The transferrin receptor CD71 regulates type II CD38, revealing tight topological compartmentalization of intracellular cyclic ADP-ribose production.

The CD38 molecule (CD38) catalyzes biogenesis of the calcium-mobilizing messenger cyclic ADP-ribose (cADPR). CD38 has dual membrane orientations, and type III CD38, with its catalytic domain facing the cytosol, has low abundance but is efficient in cyclizing cytosolic NAD to produce cADPR. The role of cell surface type II CD38 in cellular cADPR production is unknown. Here we modulated type II CD38 expression and assessed the effects of this modulation on cADPR levels. We developed a photoactivatable cross-linking probe based on a CD38 nanobody, and, combining it with MS analysis, we discovered that cell surface CD38 interacts with CD71. CD71 knockdown increased CD38 levels, and CD38 knockout reciprocally increased CD71, and both could be cocapped and coimmunoprecipitated. We constructed a chimera comprising the N-terminal segment of CD71 and a CD38 nanobody to mimic CD71's ligand property. Overexpression of this chimera induced a dramatically large decrease in CD38 via lysosomes. Remarkably, cellular cADPR levels did not decrease correspondingly. Bafilomycin-mediated blockade of lysosomal degradation greatly elevated active type II CD38 by trapping it in the lysosomes but also did not increase cADPR levels. Retention of type II CD38 in the endoplasmic reticulum (ER) by expressing an ER construct that prevented its transport to the cell surface likewise did not change cADPR levels. These results provide first and direct evidence that cADPR biogenesis occurs in the cytosol and is catalyzed mainly by type III CD38 and that type II CD38, compartmentalized in the ER or lysosomes or on the cell surface, contributes only minimally to cADPR biogenesis.

Resolving the topological enigma in Ca2+ signaling by cyclic ADP-ribose and NAADP.

Cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) are two structurally distinct messengers that mobilize the endoplasmic and endolysosomal Ca2+ stores, respectively. Both are synthesized by the CD38 molecule (CD38), which has long been thought to be a type II membrane protein whose catalytic domain, intriguingly, faces to the outside of the cell. Accordingly, for more than 20 years, it has remained unresolved how CD38 can use cytosolic substrates such as NAD and NADP to produce messengers that target intracellular Ca2+ stores. The discovery of type III CD38, whose catalytic domain faces the cytosol, has now begun to clarify this topological conundrum. This article reviews the ideas and clues leading to the discovery of the type III CD38; highlights an innovative approach for uncovering its natural existence; and discusses the regulators of its activity, folding, and degradation. We also review the compartmentalization of cADPR and NAADP biogenesis. We further discuss the possible mechanisms that promote type III CD38 expression and appraise a proposal of a Ca2+-signaling mechanism based on substrate limitation and product translocation. The surprising finding of another enzyme that produces cADPR and NAADP, sterile α and TIR motif-containing 1 (SARM1), is described. SARM1 regulates axonal degeneration and has no sequence similarity with CD38 but can catalyze the same set of multireactions and has the same cytosolic orientation as the type III CD38. The intriguing finding that SARM1 is activated by nicotinamide mononucleotide to produce cADPR and NAADP suggests that it may function as a regulated Ca2+-signaling enzyme like CD38.

A cytosolic chaperone complex controls folding and degradation of type III CD38.

Cluster of differentiation 38 (CD38) is the best-studied enzyme catalyzing the synthesis of the Ca2+ messenger cyclic ADP-ribose. It is a single-pass transmembrane protein, but possesses dual orientations. We have documented the natural existence of type III CD38 in cells and shown that it is regulated by a cytosolic activator, calcium- and integrin-binding 1 (CIB1). However, how type III CD38 can be folded correctly in the reductive cytosol has not been addressed. Using the yeast two-hybrid technique with CD38's catalytic domain (sCD38) as bait, here we identified a chaperone, Hsp70-interacting protein (Hip), that specifically interacts with both the type III CD38 and sCD38. Immunoprecipitation coupled with MS identified a chaperone complex associated specifically with sCD38. Pharmacological and siRNA-mediated knockdown of Hsp90 chaperones decreased the expression levels of both sCD38 and type III CD38, suggesting that these chaperones facilitate their folding. Moreover, knockdown of Hsc70 or DNAJA2 increased the levels of both CD38 types, consistent with the roles of these proteins in mediating CD38 degradation. Notably, Hip knockdown decreased type III CD38 substantially, but only marginally affected sCD38, indicating that Hip was selective for the former. More remarkably, DNAJA1 knockdown decreased sCD38 but increased type III CD38 levels. Mechanistically, we show that Hsc70 mediates lysosomal degradation of type III CD38, requiring the lysosomal receptor Lamp2A and the C19-motif in the C terminus of CD38. Our results indicate that folding and degradation of type III CD38 is effectively controlled in cells, providing further strong support of its physiological relevance.

