Abstract
The receptor Tyro3 together with Axl and Mer form the Axl/Tyro3 family of receptor tyrosine kinases. Members of this family play essential roles in spermatogenesis, immunoregulation, and phagocytosis. Gas6, the product of growth arrest-specific gene, activates the kinase activity of all three receptors. Here, we report the first biochemical and structural characterization of a member of this family, namely of a fragment spanning the two N-terminal Ig domains of the extracellular part of human Tyro3. Its ligand binding specificity profile is identical to the activation profile of the native receptor. The 1.95-A crystal structure suggests a common ligand-binding site in this receptor family located at the interface of the Ig domains and unusually rich in cis-prolines. Furthermore, both in the crystal and in solution we observed the ligand-independent dimerization of the receptor fragment. This homophilic interaction emphasizes previous functional reports, which hinted that in addition to signal transduction, members of this family of receptors might participate in cell adhesion.
Highlights
The receptor Tyro3 together with Axl and Mer form the Axl/Tyro3 family of receptor tyrosine kinases
Functional Characterization of Tyro3-D1D2—A fragment containing the first two Ig domains of the human Tyro3 receptor was tested by analytical size exclusion chromatography for its ability to bind to its physiological ligand Gas6 as well as to the proteins bovine and human protein S
Ligand Recognition and Signaling by the Tyro3 Receptor— All three Receptor tyrosine kinases (RTKs) of the Axl/Tyro3 family are activated by the protein Gas6, which they bind with nanomolar affinity
Summary
Protein Production—The DNA encoding for the extracellular domain of the human Tyro receptor was amplified by PCR from a cDNA pool of human adult brain, kindly obtained from Regina Reszka (Max Delbruck Center, Berlin, Germany). The Tyro3-D1D2 fragment was purified with a HR Q-Sepharose chromatography column (Amersham Biosciences) equilibrated with 20 mM Tris buffer, pH 8, and eluted with a 0 –1 M NaCl gradient. Biochemical Characterization—Analytical size exclusion chromatography of Gas6-LG, human, or bovine protein S alone or in combination with Tyro3-D1D2 was performed at 4 °C with a Superose 6 column (Amersham Biosciences) attached to an Akta Purifier (Amersham Biosciences) equilibrated with a running buffer containing 20 mM Tris, pH 8.0, 150 mM NaCl, and 5 mM CaCl2 at a flow rate of 0.5 ml/min. Radial absorbance data sets at 280 nm and at two different protein concentrations (0.2 and 0.75 mg/ml) were collected at four different angular speeds (15,000, 25,000, 35,000, and 42,000 rpm) for Tyro3-D1D2 wild type and at two different speeds (15,000 and 25,000 rpm) for mutant I59R.
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