Human Stem Cell Factor Dimer Forms a Complex with Two Molecules of the Extracellular Domain of Its Receptor, Kit

Philo, John S.; Wen, Jie; Wypych, Jette; Schwartz, Meredith G.; Mendiaz, Elizabeth A.; Langley, Keith

doi:10.1074/jbc.271.12.6895

Cited by 66 publications

(56 citation statements)

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“…Overall, the EM reconstructions of the extracellular regions of both form I and form II exhibit a nearly perfect twofold symmetry, similar to that seen in the crystal structure of the dimeric KIT extracellular region (11). Moreover, the EMderived structures of the two forms demonstrate that KIT dimerization is driven entirely by binding of the dimeric SCF molecule (6,7), and that each SCF protomer forms a complex with a single KIT molecule by binding to the three N-terminal Iglike domains D1, D2, and D3 (10,11).…”

Section: Resultssupporting

confidence: 63%

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Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers

Opatowsky

Lax

Tomé

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Using electron microscopy and fitting of crystal structures, we present the 3D reconstruction of ligand-induced dimers of intact receptor tyrosine kinase, KIT. We observe that KIT protomers form close contacts throughout the entire structure of ligand-bound receptor dimers, and that the dimeric receptors adopt multiple, defined conformational states. Interestingly, the homotypic interactions in the membrane proximal Ig-like domain of the extracellular region differ from those observed in the crystal structure of the unconstrained extracellular regions. We observe two prevalent conformations in which the tyrosine kinase domains interact asymmetrically. The asymmetric arrangement of the cytoplasmic regions may represent snapshots of molecular interactions occurring during trans autophosphorylation. Moreover, the asymmetric arrangements may facilitate specific intermolecular interactions necessary for trans phosphorylation of different KIT autophosphorylation sites that are required for stimulation of kinase activity and recruitment of signaling proteins by activated KIT.receptor tyrosine kinases | cell signaling | cancer | structure analysis | structural biology A broad range of fundamental cellular processes, including proliferation, differentiation, survival, and metabolism, are mediated by signaling pathways that are activated by the 58 members of the receptor tyrosine kinase (RTK) family of membrane receptors (1). The receptor tyrosine kinase KIT was initially discovered as the viral oncogenic protein of a feline sarcoma virus (2). Subsequent studies have demonstrated that KIT and its ligand stem cell factor (SCF) play important roles in the control of proliferation, differentiation, and survival of a variety of cell types, including germ cells, hematopoietic cells, melanocytes, intestinal pacemaker cells, and sensory neurons in the CNS (3-5).KIT and other members of type-III RTK family are composed of an extracellular ligand-binding region containing five Ig-like domains (designated D1-D5), followed by a single transmembrane (TM) spanning helix, a relatively large (35 aa) cytoplasmic juxtamembrane (JM) region, and a tyrosine kinase domain with a kinase insert region containing several autophosphorylation sites. Structural and biochemical studies have shown that the N-terminal region composed of D1, D2, and D3 of KIT extracellular region functions as a binding site for SCF, which, by virtue of its homodimeric structure, is responsible for bringing about KIT dimerization (6-11).SCF-induced KIT dimerization results in homotypic contacts between the membrane proximal Ig-like domains D4 and D5 of two neighboring KIT molecules. These homotypic contacts position the TM and the cytoplasmic regions of KIT and other RTKs at a distance and orientation that facilitate tyrosine autophosphorylation, stimulation of enzymatic activity, and cell signaling (11-13). Oncogenic gain-of-function mutations in KIT have been identified in various cancers, including gastrointestinal stromal tumors, acute myeloid leukemia, melanoma, an...

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Section: Resultssupporting

confidence: 63%

“…Structural and biochemical studies have shown that the N-terminal region composed of D1, D2, and D3 of KIT extracellular region functions as a binding site for SCF, which, by virtue of its homodimeric structure, is responsible for bringing about KIT dimerization (6)(7)(8)(9)(10)(11).…”

mentioning

confidence: 99%

Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers

Opatowsky

Lax

Tomé

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…For Kit in particular, dimerization was suggested to be independent of the bivalency of the dimeric SCF ligand (14). However, sedimentation equilibrium and size exclusion chromatography indicated that SCF and Kit form 2:2 complexes in vitro (15). The SCF concentrations used in the latter study were higher than those found in vivo.…”

mentioning

confidence: 70%

“…However, we have shown in vitro that at concentrations where rhSCF is dimeric, a complex is formed with sKit that includes two rhSCF monomers and two sKit molecules (15), and we consider it most likely that SCF dimer mediates Kit dimerization. Models by which SCF could induce receptor dimerization and consequently activate signal transduction are shown in Fig.…”

