Determinants of the membrane orientation of a calcium signaling enzyme CD38

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Highlights

  • CD38 can be expressed in two opposite membrane orientations.

  • An antibody selective for type III CD38 is produced.

  • Arginine mutation in the N-tail of CD38 to aspartate converts type II to III.

  • Serine mutation to aspartate enhances type III CD38 expression.

  • Cysteine mutation to serine enhances type II CD38 expression.

Abstract

CD38 catalyzes the synthesis of two structurally distinct messengers for Ca2 +-mobilization, cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), from cytosolic substrates, NAD and NADP, respectively. CD38 is generally thought of as a type II membrane protein with its catalytic site facing outside. We recently showed that CD38 exists, instead, in two opposite membrane orientations. The determinant for the membrane topology is unknown. Here, specific antibodies against type III CD38 were designed and produced. We show that mutating the positively charged residues in the N-terminal tail of CD38 converted its orientation to type III, with the catalytic domain facing the cytosol and it was fully active in producing intracellular cADPR. Changing the serine residues to aspartate, which is functionally equivalent to phosphorylation, had a similar effect. The mutated CD38 was expressed intracellularly and was un-glycosylated. The membrane topology could also be modulated by changing the highly conserved di-cysteine. The results indicate that the net charge of the N-terminal segment is important in determining the membrane topology of CD38 and that the type III orientation can be a functional form of CD38 for Ca2 +-signaling. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Keywords

CD38
Cyclic ADP-ribose
Membrane topology
ADP-ribosyl cyclase
Calcium signaling

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This article is part of a Special Issue entitled: 13th European Symposium on Calcium.