RESUMEN
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.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Proteínas de la Membrana/metabolismo , NAD/metabolismo , Linfocitos T/enzimología , Proteínas ADAM/metabolismo , Proteína ADAM17 , ADP Ribosa Transferasas/sangre , ADP Ribosa Transferasas/genética , Adenosina Difosfato Ribosa/metabolismo , Animales , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Citometría de Flujo , Glicosilfosfatidilinositoles/metabolismo , Células HEK293 , Humanos , Selectina L/genética , Selectina L/metabolismo , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , NAD/farmacología , Proteolisis/efectos de los fármacos , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Especificidad por Sustrato , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T/metabolismoRESUMEN
Control of immunologic tolerance and homeostasis rely on Foxp3(+)CD4(+)CD25(+) regulatory T cells (Tregs) that constitutively express the high affinity receptor for Interleukin-2, CD25. Tregs proliferate in response to injections of IL-2/anti-IL-2 antibody complexes or low doses of IL-2. However, little is known about endogenous mechanisms that regulate the sensitivity of CD25 to signaling by IL-2. Here we demonstrate that CD25 is ADP-ribosylated at Arg35 in the IL-2 binding site by ecto-ADP-ribosyltransferase ARTC2.2, a toxin-related GPI-anchored ecto-enzyme. ADP-ribosylation inhibits binding of IL-2 by CD25, IL-2- induced phosphorylation of STAT5, and IL-2-dependent cell proliferation. Our study elucidates an as-yet-unrecognized mechanism to tune IL-2 signaling. This newly found mechanism might thwart Tregs at sites of inflammation and thereby permit a more potent response of activated effector T cells.