011]) was still higher than the dissociation constants reported for the binding of mouse (0.4 nM ]) or human (0.1 nM ) CSF-1 to their cognate receptors on cells, suggesting a significant contribution of the transmembrane domain and spatial confinement of the membrane to affinity (Fig.…”