Recent studies showed that Fyn is a mediator of the LHR-induced activation of the ERK1/2 cascade in MA-10 cells. Since the LHR is a G protein-coupled receptor and the Src family of kinases can be activated by some Galpha subunits and by the non-visual arrestins we investigated the role of these signaling molecules in the LHR-provoked activation of Fyn. Small interfering RNAs (siRNAs) that target two Galpha subunits that participate in LHR signaling (Galpha(s) and Galpha(11)) and one that targets arrestin-3 were co-transfected with the hLHR in MA-10 cells. We then determined the effects of these siRNAs on the LHR-provoked activation of Fyn, the phosphorylation of FAK (a prominent Fyn substrate) and the release of EGF-like growth factors (a Fyn-mediated process). Expression of the siRNA against Galpha(s) decreased the level of Galpha(s) and LHR-stimulated cAMP production by approximately 50% but did not affect LHR-stimulated Fyn activation or FAK phosphorylation. Likewise, expression of the siRNA against Galpha(11) decreased the level of Galpha(11) and LHR-stimulated inositol phosphate production by approximately 50% but did not affect LHR-stimulated Fyn activation or FAK phosphorylation. Expression of the siRNA against arrestin-3 decreased the level of arrestin-3 and the rate of internalization of hCG by approximately 50% and it also inhibited the LHR-provoked stimulation of Fyn, the phosphorylation of FAK and the release of EGF-like growth factors. These results show that, in MA-10 cells, the hLHR activates Fyn through an arrestin-3-dependent pathway and that this pathway is a mediator of the hLHR-provoked release of EGF-like growth factors.