Signal Transduction Via the CD38/NAD⁺ Glycohydrolase

Abstract

Abstract The human cell surface CD38 molecule is a 46-kDa type-II transmembrane glycoprotein with a short N-terminal cytoplamic domain and a long Cys-rich C-terminal extracellular one. We previously demonstrated that an ecto-form NAD+ glycohydrolase (NADase) activity induced by all -trans retinoic acid in HL-60 cells is due to the extracellular domain of CD38. In the present study, we investigated a possible signal transduction mediated through CD38 in the retinoic acid-differentiated HL-60 cells with anti-CD38 monoclonal antibodies (mAbs). The addition of selected anti-CD38 mAbs to the cells induced rapid tyrosine phosphorylation of the cellular proteins with the molecular weights of 120,000, 87,000 and 77,000; the phosphorylated 120-kDa protein was identified as the c -cbl proto-oncogene product, p120^{c -cbl.} Furthermore, the phosphorylated p120^{c -cbl} associated with the 85-kDa subunit of phosphatidylinositol 3-kinase. To determine the relationship between the amino acid sequence responsible for the NADase activity and epitopes recognized by the stimulatory mAbs, we produced its carboxy-terminal deletion mutants in COS-7 cells. The mutants with less than 15 amino acids deleted from the carboxyl terminus of the 300-amino acid wild-type molecule still maintained NADase activity, but those with more than 27 amino acids deleted did not. Introduction of site-directed mutation of a cysteine residue (Cys²⁷⁵), located in the 273-285 sequence, completely abolished the NADase activity. These CD38 mutants were also used for an epitope mapping of anti CD38 mAbs. All the epitopes recognized by the mAbs inducing the tyrosine phosphorylation were mapped on the same Cys²⁷⁵-containing sequence of 273-285. Thus, the discrete carboxy-terminal sequence not only plays a key role in its ecto-NADase activity, but also contains the epitopes of the agonistic anti-CD38 mAbs for the transmembrane signaling. We also found that the agonistic mAbs markedly potentiate superoxide generation induced by the stimulation of G protein-coupled chemotactic receptors. Our results suggested that the stimulation of CD38 might generate an accessory signal(s) to enhance the G proteinmediated signaling, probably though the protein-tyrosine phosphorylation.