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The mammalian GTP-binding protein GSPT, whose carboxy-terminal sequence is homologous to the eukaryotic elongation factor EF1alpha, binds to the polypeptide chain releasing factor eRF1 to function as eRF3 in translation termination. However, the amino-terminal domain of GSPT, which contains a prion-like sequence, is not required for the binding. Instead, the amino-terminal domain is capable of binding to the carboxy-terminal domain of polyadenylate-binding protein (PABP), whose amino terminus is associating with the poly(A) tail of mRNAs, presumably for their stabilization. Interestingly, multimerization of PABP with poly(A), which is ascribed to the action of its carboxy-terminal domain, was completely inhibited by the interaction with the amino-terminal domain of GSPT. This may facilitate shortening of the poly(A) tail of mRNAs by an RNase. Thus, GSPT/eRF3 appears to function not only as a stimulator of eRF1 in the translation termination but also as an initiator of the mRNA degradation machinery. Further physiological and cell biological approaches will be necessary to show whether our current in vitro findings on GSPT/eRF3 indeed reflect its bifunctional properties in living cells.

We reported that 1) ecto-NAD+ glycohydrolase (NADase) activity induced upon differentiation of HL-60 cells is localized on the extracellular carboxyl-terminal side of CD38 and that 2) CD38 ligation by specific mAbs is followed by protein tyrosine phosphorylation in the cells. The strategy selected for identifying the relevant catalytic domains of the molecule relies upon the production in COS-7 cells of carboxyl-terminal deletion mutants of CD38. The mutants with fewer than 15 amino acids deleted at the carboxyl terminus of the 300-amino acid wild-type molecule maintained NADase activity, whereas those with more than 27 amino acids deleted did not. The general inference is that the carboxyl-terminal 273-285 sequence bears the site of enzyme activity. Introduction of site-directed mutation of a conserved cysteine residue (Cys275), located in the 273-285 sequence, completely abolished NADase activity. The second issue resolved in this work is the definition of an epitope of the agonistic anti-CD38 mAbs. To this aim, a panel of selected anti-CD38 mAbs was tested using these mutants and various CD38 fragments as the target in immunoblot analyses. All of the epitopes recognized by mAbs inducing protein tyrosine phosphorylation were mapped on an identical site containing the carboxyl-terminal sequence of 273-285. The conclusion is that the discrete carboxyl-terminal sequence identified in the present study not only plays a key role in its ecto-NADase activity, but actually constitutes the epitopes exploited by the agonistic anti-CD38 mAbs for transmembrane signaling.