Section: Discussionmentioning

confidence: 91%

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The Majority of Stem Cell Factor Exists as Monomer under Physiological Conditions

Hsu

Mendiaz

et al. 1997

Journal of Biological Chemistry

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Soluble Escherichia coli-derived recombinant human stem cell factor (rhSCF) forms a non-covalently associated dimer. We have determined a dimer association constant (K a ) of 2-4 ؋ 10 8 M ؊1 , using sedimentation equilibrium and size exclusion chromatography. SCF has been shown previously to be present at concentrations of approximately 3.3 ng/ml in human serum. Based on the dimerization K a , greater than 90% of the circulating SCF would be in the monomeric form. When 125 I-rhSCF was added to human serum and the serum analyzed by size exclusion chromatography, 72-49% of rhSCF was monomer when the total SCF concentration was in the range of 10 -100 ng/ml, consistent with the K a determination. Three SCF variants, SCF(F63C), SCF (V49L,F63L), and SCF(A165C), were recombinantly expressed in Escherichia coli, purified, and characterized. The dimer K a values, biophysical properties, and biological activities of these variants were studied. Dimerization-defective variants SCF(F63C)S-CH 2 CONH 2 and SCF(V49L,F63L) showed substantially reduced mitogenic activity, while the activity of the Cys 165 -Cys 165 disulfide-linked SCF(A165C) dimer was 10-fold higher than that of wild type rhSCF. The results suggest a correlation between dimerization affinity and biological activity, consistent with a model in which SCF dimerization mediates dimerization of its receptor, Kit, and subsequent signal transduction.Stem cell factor is a cytokine that is active toward early hematopoietic cells and also plays roles in gametogenesis, melanogenesis, and mast cell function. Its biological and other properties have been extensively reviewed (1, 2). It is found in both membrane-bound and soluble forms, with the latter being derived from a membrane-bound form by proteolytic cleavage. The soluble SCF has 165 amino acids.Both soluble Escherichia coli-derived and CHO 1 cell-derived recombinant human SCF have been reported to be non-covalently associated dimers, as determined by sedimentation equilibrium and size exclusion chromatography at protein concentrations above 0.4 mg/ml (3). In a previous paper (4), we demonstrated that SCF dimer is dissociable under non-denaturing conditions and the dissociation rate constant (k d ) of E. coli-derived rhSCF dimer is approximately 1.35 ϫ 10 Ϫ4 s Ϫ1 at pH 4.8, 25°C. In the present work, we arrive at a value of 2-4 ϫ 10 8 M Ϫ1 for the dimer association constant (K a ) of E. coli-derived rhSCF, based on several approaches including ultracentrifugation and size exclusion chromatography at low SCF concentrations. Since the SCF concentration in human serum has been found previously to be a few nanograms/ml (5), the K a value suggests that the majority of SCF in serum may be monomeric. We use 125 I-SCF as a tracer added to serum to show that this does in fact appear to be the case.The binding of ligands to cell receptors, followed by receptor dimerization, is essential for signal transduction by the family of transmembrane receptor tyrosine kinases (6 -8). The receptor for SCF on target cells is Kit (see Refs....

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Size‐Exclusion Chromatography with On‐Line Light Scattering

Arakawa

Wen

2001

CP Protein Science

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This unit describes the use of size-exclusion chromatography with on-line light scattering, UV absorbance, and refractive index detectors (SEC-LS/UV/RI) to determine: (a) the molecular weight of simple proteins containing no carbohydrates, (b) the molecular weight of glycoproteins, and (c), most importantly, the molecular weight and stoichiometry of protein-protein complexes or protein-carbohydrate complexes. Multiangle light scattering is also discussed.

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Human Stem Cell Factor Dimer Forms a Complex with Two Molecules of the Extracellular Domain of Its Receptor, Kit

Cited by 66 publications

References 49 publications

Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers

Structure, domain organization, and different conformational states of stem cell factor-induced intact KIT dimers

The Majority of Stem Cell Factor Exists as Monomer under Physiological Conditions

Size‐Exclusion Chromatography with On‐Line Light Scattering

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