Ligand induced internalization of CD38 results in intracellular Ca2+ mobilization. role of NAD+ transport across cell membranes

CD38, a transmembrane glycoprotein widely expressed in vertebrate cells, is a bifunctional ectoenzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose (cADPR). cADPR is a universal second messenger that releases calcium from intracellular stores. Since cADPR is generated by CD38 at the outer surface of many cells, where it acts intracellularly, increasing attention is paid to addressing this topological paradox. Recently, we demonstrated that CD38 is a catalytically active, unidirectional transmembrane transporter of cADPR, which then reaches its receptor-operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin-coated endocytotic vesicles upon incubating CD381 cells with either NAD1 or thiol compounds: these endocytotic vesicles can convert cytosolic NAD into cADPR despite an asymmetric unfavorable orientation that makes the active site of CD38 intravesicular. Here we demonstrate that the cADPR-generating activity of the endocytotic vesicles results in remarkable and sustained increases of intracellular free calcium concentration in different cells exposed to either NAD1, or GSH, or N-acetylcysteine. This effect of CD38-internalizing ligands on intracellular calcium levels was found to involve a two-step mechanism: 1) influx of cytosolic NAD1 into the endocytotic vesicles, mediated by a hitherto unrecognized dinucleotide transport system that is saturable, bidirectional, inhibitable by 8-N3-NAD1, and characterized by poor dinucleotide specificity, low affinity, and high efficiency; 2) intravesicular CD38-catalyzed conversion of NAD1 to cADPR, followed by outpumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigarginsensitive stores. This unknown intracellular trafficking of NAD1 and cADPR based on two distinctive and specific transmembrane carriers for either nucleotide can affect the intracellular calcium homeostasis in CD381 cells.—Zocchi, E., Usai, C., Guida, L., Franco, L., Bruzzone, S., Passalacqua, M., De Flora, A. Ligand-induced internalization of CD38 results in intracellular Ca21 mobilization: role of NAD1 transport across cell membranes. FASEB J. 13, 273–283 (1999)