Cap formation in a B-lymphocyte cell line is inhibited by pertussis toxin and phorbol ester

We have examined concanavalin A (Con A)-induced cap formation in a B-lymphocyte derived cell line, LAZ-559. Treatment with pertussis toxin (PT) or phorbol-12-myristate-13-acetate (PMA) prior to exposure of the cells to Con A abolished the capping reaction. The possible role of calcium mobilization was tested using cells pre-loaded with the fluorescent dye Quin2. Both PT and PMA caused inhibition of calcium mobilization at concentrations similar to those observed for the inhibition of capping. The possible identity of the substrate for pertussis toxin was examined by carrying out ADP-ribosylation of the isolated plasma membranes using [alpha-32P]NAD and pertussis toxin. Several bands were observed at molecular weights of 109,000, 43,000, 34,000 and 22,000. Comparative labelling with cholera toxin revealed a separate band at 42,000. The bands at 43,000 and 34,000 are PT specific. Of these, the 43,000 band comigrated with the PT substrate that has been shown to regulate capping in human neutrophils (Lad et al., 1985a, 1986b). PMA-induced phosphorylation was examined in 32P-loaded cells, and multiple bands were observed to be labelled in a dose-dependent manner, at least two of which were very similar in mobility to the PT substrate. Our results suggest that regulation of calcium mobilization and the control of capping via a PMA-sensitive, GTP-binding protein are probably general phenomena observable in multiple cell systems.