Figure 4.

Effect of MERTK deletion mutations on efferocytosis and cell growth. A, Schematics of MERTK mutants. At the top, the structure of MERTK is shown schematically. Immunoglobulin-like domains (IG1 and IG2), fibronectin type III–like domains (FN1 and FN2), the transmembrane region (TM), and the tyrosine kinase domain are boxed, with the amino acid positions indicated at the borders. Amino acid positions 530 and 560 indicate the exon–intron junction used to construct the ΔN mutant. Three tyrosine residues at positions 544, 867, and 924, and a lysine residue at position 614 are indicated. In the K614M mutant, the lysine residue at 614 was mutated to methionine. In the ΔN and ΔC mutants, the amino acid region from 531 to 559 proximal to the transmembrane region and the amino acid region from 853 to 994 in the C terminus were deleted, respectively. In the ΔNΔC mutant, both the N-terminal juxtamembrane and the C-terminal regions were deleted. B, effect of MERTK mutations on efferocytosis. TKO cell transformants (6 × 10⁴ cells) expressing WT or the indicated mutant MERTK together with TIM4 were incubated with 6 × 10⁵ pHrodo-apoptotic thymocytes at 37 °C for 60 min and then analyzed by flow cytometry for pHrodo positivity. The experiments were carried out in triplicate, and the average percentage of pHrodo-positive cells was plotted as efferocytosis with S.D. (error bars). **, p < 0.01; Student's t test. C, effect of various mutations of MERTK on apoptotic cell–promoted cell growth. Ba/F3/TIM4 cell transformants (2.5 × 10⁵ cells in 0.5 ml) expressing WT or the indicated mutant MERTK were incubated for 24, 48, or 72 h in the absence (left) or presence (right) of 2.5 × 10⁶ apoptotic thymocytes, and viable Ba/F3 cells were counted. The experiments were performed in triplicate, and average values were plotted with S.D. (error bars). The values were statistically analyzed by Student's t test against that obtained with WT MERTK.