The Plant Hormone Abscisic Acid Is a Prosurvival Factor in Human and Murine Megakaryocytes.

Abscisic acid (ABA) is a phytohormone involved in pivotal physiological functions in higher plants. Recently, ABA has been proven to be also secreted and active in mammals, where it stimulates the activity of innate immune cells, mesenchymal and hematopoietic stem cells, and insulin-releasing pancreatic ß cells through a signaling pathway involving the second messenger cyclic ADP-ribose (cADPR). In addition to behaving like an animal hormone, ABA also holds promise as a nutraceutical plant-derived compound in humans. Many biological functions of ABA in mammals are mediated by its binding to the LANCL-2 receptor protein. A putative binding of ABA to GRP78, a key regulator of endoplasmic reticulum stress, has also been proposed. Here we investigated the role of exogenous ABA in modulating thrombopoiesis, the process of platelet generation. Our results demonstrate that expression of both LANCL-2 and GRP78 is up-regulated during hematopoietic stem cell differentiation into mature megakaryocytes (Mks). Functional ABA receptors exist in mature Mks because ABA induces an intracellular Ca2+ increase ([Ca2+] i ) through PKA activation and subsequent cADPR generation. In vitro exposure of human or murine hematopoietic progenitor cells to 10 µm ABA does not increase recombinant thrombopoietin (rTpo)-dependent Mk differentiation or platelet release. However, under conditions of cell stress induced by rTpo and serum deprivation, ABA stimulates, in a PKA- and cADPR-dependent fashion, the mitogen-activated kinase ERK 1/2, resulting in the modulation of lymphoma 2 (Bcl-2) family members, increased Mk survival, and higher rates of platelet production. In conclusion, we demonstrate that ABA is a prosurvival factor for Mks in a Tpo-independent manner.

Synthesis of the Ca2+-mobilizing messengers NAADP and cADPR by intracellular CD38 enzyme in the mouse heart: Role in ß-adrenoceptor signaling.

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR) are Ca2+-mobilizing messengers important for modulating cardiac excitation-contraction coupling and pathophysiology. CD38, which belongs to the ADP-ribosyl cyclase family, catalyzes synthesis of both NAADP and cADPR in vitro However, it remains unclear whether this is the main enzyme for their production under physiological conditions. Here we show that membrane fractions from WT but not CD38-/- mouse hearts supported NAADP and cADPR synthesis. Membrane permeabilization of cardiac myocytes with saponin and/or Triton X-100 increased NAADP synthesis, indicating that intracellular CD38 contributes to NAADP production. The permeabilization also permitted immunostaining of CD38, with a striated pattern in WT myocytes, whereas CD38-/- myocytes and nonpermeabilized WT myocytes showed little or no staining, without striation. A component of ß-adrenoreceptor signaling in the heart involves NAADP and lysosomes. Accordingly, in the presence of isoproterenol, Ca2+ transients and contraction amplitudes were smaller in CD38-/- myocytes than in the WT. In addition, suppressing lysosomal function with bafilomycin A1 reduced the isoproterenol-induced increase in Ca2+ transients in cardiac myocytes from WT but not CD38-/- mice. Whole hearts isolated from CD38-/- mice and exposed to isoproterenol showed reduced arrhythmias. SAN4825, an ADP-ribosyl cyclase inhibitor that reduces cADPR and NAADP synthesis in mouse membrane fractions, was shown to bind to CD38 in docking simulations and reduced the isoproterenol-induced arrhythmias in WT hearts. These observations support generation of NAADP and cADPR by intracellular CD38, which contributes to effects of ß-adrenoreceptor stimulation to increase both Ca2+ transients and the tendency to disturb heart rhythm.

