A novel mechanism for coupling cellular intermediary metabolism to cytosolic Ca2+ signaling via CD38/ADP-ribosyl cyclase, a putative intracellular NAD+ sensor.
CD38 is an ectocyclase that converts NAD+ to the Ca2+-releasing second messenger cyclic ADP-ribose (cADPr). Here we report that in addition to CD38 ecto-catalysis, intracellularly expressed CD38 may catalyze NAD+-->cADPr conversion to cause cytosolic Ca2+ release. High levels of CD38 were found in the plasma membranes, endoplasmic reticulum, and nuclear membranes of osteoblastic MC3T3-E1 cells. More important, intracellular CD38 was colocalized with target ryanodine receptors. The cyclase also converted a NAD+ surrogate, NGD+, to its fluorescent product, cGDPr (Km approximately 5.13 microM). NAD+ also triggered a cytosolic Ca2+ signal. Similar results were obtained with NIH3T3 cells, which overexpressed a CD38-EGFP fusion protein. The Delta(-49)-CD38-EGFP mutant with a deleted amino-terminal tail and transmembrane domain appeared mainly in the mitochondria with an expected loss of its membrane localization, but the NAD+-induced cytosolic Ca2+ signal was preserved. Likewise, Ca2+ release persisted in cells transfected with the Myr-Delta(-49)-CD38-EGFP or Delta(-49)-CD38-EGFP-Fan mutants, both directed to the plasma membrane but in an opposite topology to the full-length CD38-EGFP. Finally, ryanodine inhibited Ca2+ signaling, indicating the downstream activation of ryanodine receptors by cADPr. We conclude that intracellularly expressed CD38 might link cellular NAD+ production to cytosolic Ca2+ signaling.
Sun
Li
L
Mount Sinai Bone Program, Department of Medicine, and Bronx Veterans Affairs Geriatrics Research Education and Clinical Center (GRECC), New York, New York 10029, USA.
Adebanjo
Olugbenga A
OA
Koval
Anatoliy
A
Anandatheerthavarada
Hindupur K
HK
Iqbal
Jameel
J
Wu
Xing Y
XY
Moonga
Baljit S
BS
Wu
Xue B
XB
Biswas
Gopa
G
Bevis
Peter J R
PJ
Kumegawa
Masoyoshi
M
Epstein
Solomon
S
Huang
Christopher L-H
CL
Avadhani
Narayan G
NG
Abe
Etsuko
E
Zaidi
Mone
M
D016475:3T3 Cells; D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D020013:Calcium Signaling; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000201:enzymology; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000032:analysis; D007425:Intracellular Membranes / Q000201:enzymology; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D008562:Membrane Glycoproteins; D051379:Mice; D018613:Microscopy, Confocal; D008954:Models, Biological; D009154:Mutation; D009243:NAD / Q000494:pharmacology*; D009244:NAD+ Nucleosidase / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D010006:Osteoblasts / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000032:analysis; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis
D015703:Antigens, CD; D000943:Antigens, Differentiation; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D009243:NAD; D049452:Green Fluorescent Proteins; D036541:ADP-ribosyl Cyclase; C498155:Cd38 protein, mouse; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1