Abstract ARTC2.2 is a toxin-related, GPI-anchored ADP-ribosyltransferase expressed by murine T cells. In response to NAD+ released from damaged cells during inflammation, ARTC2.2 ADP-ribosylates and thereby gates the P2X7 ion channel. This induces ectodomain shedding of metalloprotease-sensitive cell surface proteins. In this study, we show that ARTC2.2 itself is a target for P2X7-triggered ectodomain shedding. We identify the metalloprotease cleavage site 3 aa upstream of the predicted GPI anchor attachment site of ARTC2.2. Intravenous injection of NAD+ increased the level of enzymatically active ARTC2.2 in serum, indicating that this mechanism is operative also under inflammatory conditions in vivo. Radio–ADP-ribosylation assays reveal that shedding refocuses the target specificity of ARTC2.2 from membrane proteins to secretory proteins. Our results uncover nucleotide-induced membrane-proximal proteolysis as a regulatory mechanism to control the substrate specificity of ARTC2.2.