Synthesis of cyclic N (1)-pentylinosine phosphate, a new structurally reduced cADPR analogue with calcium-mobilizing activity on PC12 cells.

Cyclic N (1)-pentylinosine monophosphate (cpIMP), a novel simplified inosine derivative of cyclic ADP-ribose (cADPR) in which the N (1)-pentyl chain and the monophosphate group replace the northern ribose and the pyrophosphate moieties, respectively, was synthesized. The role played by the position of the phosphate group in the key cyclization step, which consists in the formation of a phosphodiester bond, was thoroughly investigated. We have also examined the influence of the phosphate bridge on the ability of cpIMP to mobilize Ca(2+) in PC12 neuronal cells in comparison with the pyrophosphate bridge present in the cyclic N (1)-pentylinosine diphosphate analogue (cpIDP) previously synthesized in our laboratories. The preliminary biological tests indicated that cpIMP and cpIDP induce a rapid increase of intracellular Ca(2+) concentration in PC12 neuronal cells.

Biosynthesis and Function of VIP and Oxytocin: Mechanisms of C-terminal Amidation, Oxytocin Secretion and Transport.

In this review, we provide the status of research on vasoactive intestinal peptide (VIP) and oxytocin, typical C-terminal α-amidated peptide hormones, including their precursor protein structures, processing and C-terminal α-amidation, and the recently identified mechanisms of regulation of oxytocin secretion and its transportation through the blood brain barrier. More than half of neural and endocrine peptides, such as VIP and oxytocin, have the α-amide structure at their C-terminus, which is essential for biological activities. We have studied the synthesis and function of C-terminal α-amidated peptides, including VIP and oxytocin, since the 1980s. Human VIP mRNA encoded not only VIP but also another related C-terminal α-amidated peptide, PHM-27 (peptide having amino-terminal histidine, carboxy-terminal methionine amide, and 27 amino acid residues). The human VIP/PHM-27 gene is composed of 7 exons and regulated synergistically by cyclic AMP and protein kinase C pathways. VIP has an essential role in glycemic control using transgenic mouse technology. The peptide C-terminal α-amidation proceeded through a 2-step mechanism catalyzed by 2 different enzymes encoded in a single mRNA. In the oxytocin secretion from the hypothalamus/the posterior pituitary, the CD38-cyclic ADP-ribose signal system, which was first established in the insulin secretion from pancreatic ß cells of the islets of Langerhans, was found to be essential. A possible mechanism involving RAGE (receptor for advanced glycation end-products) of the oxytocin transportation from the blood stream into the brain through the blood-brain barrier has also been suggested.

CD38, CD157, and RAGE as Molecular Determinants for Social Behavior.

Recent studies provide evidence to support that cluster of differentiation 38 (CD38) and CD157 meaningfully act in the brain as neuroregulators. They primarily affect social behaviors. Social behaviors are impaired in Cd38 and Cd157 knockout mice. Single-nucleotide polymorphisms of the CD38 and CD157/BST1 genes are associated with multiple neurological and psychiatric conditions, including autism spectrum disorder, Parkinson's disease, and schizophrenia. In addition, both antigens are related to infectious and immunoregulational processes. The most important clues to demonstrate how these molecules play a role in the brain are oxytocin (OT) and the OT system. OT is axo-dendritically secreted into the brain from OT-containing neurons and causes activation of OT receptors mainly on hypothalamic neurons. Here, we overview the CD38/CD157-dependent OT release mechanism as the initiation step for social behavior. The receptor for advanced glycation end-products (RAGE) is a newly identified molecule as an OT binding protein and serves as a transporter of OT to the brain, crossing over the blood-brain barrier, resulting in the regulation of brain OT levels. We point out new roles of CD38 and CD157 during neuronal development and aging in relation to nicotinamide adenine dinucleotide+ levels in embryonic and adult nervous systems. Finally, we discuss how CD38, CD157, and RAGE are crucial for social recognition and behavior in daily life.