A novel site contributing to growth-arrest-specific gene 6 binding to its receptors as revealed by a human monoclonal antibody

Abstract

Gas6 (growth-arrest-specific gene 6) is a vitamin K-dependent protein known to activate the Axl family of receptor tyrosine kinases. It is an important regulator of thrombosis and many other biological functions. The C-terminus of Gas6 binds to receptors and consists of two laminin-like globular domains LG1 and LG2. It has been reported that a Ca2+-binding site at the junction of LG1 and LG2 domains and a hydrophobic patch at the LG2 domain are important for receptor binding [Sasaki, Knyazev, Cheburkin, Gohring, Tisi, Ullrich, Timpl and Hohenester (2002) J. Biol. Chem. 277, 44164-44170]. In the present study, we developed a neutralizing human monoclonal antibody, named CNTO300, for Gas6. The antibody was generated by immunization of human IgG-expressing transgenic mice with recombinant human Gas6 protein and the anti-Gas6 IgG sequences were rescued from an unstable hybridoma clone. Binding of Gas6 to its receptors was partially inhibited by the CNTO300 antibody in a dose-dependent manner. To characterize further the interaction between Gas6 and this antibody, the binding kinetics of CNTO300 for recombinant Gas6 were compared with independently expressed LG1 and LG2. The CNTO300 antibody showed comparable binding affinity, yet different dependence on Ca2+, to Gas6 and LG1. No binding to LG2 was detected. In the presence of EDTA, binding of the antibody to Gas6 was disrupted, but no significant effect of EDTA on LG1 binding was evident. Further epitope mapping identified a Gas6 peptide sequence recognized by the CNTO300 antibody. This peptide sequence was found to be located at the LG1 domain distant from the Ca2+-binding site and the hydrophobic patch. Co-interaction of Gas6 with its receptor and CNTO300 antibody was detected by BIAcore analysis, suggesting a second receptor-binding site on the LG1 domain. This hypothesis was further supported by direct binding of Gas6 receptors to an independently expressed LG1 domain. Our results revealed, for the first time, a second binding site for Gas6-receptor interaction.

TAM Receptor Tyrosine Kinases: Biologic Functions, Signaling, and Potential Therapeutic Targeting in Human Cancer

“…The authors suggest that the hydrophobic residues may still affect ligand/receptor binding indirectly. Direct binding between Axl and the LG1 domain of Gas6 was first demonstrated by Fisher et al (2005). An anti-Gas6 monoclonal antibody diminished Gas6 binding to Axl and the antibody binding epitope was mapped to residues 403-414 within the J-K loop of LG1.…”

“…A large number of additional studies have investigated the interspecies affinities of Gas6 and Protein S for TAM receptors (reviewed in Hafizi and Dahlback, 2006b). Studies which evaluated the K d values for human Gas6 binding to each of the three human TAM receptors in vitro suggest that Axl and Tyro-3 bind Gas6 with roughly equal affinity while Mer affinity for Gas6 is 3-10-fold lower (Chen et al, 1997;Fisher et al, 2005).…”

“…Thus, a 2:2 ligand/receptor complex is formed. Further evidence to support two Gas6/Axl binding sites was provided by receptor binding studies, which demonstrated that Gas6 can simultaneously bind Axl-Fc and a neutralizing Gas6 antibody (Fisher et al, 2005). Receptor binding studies of an N-terminal fragment of Tyro-3 demonstrated that one site of Gas6/Tyro-3 receptor interaction is localized to the two Ig domains.…”

Molecular Pathways: MERTK Signaling in Cancer

“…The first ligand described was Gas6, identified by purification of Axl-activating conditioned media. Gas6 binds MERTK, but with 3-to 10-fold lower affinity (35,36). Subsequently, protein S was identified as a MERTK ligand that did not activate Axl (37,38).…”

Plasma concentrations of growth arrest specific protein 6 and the soluble form of its tyrosine kinase receptor Axl as markers of large abdominal aortic aneurysms

“…However, expression is low in the liver, explaining the low concentration of Gas6 (approximately 0.2 nM) in plasma [24][25][26] . The affinity between Gas6 and the Axl receptor is in the subnanomolar range suggesting that Gas6 and sAxl in plasma may form a complex 27,28 .…”