Requirement of Gamma-Carboxyglutamic Acid Modification and Phosphatidylserine Binding for the Activation of Tyro3, Axl, and Mertk Receptors by Growth Arrest-Specific 6.

Ke Geng,Pranela Rameshwar,Raymond B Birge,Sergei V Kotenko,Oleta Sandiford,Stanley G Kimani,Sushil Kumar,Vladyslav Kholodovych,Canan Kasikara,Kensaku Mizuno

doi:10.3389/fimmu.2017.01521

Ke Geng, Pranela Rameshwar + Show 8 more

Open Access

https://doi.org/10.3389/fimmu.2017.01521

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Abstract

The Tyro3, Axl, and Mertk (TAM) receptors are homologous type I receptor tyrosine kinases that have critical functions in the clearance of apoptotic cells in multicellular organisms. TAMs are activated by their endogenous ligands, growth arrest-specific 6 (Gas6), and protein S (Pros1), that function as bridging molecules between externalized phosphatidylserine (PS) on apoptotic cells and the TAM ectodomains. However, the molecular mechanisms by which Gas6/Pros1 promote TAM activation remains elusive. Using TAM/IFNγR1 reporter cell lines to monitor functional TAM activity, we found that Gas6 activity was exquisitely dependent on vitamin K-mediated γ-carboxylation, whereby replacing vitamin K with anticoagulant warfarin, or by substituting glutamic acid residues involved in PS binding, completely abrogated Gas6 activity as a TAM ligand. Furthermore, using domain and point mutagenesis, Gas6 activity also required both an intact Gla domain and intact EGF-like domains, suggesting these domains function cooperatively in order to achieve TAM activation. Despite the requirement of γ-carboxylation and the functional Gla domain, non-γ-carboxylated Gas6 and Gla deletion/EGF-like domain deletion mutants still retained their ability to bind TAMs and acted as blocking decoy ligands. Finally, we found that distinct sources of PS-positive cells/vesicles (including apoptotic cells, calcium-induced stressed cells, and exosomes) bound Gas6 and acted as cell-derived or exosome-derived ligands to activate TAMs. Taken together, our findings indicate that PS is indispensable for TAM activation by Gas6, and by inference, provides new perspectives on how PS, regulates TAM receptors and efferocytosis.

Highlights

Tyro3, Axl, and Mertk (TAMs) comprise homologous type I transmembrane receptor tyrosine kinases that are implicated as both oncogenic kinases that drive transformation and tumorigenicity of cancer cells, as well as receptors for the clearance of apoptotic cells and regulate innate immunity and dampen inflammation [1,2,3]
The expected molecular weight of these mutant growth arrest-specific 6 (Gas6) proteins is ΔG, 70 kDa; ΔE, 59 kDa; and ΔGE, 49 kDa. These plasmids were transfected into the HEK293 cells with the same method to produce mutant Gas6-Containing Media (Gas6-CM). 18 h after transfection, the cells were washed twice with PBS and the growth media was replaced with serum-free DMEM media supplemented with 4.4 μM vitamin K (Hospira) or 2 μM warfarin (Sigma) to promote, or abrogate, γ-carboxylation, respectively
We developed TAM/IFNγR1 chimeric receptor CHO reporter cell lines, whereby the extracellular and trans-membrane domains of each human and mouse TAM receptor were fused in frame to the intracellular domain of IFNγR1 to investigate the molecular mechanisms of TAM activation by endogenous ligands Gas6 and Pros1 [10]

Summary

Introduction

Axl, and Mertk (TAMs) comprise homologous type I transmembrane receptor tyrosine kinases that are implicated as both oncogenic kinases that drive transformation and tumorigenicity of cancer cells, as well as receptors for the clearance of apoptotic cells and regulate innate immunity and dampen inflammation [1,2,3]. Despite a high degree of homology between TAMs and their ligands, the ligand-inducible TAM activation follows a biochemical hierarchy whereby (i) Axl is preferentially activated by Gas with 100–1,000× higher binding affinity over Mertk and Tyro (kd in the nM range), (ii) Tyro is preferentially activated by Pros, and (iii) Merk displays lower sensitivity to both ligand proteins (in the μM range) [10,11,12] Both Tyro and Mertk become hyperactivated in the presence of phosphatidylserine (PS)-positive liposomes and apoptotic cells, implying that Mertk and Tyro may act as “PS sensors” for externalized PS on apoptotic cells and in the tumor microenvironment [13, 14]. TAMs are expressed on retinal pigmented epithelial cells (RPEs), Sertoli cells, resident brain microglia where they are involved in the uptake and clearance of rod outer segments, immature spermatogonium, and apoptotic neurons/pruned synapses, respectively, processes that appear to depend on externalized PS [19,20,21,22,23]

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