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 cycli c ADP-ribose (cADPR). cADPR is a universal second messenger that releases c alcium from intracellular stores. Since cADPR is generated by CD38 at the o uter surface of many cells, where it acts intracellularly, increasing atten tion is paid to addressing this topological paradox. Recently, we demonstra ted that CD38 is a catalytically active, unidirectional transmembrane trans porter of cADPR, which then reaches its receptor-operated intracellular cal cium stores. Moreover, CD38 was reported to undergo a selective and extensi ve internalization through non clathrin-coated endocytotic vesicles upon in cubating CD38(+) cells with either NAD(+) or thiol compounds: these endocyt otic vesicles can convert cytosolic NAD into cADPR despite an asymmetric un favorable orientation that makes the active site of CD38 intravesicular. He re we demonstrate that the cADPR-generating activity of the endocytotic ves icles results in remarkable and sustained increases of intracellular free c alcium concentration in different cells exposed to either NAD(+), or GSH, o r N-acetylcysteine. This effect of CD38-internalizing Ligands on intracellu lar calcium levels was found to involve a two-step mechanism: 1) influx of cytosolic NAD(+) into the endocytotic vesicles, mediated by a hitherto unre cognized dinucleotide transport system that is saturable, bidirectional, in hibitable by 8-N-3-NAD(+), and characterized by poor dinucleotide specifici ty, low affinity, and high efficiency; 2) intravesicular CD38-catalyzed con version of NAD+ to cADPR, followed by out-pumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigargin-sensit ive stores. This unknown intracellular trafficking of NAD(+) and cADPR base d on two distinctive and specific transmembrane carriers for either nucleot ide can affect the intracellular calcium homeostasis in CD38(+) cells.