G protein expression and second messenger formation in human granulosa cells

Abstract

The expression of heterotrimeric (αβγ subunits) GTP-binding regulatory proteins (G proteins) and the activation of G protein-linked receptors in human granulosa cells were investigated. The cells were obtained from stimulated follicles in women undergoing in vitro fertilization and were cultured in serum-supplemented medium. Immunoblotting with specific antibodies showed that granulosa cell membranes express α(S), α(i3) α(i1,2), α(q,11) and β subunits. Three antibodies against α0 failed to detect this protein. The cells responded to hCG and to prostaglandin E2 with a dose-dependent increase in cAMP formation, confirming the functional activation of Gα(S). The α2 adrenoceptor agonist, clonidine, inhibited hCG-stimulated cAMP formation and this effect was blocked with pertussis toxin, thus involving a G(i)-type protein, most likely Gα(i2). Oxytocin provoked an increase in formation of inositol phosphates and intracellular calcium concentration, which was partly pertussis toxin resistant, providing evidence of Gα(q,11) activation. However, a significant component of the response to oxytocin could be blocked by pertussis toxin, indicating G(i)-mediated phospholipase C activation (by either α(i) or βγ subunits). These data demonstrate the presence of G proteins in granulosa cells and suggest a complex regulation of hormonal signalling. The concentration of cAMP in these cells depended on the balance of Gα(S):Gα(i) activation, whereas activation of the inositol phospholipid pathway and rises in intracellular calcium involved both G(q,11) and G(i) pathways.