NADP+-Dependent internalization of recombinant CD38 in CHO cells

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Signaling

Background:

• CD38 is a type II transmembrane glycoprotein catalyzing cyclic ADP-ribose (cADPR) synthesis from NAD+.
• cADPR functions as a second messenger regulating intracellular Ca2+-induced Ca2+-release (CICR).
• Previous studies indicated CD38 internalization upon exposure to external NAD+ in Namalwa B cells.

Purpose of the Study:

• To investigate whether NADP+, a substrate analog of NAD+, can induce CD38 internalization.
• To characterize the enzymatic activity and molecular state of internalized CD38.

Main Methods:

• Recombinant rat CD38 was expressed in Chinese hamster ovary (CHO) cells.
• Cells were treated with NADP+ and analyzed for CD38 surface immunofluorescence and ADP-ribosyl cyclase activity.
• Confocal microscopy was used to visualize intracellular CD38 clustering.
• Internalized CD38 was purified and analyzed by immunoblot and enzymatic assays.

Main Results:

• NADP+ treatment of CHO cells expressing CD38 led to decreased ADP-ribosyl cyclase activity and reduced cell surface CD38 immunofluorescence.
• Confocal microscopy revealed intracellular clustering of CD38 molecules after NADP+ exposure.
• Purified internalized CD38 retained both ADP-ribosyl cyclase and cADPR hydrolase activities.
• Immunoblot analysis showed internalized CD38 exists as a 46 kDa monomer under reducing conditions.

Conclusions:

• NADP+ efficiently induces the internalization of CD38 in CHO cells.
• This NADP+-mediated CD38 internalization may play a significant role in intracellular cADPR production.
• The regulation of cADPR levels through CD38 internalization could be critical for modulating CICR.