Membrane signaling and progesterone in female and male osteoblasts. II. Direct involvement of G alpha q/11 coupled to PLC-beta 1 and PLC-beta 3

We have shown that progesterone (10 pM-10 nM) and progesterone covalently b ound to bovine serum albumin (P-CMO BSA; 100 pM-1 mu M) rapidly increased ( within 5 s) the cytosolic free Ca2+ concentration and inositol 1,4,5 trisph osphate (InsP(3)) formation in confluent female and male rat osteoblasts vi a a pertussis toxin-insensitive C-protein. The activation of G-proteins cou pled to effecters such as phospholipase C (PLC) is an early event in the si gnal transduction pathway leading to InsP(3) formation. We used antibodies against the various PLC isoforms to show that only PLC-beta 1 and PLC-beta 3 were involved in the Ca2+ mobilization and InsP(3) formation induced by b oth progestins in female and male osteoblasts, whereas PLC-beta 2, PLC-gamm a 1, and PLC-gamma 2 were not. We also used antibodies against the subunits of heterotrimeric G-proteins to show that the activation of PLC-beta 1 and PLC-beta 3 by both progestins involved the G alpha q/11 subunit, which was insensitive to pertussis toxin, whereas G alpha i, G alpha s, and G beta g amma subunits were not. The membrane effects were independent of the concen tration of nuclear progesterone receptor, because the concentration of nucl ear progesterone receptors was lower in male than in female osteoblasts. Th ese data suggest that progesterone and P-CMO BSA, which does not enter the cell, directly activate G-protein leading to the very rapid formation of se cond messengers without involving the nuclear receptor. (C) 2000 Wiley-Liss . Inc.