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

Abstract

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 CD38(+) cells with either NAD+ 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 NAD+, 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 NAD+ into the endocytotic vesicles, mediated by a hitherto unrecognized dinucleotide transport system that is saturable, bidirectional, inhibitable by 8-N3-NAD+, and characterized by poor dinucleotide specificity, low affinity, and high efficiency; 2) intravesicular CD38-catalyzed conversion of NAD+ to cADPR, followed by outpumping of the cyclic nucleotide into the cytosol and subsequent release of calcium from thapsigargin-sensitive stores. This unknown intracellular trafficking of NAD+ and cADPR based on two distinctive and specific transmembrane carriers for either nucleotide can affect the intracellular calcium homeostasis in CD38(+) cells.

Kinetic competence of the cADP-ribose‒CD38 complex as an intermediate in the CD38/NAD+ glycohydrolase-catalysed reactions: implication for CD38 signalling

“…; and (2) how does CD38, whose short intracellular domain lacks consensus sequences, cross-talk with its partners such as BCR? Related to the first question, the groups of De Flora [8] and Lee [9], based on experimental and structural data, have suggested that CD38 could function as a channel\transporter for catalytically formed cADPR leading to increased intracellular Ca# + levels. However, Lund et al [10] have shown that CD38 signalling in B-cells was independent from its catalytic activity.…”

A Self-restricted CD38-connexin 43 Cross-talk Affects NAD+ and Cyclic ADP-ribose Metabolism and Regulates Intracellular Calcium in 3T3 Fibroblasts

“…These mediate the export of "de novo" synthesized CD38 to the plasma membrane (16) and also the opposite process of CD38 endocytosis that is observed upon incubating several cells types with specific ligands, e.g. 18). In both cases, the active site of CD38 is intravesicular and therefore unavailable to cytosolic NAD ϩ .…”

“…This "topological paradox" of the CD38/cADPR system appeared to be challenging, in view of the powerful calcium releasing activity of cADPR and of the remarkably increased cytosolic calcium ([Ca 2ϩ ] i ) levels (and consequent triggering of calcium-stimulated cell functions) that are observed during both de novo expression of CD38 (16) and its ligand-induced internalization (18). Both findings clearly indicated, in the absence of obvious underlying mechanisms, that intracellularly localized CD38 (e.g.…”

Structure, function, evolution and application of bacterial Pnu-type vitamin transporters

“…The concentrations of all potential V factors (a collective term for NAD, NMN, and NR) in human body fluids are not known precisely. Although in certain microenvironments, NAD concentrations in the low micromolar range can be reached, an average NAD serum concentration of 50-60 nm has been reported (Zocchi et al, 1999). The NAD concentrations in extracellular body fluids of pigs are between 0.2 and 1 μm (O' Reilly and Niven, 2003).…”