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. 2024 Apr 17;25(8):4404. doi: 10.3390/ijms25084404

Figure 2.

Figure 2

Mertk is sequentially cleaved by ADAM17 and γ-secretase. (A) PMA-differentiated THP-1s (100 ng/mL) express Mertk and the necessary cleavage components, ADAM17 and γ-secretase (nicastrin, PEN2, Pres1, and Pres2; APH-1 not shown) Mertk expression confirmed by both N-terminal-specific (N-Mertk) and C-terminal-specific (C-Mertk) antibodies. Note the expression of an unidentified 90 kDa band. An increase in the pro-form (130 kDa) of ADAM17 is observed within 6 h of PMA treatment, though the mature form (100 kDa) remains constant. (B) PMA-differentiated THP-1s (for 72 h) were treated for 4 h with either 3 µM of GW280264X (GW; ADAM17 inhibitor), 5 µM of DAPT (γ-secretase inhibitor), or 10 µM of MG132 (proteasomal inhibitor). Both DAPT and MG132 treatment results in a band at approximately 50 kDa, with the MG132 band being slightly lower, suggesting the stabilization of mb-cMertk and cyto-cMertk. The slight difference in molecular weight is attributed to the loss of the transmembrane domain. The addition of GW280264X decreases the presence of both fragments due to the sequential nature of Mertk cleavage. In addition, GW280264X slightly increases full-length Mertk expression, demonstrating the enhanced stability of Mertk. Note the presence of the unidentified 90 kDa band. (C) THP-1 cells differentiated with 100 ng/mL of PMA for 72 h were treated with 5 µM of DAPT over the course of 4 h in serum-free media. Stabilization and the subsequent accumulation of the mb-cMertk fragment are observed robustly after 4 h. Lane 5 represents differentiated THP-1 cells after 4 h in untreated serum-free media. (D) Mertk is a substrate of ADAM17 and γ-secretase cleavage. Cleavage occurs sequentially. 1. The ADAM17 cleavage site is identified. 2. Active ADAM17 cleaves Mertk, releasing a soluble ectodomain (sMertk) and leaving a membrane-bound C-terminal fragment (mb-cMertk). 3. After ADAM17 cleavage, the γ-secretase complex identifies the remaining membrane-bound fragment of Mertk. 4. γ-secretase cleavage is initiated and releases a C-terminal fragment into the cytosol (cyto-cMertk).