Receptor Tyrosine Kinases, TYRO3, AXL, and MER, Demonstrate Distinct Patterns and Complex Regulation of Ligand-induced Activation

Wen-I Tsou,Khanh-Quynh N Nguyen,Daniel A Calarese,Scott J Garforth,Anita L Antes,Sergey V Smirnov,Steve C Almo,Raymond B Birge,Sergei V Kotenko

doi:10.1074/jbc.m114.569020

Abstract

TYRO3, AXL, and MER receptors (TAMs) are three homologous type I receptor-tyrosine kinases that are activated by endogenous ligands, protein S (PROS1) and growth arrest-specific gene 6 (GAS6). These ligands can either activate TAMs as soluble factors, or, in turn, opsonize phosphatidylserine (PS) on apoptotic cells (ACs) and serve as bridging molecules between ACs and TAMs. Abnormal expression and activation of TAMs have been implicated in promoting proliferation and survival of cancer cells, as well as in suppressing anti-tumor immunity. Despite the fact that TAM receptors share significant similarity, little is known about the specificity of interaction between TAM receptors and their ligands, particularly in the context of ACs, and about the functional diversity of TAM receptors. To study ligand-mediated activation of TAMs, we generated a series of reporter cell lines expressing chimeric TAM receptors. Using this system, we found that each TAM receptor has a unique pattern of interaction with and activation by GAS6 and PROS1, which is also differentially affected by the presence of ACs, PS-containing lipid vesicles and enveloped virus. We also demonstrated that γ-carboxylation of ligands is essential for the full activation of TAMs and that soluble immunoglobulin-like TAM domains act as specific ligand antagonists. These studies demonstrate that, despite their similarity, TYRO3, AXL, and MER are likely to perform distinct functions in both immunoregulation and the recognition and removal of ACs.

Highlights

The mechanisms by which ligands activate TAM receptors (TYRO3, AXL, and MER) are not well understood
There was no pSTAT1 detected in parental cells expressing red fluorescent protein (RFP) following treatment with either GAS6 or PROS1, whereas hamster IFN-␥ triggered strong STAT1 phosphorylation through the endogenous hamster IFN-␥ receptor, showing the integrity of the JAK-STAT pathway in the cells (Fig. 1F). These results demonstrate that STAT1 activation was mediated by chimeric TAM/␥R1 receptors expressed in reporter cell lines, and STAT1 activation in reporter cell lines can be utilized as a standardized readout to evaluate and compare biological potencies of GAS6 and PROS1 toward specific TAM receptors
Phosphatidylserine (PS) Liposomes and Apoptotic Cells Differentially Modulate Ligand-induced TAM Activation— Because ligand-inducible activation of TAMs depends on ␥-carboxylation, a biochemical modification predicted to mediate binding to anionic lipids such as PS, we investigated the effect of PS liposomes with the respect to the activation of each TAM receptor

Summary

Background

The mechanisms by which ligands activate TAM receptors (TYRO3, AXL, and MER) are not well understood. The intracellular regions of TAMs contain a tyrosine kinase domain that is highly conserved and includes a TAM-specific sequence KW(I/L)A(I/L)ES in the catalytic domain Based on these unique structural features, TYRO3, AXL, and MER form a subfamily of RTKs abbreviated as TAMs. Mice lacking all three TAMs (a TYRO3/AXL/MER triple knock-out) develop normally but demonstrate impaired ability to clear apoptotic cells (ACs) in multiple tissues, elevated levels of pro-inflammatory cytokines such as TNF-␣ and IL-6, and auto-antibody production. Our studies revealed that each TAM receptor demonstrates a unique pattern of activation by GAS6 and PROS1 that is differentially affected by the presence of ACs or PS-containing vesicles These studies suggest that, despite their similarity, TYRO3, AXL, and MER perform distinct functions in the recognition and removal of ACs

EXPERIMENTAL PROCEDURES

RESULTS

Findings

DISCUSSION