Abstract
Monocytic U937 cells were differentiated into mature macrophages in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h at 37°C. We investigated the alterations in the expression of GTP-binding proteins that take place during differentiation of these cells. A 40 KDa α-subunit of the inhibitory G-protein was identified by specific antibodies to Giα-1/2 and Giα-3 on Western blots and also by ADP-ribosylation catalyzed by pertussis toxin. The expression of the 40 KDa Giα subunit was increased 3.4 fold in differentiated cells. The expression of a 43 kDa Gsα subunit identified by Western blotting using specific antibody to Gsα and by ADP-ribosylation in the presence of cholera toxin was increased approximately 2 fold in differentiated cells. A faintly recognizable 46 KDa Gsα subunit was also increased but to a lesser extent (1.3 fold). Small molecular weight GTP-binding proteins identified by [35S]GTPγS binding on nitrocellulose blots were also increased significantly. The PMA-induced expression of Giα-1/2 and Gsα subunits was blocked to control level by both genistein and staurosporine, inhibitors of protein tyrosine kinase and protein kinase C, respectively. However, staurosporine was unable to block the PMA-induced expression of Giα-3; this was blocked only by genistein. These data suggest a role for tyrosine kinase and protein kinase C in the expression of G-proteins during differentiation of U937 cells.